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Jonas Naumann, Kresten Singer, Siddharth Shukla, Alok Maurya, Stefan Schlichter, Imre Szenti, Akos Kukovecz, Amit Rawal, Mareike Zink:

Sustainable Nonwoven Scaffolds Engineered with Recycled Carbon Fiber for Enhanced Biocompatibility and Cell Interaction: From Waste to Health

Abstract: Carbon fibers, driven by ever-increasing demand, are contributing to a continuous rise in the generation of waste and byproducts destined for landfills or incineration. Recycling carbon fibers presents a promising strategy for reducing carbon emissions and conserving resources, thus contributing to more sustainable waste management practices. Discovering applications of recycled carbon fibers (rCFs) would inevitably accelerate the targeted integration of sustainable materials, fostering a circular economy. Herein, we have engineered rCF-based needlepunched nonwoven scaffolds and their blends with polypropylene (PP) fibers, providing the first example of investigating their interactions with human lung epithelial cells (Calu-3) and murine fibroblast cells (NIH/3T3). To promote the adsorption of extracellular matrix proteins such as laminin, these three-dimensional (3D) nonwoven scaffolds are designed and developed to feature tunable porous characteristics and wetting properties. Although cell adhesion and laminin adsorption are minimal on PP fibers, cells are preferentially organized on the rCFs. These nonwovens, composed exclusively of rCFs or their blends with PP fibers, exhibit no cytotoxic effects, with both cell types showing proliferation on the scaffolds and a progressive increase in cell numbers over time. Cell viability and apoptosis assays are also employed to comprehensively evaluate biocompatibility. Thus, our study proves rCF-based nonwoven scaffolds as potential candidates for artificial lung tissue scaffolds.  DOI: 10.1021/acsabm.4c01475 Journal Homepage

ACS Appl. Bio Mater. 2025

Senol Dogan, Jenny Leopold, Daniel T. Hoffmann, Hans Kubitschke, Eliane Blauth, Carlotta Ficorella, Amalie Zschau, Jürgen Schiller, Josef A. Käs:

Identification of Lipid Droplet-Associated Genes in Breast Cancer Patients

Abstract: Lipid droplets (LDs) are known to be involved in the invasion and migration of breast cancer (BC) cells. This study aimed to identify LD-associated genes as prognostic markers in BC through comprehensive literature research and integration with lipid composition studies in BC cell lines. The GEPIA platform was used to analyze the differential expression of LD-associated genes in BC. The lipid composition of cell lines (MCF-10A, MDA-MB 436 and 231) was obtained by extraction and thin-layer chromatography coupled with mass spectrometry (MS). Additionally, cell lines were co-cultured with fatty tissue and analyzed by confocal fluorescence microscopy. A total of 143 genes were identified as LD-associated genes through literature research and were subsequently analyzed using GEPIA. Among these, three genes were found to be over-expressed and 45 under-expressed in BC. Notably, FABP7 showed a statistically significant rank for all bioinformatics criteria as a prognostic factor. Experimental results showed only minor changes from MCF-10A to both MDA-MB cell lines for apolar lipids (triacylglycerols and cholesteryl esters) compared to phospholipids (PLs). Microscopic analyses showed that MDA-MB-231 had larger LDs compared to MCF-10A after 10 days of cultivation. Our bioinformatics analysis identified 26 genes that play important roles in metastatic transition in BC via LD-related mechanisms, though these findings could be only partially confirmed by experimental lipid compositional analyses, so far.   DOI: 10.3390/lipidology1010005 Journal Homepage

Lipidology 1(1):52-74 (2024)

Eliane Blauth, Steffen Grosser, Frank Sauer, Mario Merkel, Hans Kubitschke, Enrico Warmt, Erik W. Morawetz, Philip Friedrich, Benjamin Wolf, Susanne Briest, Grit Gesine Ruth Hiller, Lars-Christian Horn, Bahriye Aktas, Josef A. Käs:

Different contractility modes control cell escape from multicellular spheroids and tumor explants

Abstract: Cells can adapt their active contractile properties to switch between dynamical migratory states and static homeostasis. Collective tissue surface tension, generated among others by the cortical contractility of single cells, can keep cell clusters compact, while a more bipolar, anisotropic contractility is predominantly used by e.g. mesenchymal cells to pull themselves into the extracellular matrix. Here, we investigate how these two contractility modes relate to cancer cell escape into the ECM. We compare multicellular spheroids from a panel of breast cancer cell lines with primary tumor explants from breast and cervical cancer patients by measuring matrix contraction and cellular spreading into ECM mimicking collagen matrices. Our results in spheroids suggest that tumor aggressiveness is associated with elevated contractile traction and reduced active tissue surface tension, allowing cancer cell escape. We show that it is not a binary switch but rather the interplay between these two contractility modes that is essential during this process. We provide further evidence in patient-derived tumor explants that these two contractility modes impact cancer cells' ability to leave cell clusters within a primary tumor. Our results indicate that cellular contractility is an essential factor during the formation of metastases and thus may be suitable as a prognostic criterion for the assessment of tumor aggressiveness.   DOI: 10.1063/5.0188186 Journal Homepage

APL Bioengineering 8, 026110 (2024)

Paul Mollenkopf, Dusan Prascevic, Martin Glaser, David M. Smith, Jörg Schnauß:

A Science Friction Story: Molecular Interactions in Semiflexible Polymer Networks

Abstract: Established model theories, developed to capture the mechanical behavior of soft, complex materials composed of semiflexible polymers, assume that entropic interactions between filaments are primarily responsible for determining the mechanical response. In recent studies, the generally accepted tube model has been challenged in terms of this basic assumption about filament-filament interactions, but also because of its predictions regarding the frequency dependence of the elastic modulus in the intermediate frequency regime. A central question is how molecular interactions and friction between network constituents influence the rheological response of isotropic entangled networks of semiflexible polymers. It has been previously shown that friction forces between aligned pairs of actin filaments are not negligible. Here, the influence of friction forces and attractive interactions on network rheology is systematically investigated by means of targeted surface modification. It is shown that these forces have a qualitative and quantitative influence on the viscoelastic properties of semiflexible polymer networks and contribute to their response to nonlinear deformations. By comparing two polymer model systems with respect to their surface compositions, a possible explanation is given about the origin of acting forces on a molecular level.   DOI: 10.1002/admi.202300623 Journal Homepage

Adv. Mat. Interfaces, 11(5), 2300623 (2024)

Xiaofan Xie, Frank Sauer, Steffen Grosser, Jürgen Lippoldt, Enrico Warmt, Amit Das, Dapeng Bi, Thomas Fuhs, Josef A. Käs:

Effect of Non-linear Strain Stiffening in eDAH and Unjamming

Abstract: In cell clusters, the prominent factors at play encompass contractility-based enhanced tissue surface tension, and cell unjamming transition. The former effect pertains to boundary effect, while the latter constitutes a bulk effect. Both effect share outcomes of inducing significant elongation in cells. This elongation is so substantial that it surpasses the limits of linear elasticity, thereby giving rise to additional effects. To investigate these effects, we employ Atomic Force Microscopy (AFM) to analyze how the mechanical properties of individual cells change under such considerable elongation. Our selection of cell lines includes MCF-10A, chosen for its pronounced demonstration of the extended differential adhesion hypothesis (eDAH), and MDA-MB-436, selected due to its manifestation of cell unjamming behavior. In the AFM analyses, we observe a common trend in both cases: as elongation increases, bot cell lines exhibit strain stiffening. Notably, this effect is more prominent in MCF-10A compared to MDA-MB-436. Subsequently, we employ AFM on a dynamic range of 1-200Hz to probe the mechanical characteristics of cell spheroids, focusing on both surface and bulk mechanics. Our findings align with the results from single cell investigations. Specifically, MCF-10A cells, characterized by strong contractile tissue tension, exhibit the greatest stiffness on their surface. Conversely, MDA-MB-436 cells, which experience significant elongation, showcase their highest stiffness within the bulk region. Consequently, the concept of single cell strain stiffening emerges as a crucial element in understanding the mechanics of multicellular spheroids (MCS), even in the case of MDA-MB-436 cells, which are comparatively softer in nature.   DOI: 10.1039/D3SM00630A Journal Homepage

Soft Matter, 20, 1996-2007 (2024)

Anne-Sophie Wegscheider, Irina Wojahn, Pablo Gottheil, Michael Spohn, Josef A. Käs, Olga Rosin, Bernhard Ulm, Peter Nollau, Christoph Wagner, Axel Niendorf, Gerrit Wolters-Eisfeld:

CD301 and LSECtin glycan-binding receptors of innate immune cells serve as prognostic markers and potential predictors of immune response in breast cancer subtypes

Abstract: Glycosylation is a prominent posttranslational modification, and alterations in glycosylation are a hallmark of cancer. Glycan-binding receptors, primarily expressed on immune cells, play a central role in glycan recognition and immune response. Here, we used the recombinant C-type glycan-binding receptors CD301, Langerin, SRCL, LSECtin, and DC-SIGNR to recognize their ligands on tissue microarrays (TMA) of a large cohort (n = 1859) of invasive breast cancer of different histopathological types to systematically determine the relevance of altered glycosylation in breast cancer. Staining frequencies of cancer cells were quantified in an unbiased manner by a computer-based algorithm. CD301 showed the highest overall staining frequency (40%), followed by LSECtin (16%), Langerin (4%) and DC-SIGNR (0.5%). By Kaplan-Meier analyses, we identified LSECtin and CD301 as prognostic markers in different breast cancer subtypes. Positivity for LSECtin was associated with inferior disease-free survival in all cases, particularly in estrogen receptor positive (ER+) breast cancer of higher histological grade. In triple negative breast cancer, positivity for CD301 correlated with a worse prognosis. Based on public RNA single-cell sequencing data of human breast cancer infiltrating immune cells, we found CLEC10A (CD301) and CLEC4G (LSECtin) exclusively expressed in distinct subpopulations, particularly in dendritic cells and macrophages, indicating that specific changes in glycosylation may play a significant role in breast cancer immune response and progression.   DOI: 10.1093/glycob/cwae003 Journal Homepage

Glycobiology, April 1;34(3) (2024)

Dimitrij Tschodu, Jürgen Lippoldt, Pablo Gottheil, Anne-Sophie Wegscheider, Josef A. Käs, Axel Niendorf:

Re-evaluation of publicly available gene-expression databases using machine-learning yields a maximum prognostic power in breast cancer

Abstract: Gene expression signatures refer to patterns of gene activities and are used to classify different types of cancer, determine prognosis, and guide treatment decisions. Advancements in high-throughput technology and machine learning have led to improvements to predict a patient's prognosis for different cancer phenotypes. However, computational methods for analyzing signatures have not been used to evaluate their prognostic power. Contention remains on the utility of gene expression signatures for prognosis. The prevalent approaches include random signatures, expert knowledge, and machine learning to construct an improved signature. We unify these approaches to evaluate their prognostic power. Re-evaluation of publicly available gene-expression data from 8 databases with 9 machine-learning models revealed previously unreported results. Gene-expression signatures are confirmed to be useful in predicting a patient's prognosis. Convergent evidence from ≈10,000 signatures implicates a maximum prognostic power. By calculating the concordance index, which measures how well patients with different prognoses can be discriminated, we show that a signature can correctly discriminate patient's prognoses no more than 80% of the time. Additionally, we show that more than 50% of the potentially available information is still missing at this value. We surmise that an accurate prognosis must incorporate molecular, clinical, histological, and other complementary factors.   DOI: 10.21203/rs.3.rs-2704246/v1 Journal Homepage

Scientific Reports 13:16402 (2023)

Thomas Fuhs, Bianca Flachmeyer, Martin Krueger, Alexandra Blietz, Wolfgang Härtig, Dominik Michalski:

Combining atomic force microscopy and fluorescence-based techniques to explore mechanical properties of naive and ischemia-affected brain regions in mice

Abstract: Knowledge of the brain's structure and function is essential for understanding processes in health and disease. Histochemical and fluorescence-based techniques have proven beneficial in characterizing brain regions and cellular compositions in pre-clinical research. Atomic force microscopy (AFM) has been introduced for mechanical tissue characterization, which may also help investigate pathophysiological aspects in disease-related models such as stroke. While combining AFM and fluorescence-based techniques, this study explored the mechanical properties of naive and ischemic brain regions in mice. Ischemia-affected regions were identified by the green signal of fluorescein isothiocyanate-conjugated albumin. A semi-automated protocol based on a brain atlas allowed regional allocations to the neocortex, striatum, thalamus, hypothalamus, hippocampus, and fiber tracts. Although AFM led to varying measurements, intra-individual analyses indicated a gradually increased tissue stiffness in the neocortex compared to subcortical areas, i.e., the striatum and fiber tracts. Regions affected by ischemia predominantly exhibited an increased tissue stiffness compared to those of the contra-lateral hemisphere, which might be related to cellular swelling. This study indicated intra-individual differences in mechanical properties among naive and ischemia-affected brain regions. The combination of AFM, semi-automated regional allocations, and fluorescence-based techniques thus qualifies for mechanical characterizations of the healthy and disease-affected brain in pre-clinical research.   DOI: 10.1038/s41598-023-39277-1 Journal Homepage

Scientific Reports, 13:12774 (2023)

Paul Mollenkopf, Dusan Prascevic, Thomas M. Bayerl, Josef A. Käs, Jörg Schnauß:

Heavy water induces bundling in entangled actin networks

Abstract: Heavy water is known to affect many different biological systems, with the most striking effects observed at the cellular level. Many dynamic processes, such as migration or invasion, but also central processes of cell proliferation are measurably inhibited by the presence of deuterium oxide (D2O). Furthermore, individual cell deformabilities are significantly decreased upon D2O treatment. In order to understand the origin of these effects, we studied entangled filamentous actin networks, a commonly used model system for the cytoskeleton, which is considered a central functional element for dynamic cellular processes. Using bulk shear rheology to extract rheological signatures of reconstituted actin networks at varying concentrations of D2O, we found a non-monotonic behavior, which is explainable by a drastic change in the actin network architecture. Applying light scattering and fluorescence microscopy, we were able to demonstrate that the presence of deuterium oxide induces bundling in reconstituted entangled networks of filamentous actin. This constitutes an entirely novel and previously undescribed actin bundling mechanism.   DOI: 10.1039/D3RA03917J Journal Homepage

RSC Adv., 13, 24795-24800 (2023)

Frank Sauer, Steffen Grosser, Mehrgan Shahryari, Alexander Hayn, Jing Guo, Jürgen Braun, Susanne Briest, Benjamin Wolf, Bahriye Aktas, Lars-Christian Horn, Ingolf Sack, Josef A. Käs:

Changes in Tissue Fluidity Predict Tumor Aggressiveness In Vivo

Abstract: Cancer progression is caused by genetic changes and associated with various alterations in cell properties, which also affect a tumor's mechanical state. While an increased stiffness has been well known for long for solid tumors, it has limited prognostic power. It is hypothesized that cancer progression is accompanied by tissue fluidization, where portions of the tissue can change position across different length scales. Supported by tabletop magnetic resonance elastography (MRE) on stroma mimicking collagen gels and microscopic analysis of live cells inside patient derived tumor explants, an overview is provided of how cancer associated mechanisms, including cellular unjamming, proliferation, microenvironment composition, and remodeling can alter a tissue's fluidity and stiffness. In vivo, state-of-the-art multifrequency MRE can distinguish tumors from their surrounding host tissue by their rheological fingerprints. Most importantly, a meta-analysis on the currently available clinical studies is conducted and universal trends are identified. The results and conclusions are condensed into a gedankenexperiment about how a tumor can grow and eventually metastasize into its environment from a physics perspective to deduce corresponding mechanical properties. Based on stiffness, fluidity, spatial heterogeneity, and texture of the tumor front a roadmap for a prognosis of a tumor's aggressiveness and metastatic potential is presented.   DOI: 10.1002/advs.202303523 Journal Homepage

Adv. Sci. 2303523 (2023)

Pablo Gottheil, Jürgen Lippoldt, Steffen Grosser, Frederic Renner, Mohamad Saibah, Dimitrij Tschodu, Anne-Kathrin Poßöel, Anne-Sophie Wegscheider, Bernhard Ulm, Kay Friedrichs, Christoph Lindner, Christoph Engel, Markus Löffler, Benjamin Wolf, Michael Höckel, Bahriye Aktas, Hans Kubitschke, Axel Niendorf, Josef A. Käs:

State of Cell Unjamming Correlates with Distant Metastasis in Cancer Patients

Abstract: Pathological morphological changes in tumor tissue enable collective cancer cell unjamming, a cellular motility transition. However, fundamental questions remain: Is unjamming essential for tumor progression? Which different unjamming states can be found in patients? Here, vital cell tracking in patient-derived solid tumor explants (N=16) reveals that states of cell unjamming can be recognized by elongated cell and nucleus shape (CeNuS) and low nucleus number density. These static variables serve as a morphodynamic link to map the broad range of morphologies and associated motility states found in histological slides of 1,380 breast cancer patients to generate a comprehensive state diagram of cancer cell unjamming. An increase in predicted cell motility in primary tumors through unjamming significantly correlates with distant metastases that may even occur a decade later. Patient risk groups are quantified via a decision boundary in the state space found by machine learning. The resulting clinical prognostic potential is evaluated using a range of quantifiers, including Harrel's concordance index. Using multivariable Cox models, we find that cell unjamming as a prognostic parameter adds a 26% information gain in the concordance index when combined with the established prognostic criteria (tumor diameter, tumor grade, lymph node status) used in the Nottingham index. Unjamming complements the information on affected lymph nodes in patients regarding metastatic risk. The derived state diagram of cancer cell unjamming reconciles conflicting observations regarding shape- or density-induced unjamming and stresses the nuclei's mechanical importance, which is not considered in current theories of cell unjamming. We conclude that cancer cell unjamming is part of the metastatic cascade; thus, an emergent physical phenomenon contributes to tumor progression.   DOI: 10.1103/PhysRevX.13.031003 Journal Homepage

Phys. Rev. X 13, 031003 (2023)

Hannah Marie Eichholz, Alissa Cornelis, Benjamin Wolf, Hanna Grubitzsch, Philip Friedrich, Ahmad Makky, Bahriye Aktas, Josef A. Käs, Holger Stepan:

Anatomy of the fetal membranes: insights from spinning disk confocal microscopy

Abstract: Purpose: The fetal membranes are essential for the maintenance of pregnancy, and their integrity until parturition is critical for both fetal and maternal health. Preterm premature rupture of the membranes (pPROM) is known to be an indicator of preterm birth, but the underlying architectural and mechanical changes that lead to fetal membrane failure are not yet fully understood. The aim of this study was to gain new insights into the anatomy of the fetal membrane and to establish a tissue processing and staining protocol suitable for future prospective cohort studies. Methods: In this proof of principle study, we collected fetal membranes from women undergoing vaginal delivery or cesarean section. Small membrane sections were then fixed, stained for nucleic acids, actin, and collagen using fluorescent probes, and subsequently imaged in three dimensions using a spinning disk confocal microscope. Results: Four fetal membranes of different types were successfully processed and imaged after establishing a suitable protocol. Cellular and nuclear outlines are clearly visible in all cases, especially in the uppermost membrane layer. Focal membrane (micro) fractures could be identified in several samples. Conclusion: The presented method proves to be well suited to determine whether and how the occurrence of membrane (micro) fractures and cellular jamming correlate with the timing of membrane rupture and the mode of delivery. In future measurements, this method could be combined with mechanical probing techniques to compare optical and mechanical sample information.   DOI: 10.1007/s00404-023-07070-0 Journal Homepage

Arch Gynecol Obstet (2023)

Martin Glaser, Paul Mollenkopf, Dusan Prascevic, Catarina Ferraz, Josef A. Käs, Jörg Schnauß, David M. Smith:

Systematic altering of semiflexible DNA-based polymer networks via tunable crosslinking

Abstract:In order to understand and predict the mechanical behaviours of complex, soft biomaterials such as cells or stimuli-responsive hydrogels, it is important to connect how the nanoscale properties of their constituent components impact those of the bulk material. Crosslinked networks of semiflexible polymers are particularly ubiquitous, being underlying mechanical components of biological systems such as cells or ECM, as well as many synthetic or biomimetic materials. Cell-derived components such as filamentous biopolymers or protein crosslinkers are readily available and well-studied model systems. However, as evolutionarily derived materials, they are constrained to a fixed set of structural parameters such as the rigidity and size of the filaments, or the valency and strength of binding of crosslinkers forming inter-filament connections. By implementing a synthetic model system based on the self-assembly of DNA oligonucleotides into nanometer-scale tubes and simple crosslinking constructs, we used the thermodynamic programmability of DNA hybridization to explore the impact of binding affinity on bulk mechanical response. Stepwise tuning the crosslinking affinity over a range from transient to thermodynamically stable shows an according change in viscoelastic behaviour from loosely entangled to elastic, consistent with models accounting for generalized inter-filament interactions. While characteristic signatures of concentration-dependent changes in network morphology found in some other natural and synthetic filament-crosslinker systems were not apparent, the presence of a distinct elasticity increase within a narrow range of conditions points towards potential subtle alterations of crosslink-filament architecture. Here, we demonstrate a new synthetic approach for gaining a deeper understanding of both biological as well as engineered hydrogel systems.   DOI: 10.1039/D2NR05615A Journal Homepage

Nanoscale, Advance Article (2023)

Jessica S. Freitag, C. Möser, Robel Belay, Basma Altattan, Nico Grasse, Bhanu Kiran Pothineni, Jörg Schnauß, David M. Smith:

Integration of functional peptides into nucleic acid-based nanostructures

Abstract:In many applications such as diagnostics and therapy development, small peptide fragments consisting of only a few amino acids are often attractive alternatives to bulky proteins. This is due to factors such as the ease of scalable chemical synthesis and numerous methods for their discovery. One drawback of using peptides is that their activity can often be negatively impacted by the lack of a rigid, 3D stabilizing structure provided by the rest of the protein. In many cases, this can be alleviated by different methods of rational templating onto nanomaterials, which provides additional possibilities to use concepts of multivalence or rational nano-engineering to enhance or even create new types of function or structure. In recent years, nanostructures made from the self-assembly of DNA strands have been used as scaffolds to create functional arrangements of peptides, often leading to greatly enhanced biological activity or new material properties. This review will give an overview of nano-templating approaches based on the combination of DNA nanotechnology and peptides. This will include both bioengineering strategies to control interactions with cells or other biological systems, as well as examples where the combination of DNA and peptides has been leveraged for the rational design of new functional materials.   DOI: 10.1039/D2NR05429A Journal Homepage

Nanoscale, Advance Article (2023)

Anna S. Morr, Marcin Nowicki, Gergely Bertalan, Rafaela Vieira Silva, Carmen Infante Duarte, Stefan Paul Koch, Philipp Boehm-Sturm, Ute Krügel, Jürgen Braun, Barbara Steiner, Josef A. Käs, Thomas Fuhs, Ingolf Sack:

Mechanical properties of murine hippocampal subregions investigated by atomic force microscopy and in vivo magnetic resonance elastography

Abstract: The hippocampus is a very heterogeneous brain structure with different mechanical properties reflecting its functional variety. In particular, adult neurogenesis in rodent hippocampus has been associated with specific viscoelastic properties in vivo and ex vivo. Here, we study the microscopic mechanical properties of hippocampal subregions using ex vivo atomic force microscopy (AFM) in correlation with the expression of GFP in presence of the nestin promoter, providing a marker of neurogenic activity. We further use magnetic resonance elastography (MRE) to investigate whether in vivo mechanical properties reveal similar spatial patterns, however, on a much coarser scale. AFM showed that tissue stiffness increases with increasing distance from the subgranular zone (p = 0.0069), and that stiffness is 39% lower in GFP than non-GFP regions (p = 0.0004). Consistently, MRE showed that dentate gyrus is, on average, softer than Ammon's horn (shear wave speed = 3.2 ± 0.2 m/s versus 4.4 ± 0.3 m/s, p = 0.01) with another 3.4% decrease towards the subgranular zone (p = 0.0001). The marked reduction in stiffness measured by AFM in areas of high neurogenic activity is consistent with softer MRE values, indicating the sensitivity of macroscopic mechanical properties in vivo to micromechanical structures as formed by the neurogenic niche of the hippocampus.   DOI: 10.1038/s41598-022-21105-7 Journal Homepage

Scientific Reports, 12:6723 (2022)

Jan Frenzel, Astrid Kupferer, Mareike Zink, Stefan G. Mayr:

Laminin Adsorption and Adhesion of Neurons and Glial Cells on Carbon Implanted Titania Nanotube Scaffolds for Neural Implant Applications

Abstract:Interfacing neurons persistently to conductive matter constitutes one of the key challenges when designing brain-machine interfaces such as neuroelectrodes or retinal implants. Novel materials approaches that prevent occurrence of loss of long-term adhesion, rejection reactions, and glial scarring are highly desirable. Ion doped titania nanotube scaffolds are a promising material to fulfill all these requirements while revealing sufficient electrical conductivity, and are scrutinized in the present study regarding their neuron-material interface. Adsorption of laminin, an essential extracellular matrix protein of the brain, is comprehensively analyzed. The implantation-dependent decline in laminin adsorption is revealed by employing surface characteristics such as nanotube diameter, ζ-potential, and surface free energy. Moreover, the viability of U87-MG glial cells and SH-SY5Y neurons after one and four days are investigated, as well as the material's cytotoxicity. The higher conductivity related to carbon implantation does not affect the viability of neurons, although it impedes glial cell proliferation. This gives rise to novel titania nanotube based implant materials with long-term stability, and could reduce undesirable glial scarring.   DOI: 10.3390/nano12213858 Journal Homepage

Nanomaterials, 12(21):3858 (2022)

Thomas Fuhs, Franziska Wetzel, Anatol W. Fritsch, Xinzhi Li, Roland Stange, Steve Pawlizak, Tobias R. Kießling, Erik Morewetz, Steffen Grosser, Frank Sauer, Jürgen Lippoldt, Frederic Renner, Sabrina Friebe, Mareike Zink, Klaus Bendrat, Jürgen Braun, Maja H. Oktay, John Condeelis, Susanne Briest, Benjamin Wolf, Lars-Christian Horn, Michael Höckel, Bahriye Aktas, M. Cristina Marchetti, M. Lisa Manning, Axel Niendorf, Dapeng Bi, Josef Käs:

Rigid tumours contain soft cancer cells

Abstract:Palpation utilizes the fact that solid breast tumours are stiffer than the surrounding tissue. However, cancer cells tend to soften, which may enhance their ability to squeeze through dense tissue. This apparent paradox proposes two contradicting hypotheses: either softness emerges from adaptation to the tumour's microenvironment or soft cancer cells are already present inside a rigid primary tumour mass giving rise to cancer cell motility. We investigate primary tumour explants from patients with breast and cervix carcinomas on multiple length scales. We find that primary tumours are highly heterogeneous in their mechanical properties on all scales from the tissue level down to individual cells. This results in a broad rigidity distribution - from very stiff cells to cells softer than those found in healthy tissue - that is shifted towards a higher fraction of softer cells. Atomic-force-microscopy-based tissue rheology reveals that islands of rigid cells are surrounded by soft cells. The tracking of vital cells confirms the coexistence of jammed and unjammed areas in tumour explants. Despite the absence of a percolated backbone of stiff cells and a large fraction of unjammed, motile cells, cancer cell clusters show a heterogeneous solid behaviour with a finite elastic modulus providing mechanical stability.   DOI: 10.1038/s41567-022-01755-0 Journal Homepage

Nature Physics, Sept. (2022)

Jonas Naumann, Nicklas Koppe, Ulrich H. Thome, Mandy Laube, Mareike Zink:

Mechanical properties of the premature lung: From tissue deformation under load to mechanosensitivity of alveolar cells

Abstract:Many preterm infants require mechanical ventilation as life-saving therapy. However, ventilation-induced overpressure can result in lung diseases. Considering the lung as a viscoelastic material, positive pressure inside the lung results in increased hydrostatic pressure and tissue compression. To elucidate the effect of positive pressure on lung tissue mechanics and cell behavior, we mimic the effect of overpressure by employing an uniaxial load onto fetal and adult rat lungs with different deformation rates. Additionally, tissue expansion during tidal breathing due to a negative intrathoracic pressure was addressed by uniaxial tension. We found a hyperelastic deformation behavior of fetal tissues under compression and tension with a remarkable strain stiffening. In contrast, adult lungs exhibited a similar response only during compression. Young's moduli were always larger during tension compared to compression, while only during compression a strong deformation-rate dependency was found. In fact, fetal lung tissue under compression showed clear viscoelastic features even for small strains. Thus, we propose that the fetal lung is much more vulnerable during inflation by mechanical ventilation compared to normal inspiration. Electrophysiological experiments with different hydrostatic pressure gradients acting on primary fetal distal lung epithelial cells revealed that the activity of the epithelial sodium channel (ENaC) and the sodium-potassium pump (Na,K-ATPase) dropped during pressures of 30 cmH2O. Thus, pressures used during mechanical ventilation might impair alveolar fluid clearance important for normal lung function.   DOI: 10.3389/fbioe.2022.964318   Journal Homepage

Front. Bioeng. Biotechnol., 16 September (2022)

Alice Abend, Chelsie Steele, Sabine Schmidt, Ronny Frank, Heinz-Georg Jahnke, Mareike Zink:

Neuronal and glial cell co-culture organization and impedance spectroscopy on nanocolumnar TiN films for lab-on-a-chip devices

Abstract:Lab-on-a-chip devices, such as multielectrode arrays (MEAs), offer great advantages to study function and behavior of biological cells, such as neurons, outside the complex tissue structure. Nevertheless, in vitro systems can only succeed if they represent realistic conditions such as cell organization as similarly found in tissues. In our study, we employ a co-culture system of neuron-like (SH-SY5Y) and glial-like (U-87 MG) cells with various neuron-glial ratios to model different brain regions with different cellular compositions in vitro. We find that cell behavior in terms of cellular organization, as well as proliferation, depends on neuron-glial cell ratio, as well as the underlying substrate material. In fact, nanocolumnar tita- nium nitride (TiN nano), which exhibits improved electric properties for neural recording on MEA, shows improved biocompatible features compared to indium tin oxide (ITO). Moreover, electrochemical impe- dance spectroscopy experiments allow us to monitor cellular processes label-free in real-time over several days with multielectrode arrays. Additionally, electrochemical impedance experiments reveal superiority of TiN with nanocolumnar surface modification in comparison with ITO. TiN nano exhi- bits enhanced relative cell signals and improved signal-to-noise ratio, especially for smaller electrode sizes, which makes nanocolumnar TiN a promising candidate for research on neural recording and stimulation.   DOI: 10.1039/d2bm01066f   Journal Homepage

Biomaterials Sciences, 10:5719 - 5730 (2022)

Friedrich Fabian Spukti, Jörg Schnauß:

Large and stable: actin aster networks formed via entropic forces

Abstract:Biopolymer networks play a major role as part of the cytoskeleton. They provide stable structures and act as a medium for signal transport. These features encourage the application of such networks as organic computation devices. While research on this topic is not advanced yet, previous results are very promising. The protein actin in particular appears advantageous. It can be arranged to various stable structures and transmit several signals. In this study aster shaped networks were self-assembled via entropic forces by the crowding agent methyl cellulose. These networks are characterised by a regular and uniquely thick bundle structure, but have so far only been accounted in droplets of 100 µm diameter. We report now regular asters in an area of a few mm2 that could be observed even after months. Such stability outside of an organism is striking and underlines the great potential actin aster networks display.   DOI: 10.3389/fchem.2022.899478   Journal Homepage

Front. Chem. 10:899478 (2022)

Henrike Herzog, Senol Dogan, Bahriye Aktas, Ivonne Nel:

Targeted Sequencing of Plasma-Derived vs. Urinary cfDNA from Patients with Triple-Negative Breast Cancer

Abstract:Circulating cell-free DNA displays vast potential to capture the entire genetic landscape of a tumor and to characterize intratumoral heterogeneity, providing a minimally invasive alternative to tissue biopsy. Several studies have demonstrated the potential of cell-free DNA in the plasma of breast cancer patients. In contrast, very little is known about the utility of urine as an even more patient-convenient analyte for these applications. In this pilot study, we investigated plasma- derived and matching urinary cell-free DNA samples obtained from 15 presurgical triple-negative breast cancer patients using a targeted sequencing approach to identify breast-cancer-related genetic alterations in both body fluids. Taken together, our results indicated that both body fluids appear to be valuable sources bearing complementary information concerning the genetic tumor profile, which might be relevant for disease monitoring and individual treatment decisions.   DOI: 10.3390/cancers14174101   Journal Homepage

Cancers 14(17):4101 (2022)

Federico Sala, Carlotta Ficorella, Roberto Osellame, Josef A. Käs, Rebeca Martinez Vázquez:

Microfluidic Lab-on-a-Chip for Studies of Cell Migration under Spatial Confinement

Abstract:Understanding cell migration is a key step in unraveling many physiological phenomena and predicting several pathologies, such as cancer metastasis. In particular, confinement has been proven to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists' analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical and spatial stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely the soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in vitro migration studies under spatial confinement in microfluidic devices.   DOI: 10.3390/bios12080604   Journal Homepage

Biosensors 12(8) 604 (2022)

Senol Dogan, Emrulla Spahiu, Anis Cilic:

Structural Analysis of microRNAs in Myeloid Cancer Reveals Consensus Motifs

Abstract:MicroRNAs (miRNAs) are short non-coding RNAs that function in post-transcriptional gene silencing and mRNA regulation. Although the number of nucleotides of miRNAs ranges from 17 to 27, they are mostly made up of 22 nucleotides. The expression of miRNAs changes significantly in cancer, causing protein alterations in cancer cells by preventing some genes from being translated into proteins. In this research, a structural analysis of 587 miRNAs that are differentially expressed in myeloid cancer was carried out. Length distribution studies revealed a mean and median of 22 nucleotides, with an average of 21.69 and a variance of 1.65. We performed nucleotide analysis for each position where Uracil was the most observed nucleotide and Adenine the least observed one with 27.8% and 22.6%, respectively. There was a higher frequency of Adenine at the beginning of the sequences when compared to Uracil, which was more frequent at the end of miRNA sequences. The purine content of each implicated miRNA was also assessed. A novel motif analysis script was written to detect the most frequent 3-7 nucleotide (3-7n) long motifs in the miRNA dataset. We detected CUG (42%) as the most frequent 3n motif, CUGC (15%) as a 4n motif, AGUGC (6%) as a 5n motif, AAGUGC (4%) as a 6n motif, and UUUAGAG (4%) as a 7n motif. Thus, in the second part of our study, we further characterized the motifs by analyzing whether these motifs align at certain consensus sequences in our miRNA dataset, whether certain motifs target the same genes, and whether these motifs are conserved within other species. This thorough structural study of miRNA sequences provides a novel strategy to study the implications of miRNAs in health and disease. A better understanding of miRNA structure is crucial to developing therapeutic settings.   DOI: 10.3390/genes13071152   Journal Homepage

Genes (13) 7, 1152 (2022)

Tina Händler, Cary Tutmarc, Jessica S. Freitag, David M. Smith, Jörg Schnauß:

Constraint Release for Reptating Filaments in Semiflexible Networks Depends on Background Fluctuations

Abstract:Entangled semiflexible polymer networks are usually described by the tube model, although this concept has not been able to explain all experimental observations. One of its major shortcomings is neglecting the thermal fluctuations of the polymers surrounding the examined test filament, such that disentanglement effects are not captured. In this study, we present experimental evidence that correlated constraint release which has been predicted theoretically occurs in entangled, but not in crosslinked semiflexible polymer networks. By tracking single semiflexible DNA nanotubes embedded both in entangled and crosslinked F-actin networks, we observed different reptation dynamics in both systems, emphasizing the need for a revision of the classical tube theory for entangled polymer solutions.   DOI: 10.3390/polym14040707   Journal Homepage

Polymers 2022, 14(4), 707

Dimitrij Tschodu, Bernhard Ulm, Klaus Bendrat, Jürgen Lippoldt, Pablo Gottheil, Josef A. Käs, Axel Niendorf:

Comparative analysis of molecular signatures reveals a hybrid approach in breast cancer: Combining the Nottingham Prognostic Index with gene expressions into a hybrid signature

Abstract:The diagnosis of breast cancer-including determination of prognosis and prediction-has been traditionally based on clinical and pathological characteristics such as tumor size, nodal status, and tumor grade. The decision-making process has been expanded by the recent introduction of molecular signatures. These signatures, however, have not reached the highest levels of evidence thus far. Yet they have been brought to clinical practice based on statistical significance in prospective as well as retrospective studies. Intriguingly, it has also been reported that most random sets of genes are significantly associated with disease outcome. These facts raise two highly relevant questions: What information gain do these signatures procure? How can one find a signature that is substantially better than a random set of genes? Our study addresses these questions. To address the latter question, we present a hybrid signature that joins the traditional approach with the molecular one by combining the Nottingham Prognostic Index with gene expressions in a data-driven fashion. To address the issue of information gain, we perform careful statistical analysis and comparison of the hybrid signature, gene expression lists of two commercially available tests as well as signatures selected at random, and introduce the Signature Skill Score-a simple measure to assess improvement on random signatures. Despite being based on in silico data, our research is designed to be useful for the decision-making process of oncologists and strongly supports association of random signatures with outcome. Although our study shows that none of these signatures can be considered as the main candidate for providing prognostic information, it also demonstrates that both the hybrid signature and the gene expression list of the OncotypeDx signature identify patients who may not require adjuvant chemotherapy. More importantly, we show that combining signatures substantially improves the identification of patients who do not need adjuvant chemotherapy.   DOI: 10.1371/journal.pone.0261035   Journal Homepage

PLoS ONE 17(2): e0261035

Iman Elbalasy, Nils Wilharm, Eric Herchenhahn, Robert Konieczny, Stefan G. Mayr, Jörg Schnauß:

From Strain Stiffening to Softening-Rheological Characterization of Keratins 8 and 18 Networks Crosslinked via Electron Irradiation

Abstract:Networks of crosslinked keratin filaments are abundant in epithelial cells and tissues, providing resilience against mechanical forces and ensuring cellular integrity. Although studies of in vitro models of reconstituted keratin networks have revealed important mechanical aspects, the mechanical properties of crosslinked keratin structures remain poorly understood. Here, we exploited the power of electron beam irradiation (EBI) to crosslink in vitro networks of soft epithelial keratins 8 and 18 (k8-k18) filaments with different irradiation doses (30 kGy, 50 kGy, 80 kGy, 100 kGy, and 150 kGy). We combined bulk shear rheology with confocal microscopy to investigate the impact of crosslinking on the mechanical and structural properties of the resultant keratin gels. We found that irradiated keratin gels display higher linear elastic modulus than the unirradiated, entangled networks at all doses tested. However, at the high doses (80 kGy, 100 kGy, and 150 kGy), we observed a remarkable drop in the elastic modulus compared to 50 kGy. Intriguingly, the irradiation drastically changed the behavior for large, nonlinear deformations. While untreated keratin networks displayed a strong strain stiffening, increasing irradiation doses shifted the system to a strain softening behavior. In agreement with the rheological behavior in the linear regime, the confocal microscopy images revealed fully isotropic networks with high percolation in 30 kGy and 50 kGy-treated keratin samples, while irradiation with 100 kGy induced the formation of thick bundles and clusters. Our results demonstrate the impact of permanent crosslinking on k8-k18 mechanics and provide new insights into the potential contribution of intracellular covalent crosslinking to the loss of mechanical resilience in some human keratin diseases. These insights will also provide inspiration for the synthesis of new keratin-based biomaterials.   DOI: 10.3390/polym14030614   Journal Homepage

Polymers 2022, 14(3), 614

Frank Sauer, Anatol Fritsch, Steffen Grosser, Steve Pawlizak, Tobias Kießling, Martin Reiss-Zimmermann, Mehrgan Shahryari, Wolf C. Müller, Karl-Titus Hoffmann, Josef A. Käs, Ingolf Sack:

Whole tissue and single cell mechanics are correlated in human brain tumors

Abstract:Biomechanical changes are critical for cancer progression. However, the relationship between the rheology of single cells measured ex-vivo and the living tumor is not yet understood. Here, we combined single-cell rheology of cells isolated from primary tumors with in vivo bulk tumor rheology in patients with brain tumors. Eight brain tumors (3 glioblastoma, 3 meningioma, 1 astrocytoma, 1 metastasis) were investigated in vivo by magnetic resonance elastography (MRE), and after surgery by the optical stretcher (OS). MRE was performed in a 3-Tesla clinical MRI scanner and magnitude modulus |G*|, loss angle φ, storage modulus G', and loss modulus G'' were derived. OS experiments measured cellular creep deformation in response to laser-induced step stresses. We used a Kelvin-Voigt model to deduce two parameters related to cellular stiffness (μKV) and cellular viscosity (ηKV) from OS measurements in a time regimen that overlaps with that of MRE. We found that single-cell μKV was correlated with |G*| (R = 0.962, p < 0.001) and G'' (R = 0.883, p = 0.004) but not G' of the bulk tissue. These results suggest that single-cell stiffness affects tissue viscosity in brain tumors. The observation that viscosity parameters of individual cells and bulk tissue were not correlated suggests that collective mechanical interactions (i.e. emergent effects or cellular unjamming) of many cancer cells, which depend on cellular stiffness, influence the mechanical dissipation behavior of the bulk tissue. Our results are important to understand the emergent rheology of active multiscale compound materials such as brain tumors and its role in disease progression.   DOI: 10.1039/D1SM01291F   Journal Homepage

Soft Matter, 17, 10744 - 10752 (2021)

Enrico Warmt, Steffen Grosser, Eliane Blauth, Xiaofan Xie, Roland Stange, Janina Tomm, Frank Sauer, Jörg Schnauß, Martin von Bergen, Josef A. Käs:

Differences in cortical contractility between healthy epithelial and cancerous mesenchymal breast cells

Abstract:Cell contractility is mainly imagined as a force dipole-like interaction based on actin stress fibers that pull on cellular adhesionsites. Here, we present a different type of contractility based on isotropic contractions within the actomyosin cortex. Measuring mechanosensitive cortical contractility of suspended cells among various cell lines allowed us to exclude effects caused by stressfibers. We found that epithelial cells display a higher cortical tension than mesenchymal cells, directly contrasting to stressfiber-mediated contractility. These two types of contractility can even be used to distinguish epithelial from mesenchymal cells.These findings from a single cell level correlate to the rearrangement effects of actomyosin cortices within cells assembled in multicellular aggregates. Epithelial cells form a collective contractile actin cortex surrounding multicellular aggregates and further generate a high surface tension reminiscent of tissue boundaries. Hence, we suggest this intercellular structure as tobe crucial for epithelial tissue integrity. In contrast, mesenchymal cells do not form collective actomyosin cortices reducing multicellular cohesion and enabling cell escape from the aggregates.   DOI: 10.1088/1367-2630/ac254e   Journal Homepage

New Journal of Physics, 23, 103020 (2021)

Eliane Blauth, Hans Kubitschke, Pablo Gottheil, Steffen Grosser, Josef A. Käs:

Jamming in Embryogenesis and Cancer Progression

Abstract:The ability of tissues and cells to move and rearrange is central to a broad range of diverse biological processes such as tissue remodeling and rearrangement in embryogenesis, cell migration in wound healing, or cancer progression. These processes are linked to a solid-like to fluid-like transition, also known as unjamming transition, a not rigorously defined framework that describes switching between a stable, resting state and an active, moving state. Various mechanisms, that is, proliferation and motility, are critical drivers for the (un)jamming transition on the cellular scale. However, beyond the scope of these fundamental mechanisms of cells, a unifying understanding remains to be established. During embryogenesis, the proliferation rate of cells is high, and the number density is continuously increasing, which indicates number-density-driven jamming. In contrast, cells have to unjam in tissues that are already densely packed during tumor progression, pointing toward a shape-driven unjamming transition. Here, we review recent investigations of jamming transitions during embryogenesis and cancer progression and pursue the question of how they might be interlinked. We discuss the role of density and shape during the jamming transition and the different biological factors driving it.   DOI: 10.3389/fphy.2021.666709   Journal Homepage

Frontiers in Physics, 17 August (2021)

Janina Burk, Michaela Melzer, Alina Hagen, Katrin Susanne Lips, Katja Trinkaus, Ariane Nimptsch, Jenny Leopold:

Phospholipid Profiles for Phenotypic Characterization of Adipose-Derived Multipotent Mesenchymal Stromal Cells

Abstract:Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LCMS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.   DOI: 10.3389/fcell.2021.784405   Journal Homepage

Front. Cell Dev. Biol. 9:784405 (2021)

Carlotta Ficorella, Hannah Marie Eichholz, Federico Sala, Rebeca Martinez Vázquez, Roberto Osellame, Josef A. Käs:

Intermediate filaments ensure resiliency of single carcinoma cells, while active contractility of the actin cortex determines their invasive potential

Abstract:During the epithelial-to-mesenchymal transition, the intracellular cytoskeleton undergoes severe reorganization which allows epithelial cells to transition into a motile mesenchymal phenotype. Among the different cytoskeletal elements, the intermediate filaments keratin (in epithelial cells) and vimentin (in mesenchymal cells) have been demonstrated to be useful and reliable histological markers. In this study, we assess the potential invasiveness of six human breast carcinoma cell lines and two mouse fibroblasts cells lines through single cell migration assays in confinement. We find that the keratin and vimentin networks behave mechanically the same when cells crawl through narrow channels and that vimentin protein expression does not strongly correlate to single cells invasiveness. Instead, we find that what determines successful migration through confining spaces is the ability of cells to mechanically switch from a substrate-dependent stress fibers based contractility to a substrate-independent cortical contractility, which is not linked to their tumor phenotype.   DOI: 10.1088/1367-2630/ac1899   Journal Homepage

New Journal of Physics, 23, 083028 (2021)

Alice Abend, Chelsie Steele, Heinz-Georg Jahnke, Mareike Zink:

Adhesion of Neurons and Glial Cells with Nanocolumnar TiNFilms for Brain-Machine Interfaces

Abstract:Coupling of cells to biomaterials is a prerequisite for most biomedical applications; e.g., neuroelectrodes can only stimulate brain tissue in vivo if the electric signal is transferred to neurons attached to the electrodes' surface. Besides, cell survival in vitro also depends on the interaction of cells with the underlying substrate materials; in vitro assays such as multielectrode arrays determine cellular behavior by electrical coupling to the adherent cells. In our study, we investigated the interaction of neurons and glial cells with different electrode materials such as TiN and nanocolumnar TiN surfaces in contrast to gold and ITO substrates. Employing single-cell force spectroscopy, we quantified short-term interaction forces between neuron-like cells (SH-SY5Y cells) and glial cells (U-87 MG cells) for the different materials and contact times. Additionally, results were compared to the spreading dynamics of cells for different culture times as a function of the underlying substrate. The adhesion behavior of glial cells was almost independent of the biomaterial and the maximum growth areas were already seen after one day; however, adhesion dynamics of neurons relied on culture material and time. Neurons spread much better on TiN and nanocolumnar TiN and also formed more neurites after three days in culture. Our designed nanocolumnar TiN offers the possibility for building miniaturized microelectrode arrays for impedance spectroscopy without losing detection sensitivity due to a lowered self-impedance of the electrode. Hence, our results show that this biomaterial promotes adhesion and spreading of neurons and glial cells, which are important for many biomedical applications in vitro and in vivo.   DOI: 10.3390/ijms22168588   Journal Homepage

Int. Journal of Molecular Sciences, 22, 8588 (2021)

Daniel T. Hoffmann, Johannes Dietrich, Stefan Mändl, Mareike Zink, Stefan G. Mayr:

Nanoporous Morphogenesis in Amorphous Carbon Layers: Experiments and Modeling on Energetic Ion Induced Self-Organization

Abstract:Nanoporous amorphous carbon constitutes a highly relevant material for a multitude of applications ranging from energy to environmental and biomedical systems. In the present work, it is demonstrated experimentally how energetic ions can be utilized to tailor porosity of thin sputter deposited amorphous carbon films. The physical mechanisms underlying self-organized nanoporous morphogenesis are unraveled by employing extensive molecular dynamics and phase field models across different length scales. It is demonstrated that pore formation is a defect induced phenomenon, in which vacancies cluster in a spinodal decomposition type of self-organization process, while interstitials are absorbed by the amorphous matrix, leading to additional volume increase and radiation induced viscous flow. The proposed modeling framework is capable to reproduce and predict the experimental observations from first principles and thus opens the venue for computer assisted design of nanoporous frameworks.   DOI: 10.1002/adts.202100093   Journal Homepage

Advanced Theory and Simulation 2100093 (2021)

Jörg Schnauß, Tom Kunschmann, Steffen Grosser, Paul Mollenkopf, Tobias Zech, Jessica S. Freitag, Dusan Prascevic, Roland Stange, Luisa S. Röttger, Susanne Rönicke, David M. Smith, Thomas M. Bayerl, Josef A. Käs:

Cells in Slow Motion: Apparent Undercooling Increases Glassy Behavior at Physiological Temperatures

Abstract:Solvent conditions are unexpectedly sufficient to drastically and reversibly slow down cells. In vitro on the molecular level, protein-solvent interactions drastically change in the presence of heavy water (D2O) and its stronger hydrogen bonds. Adding D2O to the cell medium of living cells increases the molecular intracellular viscosity. While cell morphology and phenotype remain unchanged, cellular dynamics transform into slow motion in a changeable manner. This is exemplified in the slowdown of cell proliferation and migration, which is caused by a reversible gelation of the cytoplasm. In analogy to the time-temperature superposition principle, where temperature is replaced by D2O, an increase in viscosity slows down the effective time. Actin networks, crucial structures in the cytoplasm, switch from a power-law-like viscoelastic to a more rubber-like elastic behavior. The resulting intracellular resistance and dissipation impair cell movement. Since cells are highly adaptive non-equilibrium systems, they usually respond irreversibly from a thermodynamic perspective. D2O induced changes, however, are fully reversible and their effects are independent of signaling as well as expression. The stronger hydrogen bonds lead to glass-like, drawn-out intramolecular dynamics, which may facilitate longer storage times of biological matter, for instance, during transport of organ transplants.   DOI: 10.1002/adma.202101840   Journal Homepage

Advanced Materials 2101840 (2021)

Tina Händler, Cary Tutmarc, Martin Glaser, Jessica S. Freitag, David M. Smith, Jörg Schnauß:

Measuring structural parameters of crosslinked and entangled semiflexible polymer networks with single-filament tracing

Abstract:Single-filament tracing has been a valuable tool to directly determine geometrical and mechanical properties of entangled polymer networks. However, systematically verifying how the stiffness of the tracer filament or its molecular interactions with the surrounding network impacts the measurement of these parameters has not been possible with the established experimental systems. Here, we use mechanically programmable DNA nanotubes embedded in crosslinked and entangled F-actin networks, as well as in synthetic DNA networks, in order to measure fundamental, structural network properties like tube width and mesh size with respect to the stiffness of the tracers. While we confirm some predictions derived from models based purely on steric interactions, our results indicate that these models should be expanded to account for additional inter-filament interactions, thus describing the behavior of real polymer networks.   DOI: 10.1103/PhysRevE.103.062501   Journal Homepage

Physical Review E, 103, 062501 (2021)

Federico Sala, Carlotta Ficorella, Rebeca Martinez Vázquez, Hannah Marie Eichholz, Josef A. Käs, Roberto Osellame:

Rapid Prototyping of 3D Biochips for Cell Motility Studies Using Two-Photon Polymerization

Abstract:The study of cellular migration dynamics and strategies plays a relevant role in the understanding of both physiological and pathological processes. An important example could be the link between cancer cell motility and tumor evolution into metastatic stage. These strategies can be strongly influenced by the extracellular environment and the consequent mechanical constrains. In this framework, the possibility to study the behavior of single cells when subject to specific topological constraints could be an important tool in the hands of biologists. Two-photon polymerization is a sub-micrometric additive manufacturing technique that allows the fabrication of 3D structures in biocompatible resins, enabling the realization of ad hoc biochips for cell motility analyses, providing different types of mechanical stimuli. In our work, we present a new strategy for the realization of multilayer microfluidic lab-on-a-chip constructs for the study of cell motility which guarantees complete optical accessibility and the possibility to freely shape the migration area, to tailor it to the requirements of the specific cell type or experiment. The device includes a series of micro-constrictions that induce different types of mechanical stress on the cells during their migration. We show the realization of different possible geometries, in order to prove the versatility of the technique. As a proof of concept, we present the use of one of these devices for the study of the motility of murine neuronal cancer cells under high physical confinement, highlighting their peculiar migration mechanisms.   DOI: 10.3389/fbioe.2021.664094   Journal Homepage

Front. Bioeng. Biotechnol. 13 April (2021)

Bianca Mages, Thomas Fuhs, Susanne Aleithe, Alexandra Blietz, Constance Hobusch, Wolfgang Härtig, Stefan Schob, Martin Krueger, Dominik Michalski:

The Cytoskeletal Elements MAP2 and NF-L Show Substantial Alterations in Different Stroke Models While Elevated Serum Levels Highlight Especially MAP2 as a Sensitive Biomarker in Stroke Patients

Abstract:In the setting of ischemic stroke, the neurofilament subunit NF-L and the microtubule-associated protein MAP2 have proven to be exceptionally ischemia-sensitive elements of the neuronal cytoskeleton. Since alterations of the cytoskeleton have been linked to the transition from reversible to irreversible tissue damage, the present study investigates underlying time- and region-specific alterations of NF-L and MAP2 in different animal models of focal cerebral ischemia. Although NF-L is increasingly established as a clinical stroke biomarker, MAP2 serum measurements after stroke are still lacking. Therefore, the present study further compares serum levels of MAP2 with NF-L in stroke patients. In the applied animal models, MAP2-related immunofluorescence intensities were decreased in ischemic areas, whereas the abundance of NF-L degradation products accounted for an increase of NF-L-related immunofluorescence intensity. Accordingly, Western blot analyses of ischemic areas revealed decreased protein levels of both MAP2 and NF-L. The cytoskeletal alterations are further reflected at an ultrastructural level as indicated by a significant reduction of detectable neurofilaments in cortical axons of ischemia-affected areas. Moreover, atomic force microscopy measurements confirmed altered mechanical properties as indicated by a decreased elastic strength in ischemia-affected tissue. In addition to the results from the animal models, stroke patients exhibited significantly elevated serum levels of MAP2, which increased with infarct size, whereas serum levels of NF-L did not differ significantly. Thus, MAP2 appears to be a more sensitive stroke biomarker than NF-L, especially for early neuronal damage. This perspective is strengthened by the results from the animal models, showing MAP2-related alterations at earlier time points compared to NF-L. The profound ischemia-induced alterations further qualify both cytoskeletal elements as promising targets for neuroprotective therapies.   DOI: 10.1007/s12035-021-02372-3   Journal Homepage

Mol Neurobiol, 58, 4051 - 4069 (2021)

Paul Heine, Jürgen Lippoldt, Gudur Ashrith Reddy, Parag Katira, Josef A. Käs:

Anomalous cell sorting behavior in mixed monolayers discloses hidden system complexities

Abstract:In tissue development, wound healing and aberrant cancer progression cell-cell interactions drive mixing and segregation of cellular composites. However, the exact nature of these interactions is unsettled. Here we study the dynamics of packed, heterogeneous cellular systems using wound closure experiments. In contrast to previous cell sorting experiments, we find non-universal sorting behavior. For example, monolayer tissue composites with two distinct cell types that show low and high neighbor exchange rates (i.e., MCF-10A & MDA-MB-231) produce segregated domains of each cell type, contrary to conventional expectation that the construct should stay jammed in its initial configuration. On the other hand, tissue compounds where both cell types exhibit high neighbor exchange rates (i.e., MDA-MB-231 & MDA-MB-436) produce highly mixed arrangements despite their differences in intercellular adhesion strength. The anomalies allude to a complex multi-parameter space underlying these sorting dynamics, which remains elusive in simpler systems and theories merely focusing on bulk properties. Using cell tracking data, velocity profiles, neighborhood volatility, and computational modeling, we classify asymmetric interfacial dynamics. We indicate certain understudied facets, such as the effects of cell death & division, mechanical hindrance, active nematic behavior, and laminar & turbulent flow as their potential drivers. Our findings suggest that further analysis and an update of theoretical models, to capture the diverse range of active boundary dynamics which potentially influence self-organization, is warranted.   DOI: 10.1088/1367-2630/abf273   Journal Homepage

New Journal of Physics, 23, 043034 (2021)

Ivonne Nel, Erik W. Morawetz, Dimitrij Tschodu, Josef A. Käs, Bahriye Aktas:

The Mechanical Fingerprint of Circulating Tumor Cells (CTCs) in Breast Cancer Patients

Abstract:Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells - possible CTCs - were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to nonhematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin-Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.   DOI: 10.3390/cancers13051119   Journal Homepage

Cancers, 13 (5), 1119 (2021)

Iman Elbalasy, Paul Mollenkopf, Cary Tutmarc, Harald Herrmann, Jörg Schnauß

Keratins determine network stress responsiveness in reconstituted actin-keratin filament systems

Abstract:The cytoskeleton is a major determinant of cell mechanics, a property that is altered during many pathological situations. To understand these alterations, it is essential to investigate the interplay between the main filament systems of the cytoskeleton in the form of composite networks. Here, we investigate the role of keratin intermediate filaments (IFs) in network strength by studying in vitro reconstituted actin and keratin 8/18 composite networks via bulk shear rheology. We co-polymerized these structural proteins in varying ratios and recorded how their relative content affects the overall mechanical response of the various composites. For relatively small deformations, we found that all composites exhibited an intermediate linear viscoelastic behavior compared to that of the pure networks. In stark contrast, the composites displayed increasing strain stiffening behavior as a result of increased keratin content when larger deformations were imposed. This strain stiffening behavior is much more pronounced than the behavior encountered with vimentin IF as a composite network partner for actin. Our results provide new insights into the mechanical interplay between actin and keratin in which keratin provides reinforcement to actin. This interplay may contribute to the overall integrity of cells, providing an explanation for the stability of stressed epithelial tissues due to their high keratin contents.   DOI: 10.1039/D0SM02261F   Journal Homepage

Soft Matter, 17, 3954 - 3962 (2021)

Steffen Grosser, Jürgen Lippoldt, Linda Oswald, Matthias Merkel, Daniel M. Sussman, Frederic Renner, Pablo Gottheil, Erik W. Morawetz, Thomas Fuhs, Xiaofan Xie, Steve Pawlizak, Anatol W. Fritsch, Benjamin Wolf, Lars-Christian Horn, Susanne Briest, Bahriye Aktas, M. Lisa Manning, Josef A. Käs

Cell and Nucleus Shape as an Indicator of Tissue Fluidity in Carcinoma

Abstract:Tissue, cell, and nucleus morphology change during tumor progression. In 2D confluent cell cultures, different tissue states, such as fluid (unjammed) and solid (jammed) are correlated with cell shapes. These results do not have to apply a priori to three dimensions. Cancer cell motility requires and corresponds to a fluidization of the tumor tissue on the bulk level. Here we investigate bulk tissue fluidity in 3D and determine how it correlates with cell and nucleus shape. In patient samples of mamma and cervix carcinoma we find areas where cells can move or are immobile. We compare 3D cell spheroids composed of cells from a cancerous and a non-cancerous cell line. Through bulk mechanical spheroid-fusion experiments and single live-cell tracking, we show that the cancerous sample is fluidized by active cells moving through the tissue. The healthy, epithelial sample with immobile cells behaves more solid-like. 3D-segmentations of the samples show that the the degree of tissue fluidity correlates with elongated cell and nucleus shapes. This links cell shapes to cell motility and bulk mechanical behavior. We find two active states of matter in solid tumors: an amorphous glass-like state with characteristics of 3D cell jamming, and a disordered fluid state. Individual cell and nucleus shape may serve as marker for metastatic potential to foster personalized cancer treatment.   DOI: 10.1103/PhysRevX.11.011033   Journal Homepage

Physical Review X, 11, 011033 (2021)

Sheilan Sinjari, Jessica S. Freitag, Christoph Herold, Oliver Otto, David M. Smith, Harald D. H. Stöver:

Tunable polymer microgel particles and their study using microscopy and real-time deformability cytometry

Abstract: We report the preparation and mechanical properties of highly swellable, spherical polymer microgels synthesized by precipitation copolymerization of divinylbenzene-55 (DVB), 4-methylstyrene (4MS), and maleic anhydride (MA) at different cross-linker contents, in a range of methylethylketone (MEK) and heptane solvent mixtures. Microgels were characterized by optical and confocal microscopy, and their mechanical properties tested using real-time deformability cytometry (RT-DC), a technique developed to analyze cell properties by measuring deformation under shear stress. Hydrolysis of anhydride groups gave microgels with diameters ranging from 10 to 22 μ when swollen in saline, depending on vol% MEK and cross-linker loading. Young's moduli of the microgels could be tuned from 0.8 to 10 kPa by adjusting cross-linker content and MEK/heptane solvent composition, showing an inverse relationship between the effects of vol% MEK and %DVB on microgel properties. These microgels also show strain-stiffening in response to increasing shear stresses. Extension of the RT-DC method to the study of polymer colloids thus enables high-throughput analysis of microgels with tunable mechanical characteristics.   DOI: 10.1002/pol.20200274   Journal Homepage

Journal of Polymer Sciences, vol. 58, iss. 17, p. 2317 - 2326 (2020)

Mesut Karatas, Senol Dogan, Emrulla Spahiu, Adna Asic, Larisa Besic, Yusuf Turan:

Enzyme kinetics and inhibition parameters of human leukocyte glucosylceramidase

Abstract:Glucosylceramidase (GCase) is a lysosomal enzyme that catalyzes the cleavage of β-glucosidic linkage of glucocerebroside (GC) into glucose and ceramide; thereby, plays an essential function in the degradation of complex lipids and the turnover of cellular membranes. The growing list of 460 mutations in the gene coding for it-glucosylceramidase beta acid 1 (GBA1)-is reported to abolish its catalytic activity and decrease its enzyme stability, associating it with severe health conditions such as Gaucher disease (GD), Parkinson Disease (PD) and Dementia with Lewy bodies (DLB). Although the three-dimensional structure of wild type glucosylceramidase is elucidated, little is known about its features in human cells. Moreover, alternative sources of GCase that prove to be effective in the treatment of diseases with enzyme treatment therapies, impose the need for a simple and cost-effective procedure to study the enzyme behavior. This work, for the first time, shows a well-established, yet simple, cost- and time-efficient protocol for the study of GCase enzyme in human leukocytes by the artificial substrate p-Nitrophenyl-β-D-glucopyranoside (PNPG). Characterization of the enzyme in human leukocytes for activation parameters (optimal pH, Km, and Vmax) and enzyme inhibition was done. The results indicate that the optimum pH of GCase enzyme with PNPG is 5.0. The Km and Vmax values are 12.6mM and 333 U/mg, respectively. Gluconolactone competitively inhibits GCase, with a Ki value of 0.023 mM and IC50 of 0.047 mM. Glucose inhibition is uncompetitive with a Ki of 1.94 mM and IC50 of 55.3 mM. This is the first report for the inhibitory effect of glucose, δ-gluconolactone on human leukocyte GCase activity.   DOI: 10.1016/j.heliyon.2020.e05191   Journal Homepage

Heliyon, vol. 6, iss. 11, e05191 (2020)

Alice Abend, Chelsie Steele, Sabine Schmidt, Ronny Frank, Heinz-Georg Jahnke, Mareike Zink:

Proliferation and Cluster Analysis of Neurons and Glial Cell Organization on Nanocolumnar TiN Substrates

Abstract: Biomaterials employed for neural stimulation, as well as brain/machine interfaces, offer great perspectives to combat neurodegenerative diseases, while application of lab-on-a-chip devices such as multielectrode arrays is a promising alternative to assess neural function in vitro. For bioelectronic monitoring, nanostructured microelectrodes are required, which exhibit an increased surface area where the detection sensitivity is not reduced by the self-impedance of the electrode. In our study, we investigated the interaction of neurons (SH-SY5Y) and glial cells (U-87 MG) with nanocolumnar titanium nitride (TiN) electrode materials in comparison to TiN with larger surface grains, gold, and indium tin oxide (ITO) substrates. Glial cells showed an enhanced proliferation on TiN materials; however, these cells spread evenly distributed over all the substrate surfaces. By contrast, neurons proliferated fastest on nanocolumnar TiN and formed large cell agglomerations. We implemented a radial autocorrelation function of cellular positions combined with various clustering algorithms. These combined analyses allowed us to quantify the largest cluster on nanocolumnar TiN; however, on ITO and gold, neurons spread more homogeneously across the substrates. As SH-SY5Y cells tend to grow in clusters under physiologic conditions, our study proves nanocolumnar TiN as a potential bioactive material candidate for the application of microelectrodes in contact with neurons. To this end, the employed K-means clustering algorithm together with radial autocorrelation analysis is a valuable tool to quantify cell-surface interaction and cell organization to evaluate biomaterials' performance in vitro.   DOI: 10.3390/ijms21176249   Journal Homepage

Int. Journal of Molecular Sciences, vol. 21, iss. 17 (2020)

Olga Ilina, Pavlo G. Gritsenko, Simon Syga, Jürgen Lippoldt, Caterina A. M. La Porta, Oleksandr Chepizhko, Steffen Grosser, Manon Vullings, Gert-Jan Bakker, Jörn Starruβ, Peter Bult, Stefano Zapperi, Josef A. Käs, Andreas Deutsch, Peter Friedl:

Cell-Cell Adhesion and 3D Matrix Confinement Determine Jamming Transitions in Breast Cancer Invasion

Abstract: Plasticity of cancer invasion and metastasis depends on the ability of cancer cells to switch between collective and single-cell dissemination, controlled by cadherin-mediated cell-cell junctions. In clinical samples, E-cadherin-expressing and -deficient tumours both invade collectively and metastasize equally, implicating additional mechanisms controlling cell-cell cooperation and individualization. Here, using spatially defined organotypic culture, intravital microscopy of mammary tumours in mice and in silico modelling, we identify cell density regulation by three-dimensional tissue boundaries to physically control collective movement irrespective of the composition and stability of cell-cell junctions. Deregulation of adherens junctions by downregulation of E-cadherin and p120-catenin resulted in a transition from coordinated to uncoordinated collective movement along extracellular boundaries, whereas single-cell escape depended on locally free tissue space. These results indicate that cadherins and extracellular matrix confinement cooperate to determine unjamming transitions and stepwise epithelial fluidization towards, ultimately, cell individualization.  DOI:10.1038/s41556-020-0552-6   Journal Homepage

Nature Cell Biology, August, 1-13 (2020)

Kantida Juncheed, Bernd Kohlstrunk, Sabrina Friebe, Valentina Dallacasagrande, Patric Maurer, Andreas Reichenbach, Stefan G. Mayr, Mareike Zink:

Employing Nanostructured Scaffolds to Investigate the Mechanical Properties of Adult Mammalian Retinae Under Tension

Abstract: Numerous eye diseases are linked to biomechanical dysfunction of the retina. However, the underlying forces are almost impossible to quantify experimentally. Here, we show how biomechanical properties of adult neuronal tissues such as porcine retinae can be investigated under tension in a home-built tissue stretcher composed of nanostructured TiO2 scaffolds coupled to a selfdesigned force sensor. The employed TiO2 nanotube scaffolds allow for organotypic long-term preservation of adult tissues ex vivo and support strong tissue adhesion without the application of glues, a prerequisite for tissue investigations under tension. In combination with finite element calculations we found that the deformation behavior is highly dependent on the displacement rate which results in Young’s moduli of (760-1270) Pa. Image analysis revealed that the elastic regime is characterized by a reversible shear deformation of retinal layers. For larger deformations, tissue destruction and sliding of retinal layers occurred with an equilibration between slip and stick at the interface of ruptured layers, resulting in a constant force during stretching. Since our study demonstrates how porcine eyes collected from slaughterhouses can be employed for ex vivo experiments, our study also offers new perspectives to investigate tissue biomechanics without excessive animal experiments.   DOI: 10.3390/ijms21113889   Journal Homepage

Int. Journal of Molecular Sciences, vol. 21, iss. 11 (2020)

Stefano Siccardi, Andrew Adamatzky, Jack Tuszyński, Florian Huber, Jörg Schnauß:

Actin networks voltage circuits

Abstract: Filaments of the cellular protein actin can form bundles, which can conduct ionic currents as well as mechanical and voltage solitons. These inherent properties can be utilized to generate computing circuits solely based on self-assembled actin bundle structures. Starting with experimentally observed networks of actin bundles, we model their network structure in terms of edges and nodes. We compute and discuss the main electrical parameters, considering the bundles as electrical wires with either low or high filament densities. A set of equations describing the network is solved with several initial conditions. Input voltages, which can be considered as information bits, are applied in a set of points and output voltages are computed in another set of positions. We consider both an idealized situation, where point-like electrodes can be inserted in any points of the bundles and a more realistic case, where electrodes lay on a surface and have typical dimensions available in the industry. We find that in both cases such a system can implement the main logical gates and a finite state machine.   DOI: 10.1103/PhysRevE.101.052314   Journal Homepage

Physical Review E 101, 052314 (2020)

Paul Van Liedekerke, Johannes Neitsch, Tim Johann, Enrico Warmt, Ismael Gonzales Valverde, Steffen Grosser, Stefan Hoehme, Josef Käs, Dirk Drasdo:

A quantitative high-resolution computational mechanics cell model for growing and regenerating tissues

Abstract:Mathematical models are increasingly designed to guide experiments in biology, biotechnology, as well as to assist in medical decision making. They are in particular important to understand emergent collective cell behavior. For this purpose, the models, despite still abstractions of reality, need to be quantitative in all aspects relevant for the question of interest. The focus in this paper is to study the regeneration of liver after drug-induced depletion of hepatocytes, in which surviving dividing and migrating hepatocytes must squeeze through a blood vessel network to fill the emerged lesions. Here, the cells' response to mechanical stress might significantly impact on the regeneration process. We present a 3D high-resolution cell-based model integrating information from measurements in order to obtain a refined quantitative understanding of the cell-biomechanical impact on the closure of drug-induced lesions in liver. Our model represents each cell individually, constructed as a physically scalable network of viscoelastic elements, capable of mimicking realistic cell deformation and supplying information at subcellular scales. The cells have the capability to migrate, grow and divide, and infer the nature of their mechanical elements and their parameters from comparisons with optical stretcher experiments. Due to triangulation of the cell surface, interactions of cells with arbitrarily shaped (triangulated) structures such as blood vessels can be captured naturally. Comparing our simulations with those of so-called center-based models, in which cells have a rigid shape and forces are exerted between cell centers, we find that the migration forces a cell needs to exert on its environment to close a tissue lesion, is much smaller than predicted by center-based models. This effect is expected to be even more present in chronic liver disease, where tissue stiffens and excess collagen narrows pores for cells to squeeze through.   DOI: 10.1007/s10237-019-01204-7   Journal Homepage

Biomechanics and Modelling in Mechanobiology, 19, 189 - 220 (2020)

Jörg Schnauß, B. U. Sebastian Schmidt, Christina B. Brazel, Senol Dogan, Wolfgang Losert, Ulf Anderegg, Josef A. Käs:

Influence of Hyaluronic Acid Binding on the Actin Cortex measured by Optical Forces

Abstract: Melanoma cells are often surrounded by hyaluronic acid (HA) rich environments, which are considered to promote tumor progression and metastasis. Induced effects in compound materials consisting of cells embedded in an extracellular matrix (ECM) have been studied, however, alterations of the single cells have never been addressed. Here, we explicitly addressed single cell properties and measured HA-induced biomechanical changes via deformations induced solely by optical forces. With the optical stretcher setup, cells were deformed after culturing them either in the presence or absence of HA revealing the crucial interplay of HA with the CD44 receptor. To assess the role of CD44 in transducing effects of HA, we compared a CD44 expressing variant of the melanoma cell line RPM-MC to its natural CD44-negative counterpart. Our measurements revealed a significant stiffness change, which we attribute to changes of the actin cytoskeleton.   DOI: 10.1002/jbio.20190215   Journal Homepage

Journal of Biophotonics, e201960215 (2020)

Senol Dogan:

Genomic Characterization of Mixed Lineage Leukemias

Abstract: Developing new technologies and genomic techniques generate big data, which is very helpful to evaluate extensively hematologic malignancies. Besides wet lab results, such as gene and microRNA expression, methylation profile, next-generation sequencing data, whole-ex-ome and RNA-seq, FISH analysis, SNP arrays, new computer algorithm, and programs have allowed for improving the understanding transformation oncohematological disorder mechanism. In addition to that, the whole wet and dry lab results permit us to develop a new prognostic algorithm, which can assist to decide the most appropriate treatment. Furthermore, finding and analyzing new biomarkers drive the geneticists to produce therapy target and personalized medicine. The novel and effective contribution of the new technologies in the context of leukemia of both AML, Acute Myeloid Leukemia and ALL, Acute Lymphoblastic Leukemia and genomic characterization of de novo mixed-lineage leukemia are described in this review.   DOI: 10.32113/wcrj_20203_1516   Journal Homepage

WCRJ 7, e1516 (2020)

Yang Shen, B. U. Sebastian Schmidt, Hans Kubitschke, Erik W. Morawetz, Benjamin Wolf, Josef A. Käs, Wolfgang Losert:

Detecting heterogeneity in and between breast cancer cell lines

Abstract: Cellular heterogeneity in tumor cells is a well-established phenomenon. Genetic and phenotypic cell-to-cell variability have been observed in numerous studies both within the same type of cancer cells and across different types of cancers. Another known fact for metastatic tumor cells is that they tend to be softer than their normal or non-metastatic counterparts. However, the heterogeneity of mechanical properties in tumor cells are not widely studied. Here we analyzed single-cell optical stretcher data with machine learning algorithms on three different breast tumor cell lines and show that similar heterogeneity can also be seen in mechanical properties of cells both within and between breast tumor cell lines. We identified two clusters within MDA-MB-231 cells, with cells in one cluster being softer than in the other. In addition, we show that MDA-MB-231 cells and MDA-MB-436 cells which are both epithelial breast cancer cell lines with a mesenchymal-like phenotype derived from metastatic cancers are mechanically more different from each other than from non-malignant epithelial MCF-10A cells. Since stiffness of tumor cells can be an indicator of metastatic potential, this result suggests that metastatic abilities could vary within the same monoclonal tumor cell line.   DOI: 10.1186/s41236-020-0010-1   Journal Homepage

Cancer Convergence 4, Article number: 1 (2020)

Kaspar-Josche Streitberger, Ledia Lilaj, Felix Schrank, Jürgen Braun, Karl-Titus Hoffmann, Martin Reiss-Zimmermann, Josef A. Käs, Ingolf Sack:

How tissue fluidity influences brain tumor progression

Abstract: Mechanical properties of biological tissues and, above all, their solid or fluid behavior influence the spread of malignant tumors. While it is known that solid tumors tend to have higher mechanical rigidity, allowing them to aggressively invade and spread in solid surrounding healthy tissue, it is unknown how softer tumors can grow within a more rigid environment such as the brain. Here, we use in vivo magnetic resonance elastography (MRE) to elucidate the role of anomalous fluidity for the invasive growth of soft brain tumors, showing that aggressive glioblastomas (GBMs) have higher water content while behaving like solids. Conversely, our data show that benign meningiomas (MENs), which contain less water than brain tissue, are characterized by fluid-like behavior. The fact that the 2 tumor entities do not differ in their soft properties suggests that fluidity plays an important role for a tumor's aggressiveness and infiltrative potential. Using tissue-mimicking phantoms, we show that the anomalous fluidity of neurotumors physically enables GBMs to penetrate surrounding tissue, a phenomenon similar to Saffman-Taylor viscous-fingering instabilities, which occur at moving interfaces between fluids of different viscosity. Thus, targeting tissue fluidity of malignant tumors might open horizons for the diagnosis and treatment of cancer.   DOI: 10.1073/pnas.1913511116   Journal Homepage

PNAS January 7, 2020 117 (1) 128-134

Senol Dogan, Emrulla Spahiu:

Engaging Students in Science Using Project Olympiads: A case study in Bosnia and Herzegovina

Abstract: Making science enjoyable inspires students to learn more. Out-of-class activities such as science fairs and Olympiads, serve as reasonable informal learning environments that demand attention. The association of students' involvement in these activities with increased student interest in science followed by the selection of science-related careers, should motivate all in-charge stakeholders. In this work, we analysed the outcomes of the Bosnia Science Olympiad (BSO) as the first national Science Olympiad inBosnia and Herzegovina (BiH), aiming the improvement of science education and bringing different ethnic groups under the umbrella of science, in a post-conflict area. The two-day endeavour held in Sarajevo includes competition in four science-related categories (Environment, Engineering, Have an Idea, Web Design) and social activities.In this work, the comprehensive data, including participants' gender, their ethnic background, cities, schools, and supervisors, over fiveyears, was analysed. The number of participating high-school students increased from 78 to 143, of supervisors from 21 to 95, and of schools from 7 to 15, reaching a wide demographic acceptance to cover all ethnic regions in BiH. The relationship between gender and the selection of a category, shows bias of male participants towards Web Design (21%) and Engineering (40%), and of female students towards "Have an Idea" (40%) and Environment (44%) categories. The contribution of BSO choosing a science career, getting socialized without prejudices, and the improvement of students' self-confidence, were as well addressed. Our work demonstrates a model work to successfully promote science in post-conflict settings.   DOI: 10.31756/jrsmte.412   Journal Homepage

J. of Res. in Sci. Math. and Tech. Edu. (2020)

Jörg Schnauß:

Audience Response Systeme und Online Self-Assessments zur Aktivierung und Evaluationdes Plenums

Abstract:Der vorliegende Beitrag beleuchtet als Teil eines Blended-Learning Ansatzes vorrangig den Einsatz von Live-Umfragen (ARS - Audience Responce Systems) im Vorlesungsrahmen. Gerade naturwissenschaftlich geprägte Studiengänge (hier die Fachrichtung Physik) sind häufig durch Frontalunterricht geprägt. Das maßgebliche Ziel des Projektes war es, das Format durch gezielte Einbindung der Studierenden aufzulockern und die Diskussionskultur in der Lehrveranstaltung zu stärken. Einhergehend mit der Aktivierung erhalten die Lernenden eine unmittelbare Rückmeldung zu ihrem Wissensstand und die/ der Lehrende ein Feedback zu möglichen Wissenslücken. Die Live-Umfragen fanden über die Online- Plattform invote.de in Form von Single-Choice-Fragen statt. Erweitert wurde dieser Ansatz, indem diese Inhalte ebenfalls für eine asynchrone Wissensvermittlung im Lernmanagement-System (LMS) Moodle implementiert und mit Feedback flankiert wurden. Dies führte im Vergleich zu früheren Iterationen der Lehrveranstaltung zu einem höheren Aktivitätslevel des Plenums und fachlich fundierten Diskussionen. In Evaluationen zum Ende des Semesters sowie in persönlichen Gesprächen mit den Studierenden, wurde der Einsatz der Fragen in synchroner sowie asynchroner Form explizit als Zugewinn für die Qualität der Lehrveranstaltung herausgestellt.   Herausgeber Hochschuldidaktisches Zentrum Sachsen

Jahrgang 2020, Seiten 53-57, ISSN: 2195-0334

Nils Wilharm, Tony Fischer, Florian Ott, Robert Konieczny, Mareike Zink, Annette G. Beck-Sickinger, Stefan G. Mayr:

Energetic electron assisted synthesis of highly tunable temperature-responsive collagen/elastin gels for cyclic actuation: macroscopic switching and molecular origins

Abstract:Thermoresponsive bio-only gels that yield sufficiently large strokes reversibly and without large hysteresis at a well-defined temperature in the physiological range, promise to be of value in biomedical application. Within the present work we demonstrate that electron beam modification of a blend of natural collagen and elastin gels is a route to achieve this goal, viz. to synthesize a bioresorbable gel with largely reversible volume contractions as large as 90% upon traversing a transition temperature that can be preadjusted between 36°C and 43°C by the applied electron dose. Employing circular dichroism and temperature depending confocal laser scanning microscopy measurements, we furthermore unravel the mechanisms underlying this macroscopic behavior on a molecular and network level, respectively and suggest a stringent picture to account for the experimental observations.   DOI: 10.1038/s41598-019-48830-w   Journal Homepage

Scientific Reports 9, Article number: 12363 (2019)

Andrew Adamatzky, Jörg Schnauß, Florian Huber:

Actin droplet machine

Abstract:The actin droplet machine is a computer model of a three-dimensional network of actin bundles developed in a droplet of a physiological solution, which implements mappings of sets of binary strings. The actin bundle network is conductive to travelling excitations, i.e. impulses. The machine is interfaced with an arbitrary selected set of k electrodes through which stimuli, binary strings of length k represented by impulses generated on the electrodes, are applied and responses are recorded. The responses are recorded in a form of impulses and then converted to binary strings. The machine’s state is a binary string of length k: if there is an impulse recorded on the ith electrode, there is a ’1’ in the ith position of the string, and ’0’ otherwise. We present a design of the machine and analyse its state transition graphs. We envisage that actin droplet machines could form an elementary processor of future massive parallel computers made from biopolymers.   DOI: 10.1098/rsos.191135   Journal Homepage

Royal Society Open Science 6:191135 (2019)

Andrew Adamatzky, Florian Huber, Jörg Schnauß:

Computing on actin bundles network

Abstract:Actin filaments are conductive to ionic currents, mechanical and voltage solitons. These travelling localisations can be utilised to generate computing circuits from actin networks. The propagation of localisations on a single actin filament is experimentally unfeasible to control. Therefore, we consider excitation waves propagating on bundles of actin filaments. In computational experiments with a two-dimensional slice of an actin bundle network we show that by using an arbitrary arrangement of electrodes, it is possible to implement two-inputs-one-output circuits.   DOI: 10.1038/s41598-019-51354-y   Journal Homepage

Scientific Reports 9, Article number: 15887 (2019)

Mehrgan Shahryari, Heiko Tzschätzsch, Jing Guo, Stephan R. Marticorena Garcia, Georg Böning, Uli Fehrenbach, Lisa Stencel, Patrick Asbach, Bernd Hamm, Josef A. Käs, Jürgen Braun, Timm Denecke, Ingolf Sack:

Tomoelastography Distinguishes Noninvasively between Benign and Malignant Liver Lesions

Abstract: Patients with increased liver stiffness have a higher risk of developing cancer, however, the role of fluid-solid tissue interactions and their contribution to liver tumor malignancy remains elusive. Tomoelastography is a novel imaging method for mapping quantitatively the solid-fluid tissue properties of soft tissues in vivo. It provides high resolution and thus has clear clinical applications. In this work we used tomoelastography in 77 participants, with a total of 141 focal liver lesions of different etiologies, to investigate the contributions of tissue stiffness and fluidity to the malignancy of liver tumors. Shear-wave speed (c) as surrogate for tissue stiffness and phase-angle (φ) of the complex shear modulus reflecting tissue fluidity were abnormally high in malignant tumors and allowed them to be distinguished from nontumorous liver tissue with high accuracy [c: AUC = 0.88 with 95% confidence interval (CI) = 0.83-0.94; φ: AUC = 0.95, 95% CI = 0.92-0.98]. Benign focal nodular hyperplasia and hepatocellular adenoma could be distinguished from malignant lesions on the basis of tumor stiffness (AUC = 0.85, 95% CI = 0.72-0.98; sensitivity = 94%, 95% CI = 89-100; and specificity = 85%, 95% CI = 62-100), tumor fluidity (AUC = 0.86, 95% CI = 0.77-0.96; sensitivity = 83%, 95% CI = 72-93; and specificity = 92%, 95% CI = 77-100) and liver stiffness (AUC = 0.84, 95% CI = 0.74-0.94; sensitivity = 72%, 95% CI = 59-83; and specificity = 88%, 95% CI = 69-100), but not on the basis of liver fluidity. Together, hepatic malignancies are characterized by stiff, yet fluid tissue properties, whereas surrounding nontumorous tissue is dominated by solid properties. Tomoelastography can inform noninvasively on the malignancy of suspicious liver lesions by differentiating between benign and malignant lesions with high sensitivity based on stiffness and with high specificity based on fluidity.   DOI: 10.1158/0008-5472.CAN-19-2150   Journal Homepage

Cancer Research vol. 79 iss. 22 (2019)

Emir Sehovic, Adem Hadrovic, Senol Dogan:

Detection and analysis of stable and flexible genes towards a genome signature framework in cancer

Abstract: Comparison and detection of stable cancer genes across cancer types is of interest. The gene expression data of 6 different cancer types (colon, breast, lung, ovarian, brain and renal) and a control group from The Cancer Genome Atlas (TCGA) database were used in this study. The comparison of gene expression data together with the calculation standard deviations of such data was completed using a statistical model for the detection of stable genes. Genes having similar expression (referred as flexible genes) pattern to the control group in four out of six cancer types are PATE, NEUROD4 and TRAFD1. Moreover, 13 genes showed low difference compared to the control group with low standard deviation across cancer types (referred as stable genes). Among them, genes GDF2, KCNT1 and RNF151 showed consistent low expression while ODF4, OR5I1, MYOG and OR2B11 showed consistent high expression. Thus, the detection and analysis of stable and flexible cancer genes help towards the design and development of a framework (outline) for specific genome signature (biomarker) in cancer.  DOI: 10.6026/97320630015772   Journal Homepage

Bioinformation 15(10): 772 - 779 (2019)

Charlotte Kielar, Yang Xin, Xiaodan Xu, Sigi Zhu, Nelli Gorin, Guido Grundmeier, Christin Möser, David M. Smith, Adrian Keller:

Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability

Abstract: DNA origami nanostructures are widely employed in various areas of fundamental and applied research. Due to the tremendous success of the DNA origami technique in the academic field, considerable efforts currently aim at the translation of this technology from a laboratory setting to real-world applications, such as nanoelectronics, drug delivery, and biosensing. While many of these real-world applications rely on an intact DNA origami shape, they often also subject the DNA origami nanostructures to rather harsh and potentially damaging environmental and processing conditions. Furthermore, in the context of DNA origami mass production, the long-term storage of DNA origami nanostructures or their pre-assembled components also becomes an issue of high relevance, especially regarding the possible negative effects on DNA origami structural integrity. Thus, we investigated the effect of staple age on the self-assembly and stability of DNA origami nanostructures using atomic force microscopy. Different harsh processing conditions were simulated by applying different sample preparation protocols. Our results show that staple solutions may be stored at -20 °C for several years without impeding DNA origami self-assembly. Depending on DNA origami shape and superstructure, however, staple age may have negative effects on DNA origami stability under harsh treatment conditions. Mass spectrometry analysis of the aged staple mixtures revealed no signs of staple fragmentation. We, therefore, attribute the increased DNA origami sensitivity toward environmental conditions to an accumulation of damaged nucleobases, which undergo weaker base-pairing interactions and thus lead to reduced duplex stability.   DOI: 10.3390/molecules24142577   Journal Homepage

Molecules 24 (14):2577 (2019)

Hans Kubitschke, Benjamin Wolf, Erik Morawetz, Lars-Christian Horn, Bahriye Aktas, Ulrich Behn, Michael Höckel, Josef Käs:

Roadmap to Local Tumour Growth: Insights from Cervical Cancer

Abstract:Wide tumour excision is currently the standard approach to surgical treatment of solid cancers including carcinomas of the lower genital tract. This strategy is based on the premise that tumours exhibit isotropic growth potential. We reviewed and analysed local tumour spreading patterns in 518 patients with cancer of the uterine cervix who underwent surgical tumour resection. Based on data obtained from pathological examination of the surgical specimen, we applied computational modelling techniques to simulate local tumour spread in order to identify parameters influencing preferred infiltration patterns and used area-proportional Euler diagrams to detect and confirm ordered patterns of tumour spread. Some anatomical structures, e.g. tissues of the urinary bladder, were significantly more likely to be infiltrated than other structures, e.g. the ureter and the rectum. Computational models assuming isotropic growth could not explain these infiltration patterns. Introducing ontogenetic distance of a tissue relative to the uterine cervix as a parameter led to accurate predictions of the clinically observed infiltration likelihoods. The clinical data indicates that successive infiltration likelihoods of ontogenetically distant tissues are nearly perfect subsets of ontogenetically closer tissues. The prevailing assumption of isotropic tumour extension has significant shortcomings in the case of cervical cancer. Rather, cervical cancer spread seems to follow ontogenetically defined trajectories.   DOI: 10.1038/s41598-019-49182-1   Journal Homepage

Scientific Reports 9, Article number: 12768 (2019)

Carlotta Ficorella, Rebeca Martinez Vàzques, Paul Heine, Eugenia Lepera, Jing Cao, Enrico Warmt, Roberto Osellame, Josef Käs:

Normal epithelial and triple-negative breast cancer cells show the same invasion potential in rigid spatial confinement

Abstract:The extra-cellular microenvironment has a fundamental role in tumor growth and progression, strongly affecting the migration strategies adopted by single cancer cells during metastatic invasion. In this study, we use a novel microfluidic device to investigate the ability of mesenchymal and epithelial breast tumor cells to fluidize and migrate through narrowing microstructures upon chemoattractant stimulation. We compare the migration behavior of two mesenchymal breast cancer cell lines and one epithelial cell line, and find that the epithelial cells are able to migrate through the narrowest microconstrictions as the more invasive mesenchymal cells. In addition, we demonstrate that migration of epithelial cells through a highly compressive environment can occur in absence of a chemoattractive stimulus, thus evidencing that they are just as prone to react to mechanical cues as invasive cells.   DOI: 10.1088/1367-2630/ab3572   Journal Homepage

New Journal of Physics, 21 (2019)

Tom Golde, Martin Glaser, Cary Tutmarc, Iman Elbalasy, Constantin Huster, Gaizka Busteros, David M. Smith, Harald Herrmann, Josef A. Käs, Jörg Schnauß:

The role of stickiness in the rheology of semiflexible polymers

Abstract:Semiflexible polymers form central structures in biological material. Modelling approaches usually neglect influences of polymer-specific molecular features aiming to describe semiflexible polymers universally. Here, we investigate the influence of molecular details on networks assembled from filamentous actin, intermediate filaments, and synthetic DNA nanotubes. In contrast to prevalent theoretical assumptions, we find that bulk properties are affected by various inter-filament interactions. We present evidence that these interactions can be merged into a single parameter in the frame of the glassy wormlike chain model. The interpretation of this parameter as a polymer specific stickiness is consistent with observations from macro-rheological measurements and reptation behaviour. Our findings demonstrate that stickiness should generally not be ignored in semiflexible polymer models.   DOI: 10.1039/C9SM00433E  Journal Homepage

Soft Matter, 15 (2019) 4865-4872

Frank Sauer, Linda Oswald, Angela Ariza de Schellenberger, Heiko Tzschätzsch, Felix Schrank, Tony Fischer, Jürgen Braun, Claudia T. Mierke, Rustem Valiullin, Ingolf Sack, Josef A. Käs:

Collagen networks determine viscoelastic properties of connective tissues yet do not hinder diffusion of the aqueous solvent

Abstract:Collagen accounts for the major extracellular matrix (ECM) component in many tissues and provides mechanical support for cells. Magnetic Resonance (MR) Imaging, MR based diffusion measurements and MR Elastography (MRE) are considered sensitive to the microstructure of tissues including collagen networks of the ECM. However, little is known whether water diffusion interacts with viscoelastic properties of tissues. This study combines highfield MR based diffusion measurements, novel compact tabletop MRE and confocal microscopy in collagen networks of different cross-linking states (untreated collagen gels versus additional treatment with glutaraldehyde). The consistency of bulk rheology and MRE within a wide dynamic range is demonstrated in heparin gels, a viscoelastic standard for MRE. Additional crosslinking of collagen led to an 8-fold increased storage modulus, a 4-fold increased loss modulus and a significantly decreased power law exponent, describing multi-relaxational behavior, corresponding to a pronounced transition from viscous-soft to elastic-rigid properties. Collagen network changes were not detectable by MR based diffusion measurements and microscopy which are sensitive to the micrometer scale. The MRE-measured shear modulus is sensitive to collagen fiber interactions which take place on the intrafiber level such as fiber stiffness. The insensitivity of MR based diffusion measurements to collagen hydrogels of different cross-linking states alludes that congeneric collagen structures in connective tissues do not hinder extracellular diffusive water transport. Furthermore, the glutaraldehyde induced rigorous changes in viscoelastic properties indicate that intrafibrillar dissipation is the dominant mode of viscous dissipation in collagen-dominated connective tissue.   DOI: 10.1039/C8SM02264J  Journal Homepage

Soft Matter, 15 (2019) 3055-3064

Stefanie Riedel, Philine Hietschold, Catharina Krömmelbein, Tom Kunschmann, Robert Konieczny, Wolfgang Knolle, Claudia T. Mierke, Mareike Zink, Stefan G. Mayr:

Design of biomimetic collagen matrices by reagent-free electron beam induced crosslinking: Structure-property relationships and cellular response

Abstract: Novel strategies to mimic mammalian extracellular matrix (ECM) invitro are desirable to study cell behavior, diseases and new agents in drug delivery. Even though collagen represents the major constituent of mammalian ECM, artificial collagen hydrogels with characteristic tissue properties such as network size and stiffness are difficult to design without application of chemicals which might be even cytotoxic. In our study we investigate how high energy electron induced crosslinking can be utilized to precisely tune collagen properties for ECM modelsystems. Constituting a minimally invasive approach, collagen residues remain intact in the course of high energy electron treatment. Quantification of the 3D pore size of the collagen network as a function of irradiation dose shows an increase in density leading to decreased pore size. Rheological measurements indicate elevated storage and loss moduli correlating with an increase in crosslinking density. In addition, cell tests show well maintained viability of NIH 3T3 cells for irradiated collagen gels indicating excellent cellular acceptance. With this, our investigations demonstrate that electron beam crosslinked collagen matrices have a high potential as precisely tunable ECM-mimetic systems with excellent cytocompatibility.   DOI: 10.1016/j.matdes.2019.107606  Journal Homepage

Materials and Design, 168 (2019) 107606

Astrid Weidt, Stefan G. Mayr, Mareike Zink:

Influence of Topological Cues on Fibronectin Adsorption and Contact Guidance of Fibroblasts on Microgrooved Titanium

Abstract:The choice of suitable nano- and microstructures of biomaterials is crucial for successful implant integration within the human body. In particular, surface characteristics affect the adsorption of various extra cellular matrix proteins. This work illustrates the interaction of protein adsorption and early cell adhesion on bulk microstructured titanium surfaces with parallel grooves of 27 to 35 μm widths and 15 to 19 μm depths, respectively. In contact with low concentrated fibronectin solutions, distinct adsorption patterns are observed on the edges of the ridges. Moreover, NIH/3T3 fibroblasts cultured in serum-free medium for 1 h, 3 h, and 1 day show enhanced early cell adhesion on fibronectin coated samples compared to uncoated ones. In fact, early adhesion and cell contacts occur mainly on the groove edges where fibronectin adsorption was preferentially detected. Such adsorption patterns support cellular contact guidance on short time scales since the adsorbed fibronectin proteins acted as a chemical boundary superimposing the topographical cues of the grooved microstructure. In fibronectin-free conditions, this chemical boundary is absent after cell seeding and initial cell-surface interaction. Here, cellular fibronectin released by the fibroblasts adsorbs along the grooves after 3 h and contact guidance occurs delayed. After 1 day, cell adhesion and cell morphology on uncoated and fibronectin coated titanium microgrooves were nearly equilibrated. Thus, surface structures can promote directed adsorption of low concentrated fibronectin, which, furthermore, facilitates early cell adhesion. These results give rise to new developments in surface engineering of biomedical implants for improved osseointegration.   DOI: 10.1021/acsabm.8b00667  Journal Homepage

ACS Appl. Bio Mater.,  (2019) 2,3, 1066-1077

Claudia T. Mierke, Frank Sauer, Steffen Grosser, Stefanie Puder, Tony Fischer, Josef A. Käs:

The two faces of enhanced stroma: Stroma acts as a tumor promoter and a steric obstacle

Abstract: In addition to genetic, morphological and biochemical alterations in cells, a key feature of the malignant progression of cancer is the stroma, including cancer cell motility as well as the emergence of metastases. Our current knowledge with regard to the biophysically driven experimental approaches of cancer progression indicates that mechanical aberrations are major contributors to the malignant progression of cancer. In particular, the mechanical probing of the stroma is of great interest. However, the impact of the tumor stroma on cellular motility, and hence the metastatic cascade leading to the malignant progression of cancer, is controversial as there are two different and opposing effects within the stroma. On the one hand, the stroma can promote and enhance the proliferation, survival and migration of cancer cells through mechanotransduction processes evoked by fiber alignment as a result of increased stroma rigidity. This enables all types of cancer to overcome restrictive biological capabilities. On the other hand, as a result of its structural constraints, the stroma acts as a steric obstacle for cancer cell motility in dense three-dimensional extracellular matrices, when the pore size is smaller than the cell´s nucleus. The mechanical properties of the stroma, such as the tissue matrix stiffness and the entire architectural network of the stroma, are the major players in providing the optimal environment for cancer cell migration. Thus, biophysical methods determining the mechanical properties of the stroma, such as magnetic resonance elastography, are critical for the diagnosis and prediction of early cancer stages. Fibrogenesis and cancer are tightly connected, as there is an elevated risk of cancer on cystic fibrosis or, subsequently, cirrhosis. This also applies to the subsequent metastatic process.   DOI: 10.1002/nbm.3831  Journal Homepage

NMR Biomed.,  vol. 10, e3831 (2018)

Tom Golde, Constantin Huster, Martin Glaser, Tina Händler, Harald Herrmann, Josef A. Käs and Jörg Schnauß:

Glassy dynamics in composite biopolymer networks

Abstract: The cytoskeleton is a highly interconnected meshwork of strongly coupled subsystems providing mechanical stability as well as dynamic functions to cells. To elucidate the underlying biophysical principles, it is central to investigate not only one distinct functional subsystem but rather their interplay as composite biopolymeric structures. Two of the key cytoskeletal elements are actin and vimentin filaments. Here, we show that composite networks reconstituted from actin and vimentin can be described by a superposition of two non-interacting scaffolds. Arising effects are demonstrated in a scale-spanning frame connecting single filament dynamics to macro-rheological network properties. The acquired results of the linear and non-linear bulk mechanics can be captured within an inelastic glassy wormlike chain model. In contrast to previous studies, we find no emergent effects in these composite networks. Thus, our study paves the way to predict the mechanics of the cytoskeleton based on the properties of its single structural components.   DOI: 10.1039/C8SM01061G  Journal Homepage

Soft Matter,  vol. 14, 7970-7978 (2018)

Mehmet Volkan Cakir, Uta Allenstein, Mareike Zink, Stefan G. Mayr:

Early adhesion of cells to ferromagnetic shape memory alloys functionalized with plasma assembled biomolecules - a single cell force spectroscopy study

Abstract: Biomaterial performance and integration of prostheses in vivo strongly depend on the ability of cells to adhere. Plasma-assisted functionalization of smart metals with biopolymers, including plasma polymerized l-lysine (PPLL), constitutes a recently-developed promising approach to synthesize highly flexible, yet robust and strongly adherent protein coatings that support cell-biomaterial interaction. In the present study we employ single cell force spectroscopy to demonstrate that PPLL coatings promote early adhesion of fibroblast cells on the ferromagnetic shape memory alloy FePd - a promising magnetically switchable biomaterial. By varying the contact time of a cell with the substrate surface, we show that the forces and work needed to fully detach a cell increase with time and quantify bioactivity of the material. In contrast to glass and PPLL-coated glass, cell detachment from FePd requires much larger work, while a PPLL biofunctionalization further improves cell adhesion and binding affinity by an increased detachment work on short time scales. Together with a time-dependent bond model we postulate a transition from unspecific to specific cell adhesion on FePd and PPLL-coated FePd, while on glass detachment forces are lower and level off to a saturation regime on short times prior to the expected time necessary for specific integrin-based bond formation.   DOI: 10.1016/j.matdes.2018.012  Journal Homepage

Materials & Design, vol. 158, 19-27  (2018)

Felix Meinhövel, Roland Stange, Jörg Schnauß, Michael Sauer, Josef A. Käs, Torsten W. Remmerbach:

Changing cell mechanics - a precondition for malignant transformation of oral squamous carcinoma cells

Abstract: Oral squamous cell carcinomas (OSCC) are the sixth most common cancer and the diagnosis is often belated for a curative treatment. The reliable and early differentiation between healthy and diseased cells is the main aim of this study in order to improve the quality of the treatment and to understand tumour pathogenesis. Here, the optical stretcher is used to analyse mechanical properties of cells and their potential to serve as a marker for malignancy. Stretching experiments revealed for the first time that cells of primary OSCCs were deformed by 2.9% rendering them softer than cells of healthy mucosa which were deformed only by 1.9%. Furthermore, the relaxation behaviour of the cells revealed that these malignant cells exhibit a faster contraction than their benign counterparts. This suggests that deformability as well as relaxation behaviour can be used as distinct parameters to evaluate emerging differences between these benign and malignant cells. Since many studies in cancer research are performed with cancer cell lines rather than primary cells, we have compared the deformability and relaxation of both types, showing that long time culturing leads to softening of cells. The higher degree of deformability and relaxation behaviour can enable cancer cells to traverse tissue emphasizing that changes in cell architecture may be a potential precondition for malignant transformation. Respecting the fact that even short culture times have an essential effect on the significance of the results, the use of primary cells for further research is recommended. The distinction between malignant and benign cells would enable an early confirmation of cancer diagnoses by testing cell samples of suspect oral lesions.   DOI: 10.1088/2057-1739/aac72d  Journal Homepage

Convergent Science Physical Oncology, vol. 4, 034001  (2018)

Jessica Lorenz, Jörg Schnauß, Martin Glaser, Martin Sajfutdinow, Carsten Schuldt, Josef A. Käs, David M. Smith:

Synthetic Transient Crosslinks Program the Mechanics of Soft, Biopolymer-Based Materials

Abstract: Actin networks are adaptive materials enabling dynamic and static functions of living cells. A central element for tuning their underlying structural and mechanical properties is the ability to reversibly connect, i.e., transiently crosslink, filaments within the networks. Natural crosslinkers, however, vary across many parameters. Therefore, systematically studying the impact of their fundamental properties like size and binding strength is unfeasible since their structural parameters cannot be independently tuned. Herein, this problem is circumvented by employing a modular strategy to construct purely synthetic actin crosslinkers from DNA and peptides. These crosslinkers mimic both intuitive and noncanonical mechanical properties of their natural counterparts. By isolating binding affinity as the primary control parameter, effects on structural and dynamic behaviors of actin networks are characterized. A concentration-dependent triphasic behavior arises from both strong and weak crosslinkers due to emergent structural polymorphism. Beyond a certain threshold, strong binding leads to a nonmonotonic elastic pulse, which is a consequence of self-destruction of the mechanical structure of the underlying network. The modular design also facilitates an orthogonal regulatory mechanism based on enzymatic cleaving. This approach can be used to guide the rational design of further biomimetic components for programmable modulation of the properties of biomaterials and cells.   DOI: 10.1002/adma.201706092  Journal Homepage

Advanced Materials 1706092  (2018)

Christin Möser, Jessica S. Lorenz, Martin Sajfutdinow, David M. Smith:

Pinpointed Stimulation of EphA2 Receptors via DNA-Templated Oligovalence

Abstract: DNA nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. Here, the efficacy of SWL, an ephrin-mimicking peptide that binds specifically to EphrinA2 (EphA2) receptors, increased by presenting up to three of these peptides on small DNA nanostructures in an oligovalent manner. Ephrin signaling pathways play crucial roles in tumor development and progression. Moreover, Eph receptors are potential targets in cancer diagnosis and treatment. Here, the quantitative impact of SWL valency on binding, phosphorylation (key player for activation) and phenotype regulation in EphA2-expressing prostate cancer cells was demonstrated. EphA2 phosphorylation was significantly increased by DNA trimers carrying three SWL peptides compared to monovalent SWL. In comparison to one of EphA2´s natural ligands ephrin-A1, which is known to bind promiscuously to multiple receptors, pinpointed targeting of EphA2 by oligovalent DNA-SWL constructs showed enhanced cell retraction. Overall, we show that DNA scaffolds can increase the potency of weak signaling peptides through oligovalent presentation and serve as potential tools for examination of complex signaling pathways.   DOI: 10.3390/ijms19113482  Journal Homepage

Int. J. Mol. Sci. 19 (11), 3482  (2018)

Martin Sajfutdinow, William M. Jacobs, Aleks Reinhardt, Christoph Schneider, David M. Smith:

Direct observation and rational design of nucleation behavior in addressable self-assembly

Abstract: To optimize a self-assembly reaction, it is essential to understand the factors that govern its pathway. Here, we examine the influence of nucleation pathways in a model system for addressable, multicomponent self-assembly based on a prototypical “DNA-brick” structure. By combining temperature-dependent dynamic light scattering and atomic force microscopy with coarse-grained simulations, we show how subtle changes in the nucleation pathway profoundly affect the yield of the correctly formed structures. In particular, we can increase the range of conditions over which self-assembly occurs by using stable multisubunit clusters that lower the nucleation barrier for assembling subunits in the interior of the structure. Consequently, modifying only a small portion of a structure is sufficient to optimize its assembly. Due to the generality of our coarse-grained model and the excellent agreement that we find with our experimental results, the design principles reported here are likely to apply generically to addressable, multicomponent self-assembly.   DOI: 10.1073/pnas.1806010115  Journal Homepage

PNAS June 26, 115 (26) E5877-E5886  (2018)

Megan C. Engel, David M. Smith, Markus A. Jobst, Martin Sajfutdinow, Tim Liedl, Flavio Romano, Lorenzo Rovigatti, Ard A. Louis, Jonathan P. K. Doye:

Force-Induced Unravelling of DNA Origami

Abstract: The mechanical properties of DNA nanostructures are of widespread interest as applications that exploit their stability under constant or intermittent external forces become increasingly common. We explore the force response of DNA origami in comprehensive detail by combining AFM single molecule force spectroscopy experiments with simulations using oxDNA, a coarse-grained model of DNA at the nucleotide level, to study the unravelling of an iconic origami system: the Rothemund tile. We contrast the force-induced melting of the tile with simulations of an origami 10-helix bundle. Finally, we simulate a recently proposed origami biosensor, whose function takes advantage of origami behavior under tension. We observe characteristic stick-slip unfolding dynamics in our force-extension curves for both the Rothemund tile and the helix bundle and reasonable agreement with experimentally observed rupture forces for these systems. Our results highlight the effect of design on force response: we observe regular, modular unfolding for the Rothemund tile that contrasts with strain-softening of the 10-helix bundle which leads to catastropic failure under monotonically increasing force. Further, unravelling occurs straightforwardly from the scaffold ends inward for the Rothemund tile, while the helix bundle unfolds more nonlinearly. The detailed visualization of the yielding events provided by simulation allows preferred pathways through the complex unfolding free-energy landscape to be mapped, as a key factor in determining relative barrier heights is the extensional release per base pair broken. We shed light on two important questions: how stable DNA nanostructures are under external forces and what design principles can be applied to enhance stability.   DOI: 10.1021/acsnano.8b01844  Journal Homepage

ACS Nano 12(7), 6734-6747  (2018)

2017
 

Dan Strehle, Paul Mollenkopf, Martin Glaser, Tom Golde, Carsten Schuldt, Josef A. Käs, Jörg Schnauß:

Single Actin Bundle Rheology

Abstract: Bundled actin structures play an essential role in the mechanical response of the actin cytoskeleton in eukaryotic cells. Although responsible for crucial cellular processes, they are rarely investigated in comparison to single filaments and isotropic networks. Presenting a highly anisotropic structure, the determination of the mechanical properties of individual bundles was previously achieved through passive approaches observing bending deformations induced by thermal fluctuations. We present a new method to determine the bending stiffness of individual bundles, by measuring the decay of an actively induced oscillation. This approach allows us to systematically test anisotropic, bundled structures. Our experiments revealed that thin, depletion force-induced bundles behave as semiflexible polymers and obey the theoretical predictions determined by the wormlike chain model. Thickening an individual bundle by merging it with other bundles enabled us to study effects that are solely based on the number of involved filaments. These thicker bundles showed a frequency-dependent bending stiffness, a behavior that is inconsistent with the predictions of the wormlike chain model. We attribute this effect to internal processes and give a possible explanation with regard to the wormlike bundle theory.  DOI: 10.3390/molecules22101804  Journal Homepage

Molecules, Volume 22, 1804  (2017)

 

Jörg Schnauß, Martin Glaser, Jessica S. Lorenz, Carsten Schuldt, Christin Möser, Martin Sajfutinow, Tina Händler, Josef A. Käs, David M. Smith:

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Abstract: Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.  DOI: 10.3791/56056  Journal Homepage

Journal of Visualized Experiments, 56056  (2017)

 

Linda Oswald, Steffen Grosser, David M. Smith, J Käs:

Jamming transitions in cancer

Abstract:The traditional picture of tissues, where they are treated as liquids defined by properties such as surface tension or viscosity has been redefined during the last few decades by the more fundamental question: Under which conditions do tissues display liquid-like or solid-like behaviour? As a result, basic concepts arising from the treatment of tissues as solid matter, such as cellular jamming and glassy tissues, have shifted into the current focus of biophysical research. Here, we review recent works examining the phase states of tissue with an emphasis on jamming transitions in cancer. When metastasis occurs, cells gain the ability to leave the primary tumour and infiltrate other parts of the body. Recent studies have shown that a linkage between an unjamming transition and tumour progression indeed exists, which could be of importance when designing surgery and treatment approaches for cancer patients.  DOI: 10.1088/1361-6463/aa8e83  Journal Homepage

Journal of Physics D: Applied Physics, Volume 50, 483001  (2017)

 

Hans Kubitschke, Jörg Schnauß, K. David Nnetu, Enrico Warmt, Roland Stange, Josef A. Käs:

Actin and microtubule networks contribute differently to cell response for small and large strains

Abstract: Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actinfilaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing thesefilaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.  DOI: 10.1088/1367-2630/aa7658  Journal Homepage

New Journal of Physics, Volume 19, 093003  (2017)

 

Erik W. Morawetz, Roland Stange, Tobias R. Kießling, Jörg Schnauß, Josef A Käs:

Optical stretching in continuous flows

Abstract: Rheology of living cells has developed an increasing need for high throughput measurements. Diseases such as cancer heavily remodel the cytoskeleton and impinge on cellular functions. Cells affected by such diseases show altered rheologic responses on many different levels rendering cells' mechanical fingerprints - a potential target for diagnostics. To counteract naturally occurring distributions of properties in samples of living cells and foster the validity of experiments, high numbers of single cell measurements are necessary. Here, we present the 'in flow optical stretcher' (IFOS), a concept of non-invasive optical cytometry capable of high throughput rates, while working in a regime of long measurement times and low frequencies. The setup deforms whole cells in a continuous flow by optical forces, bypassing steps of cell positioning that are unavoidable in state-of-the-art optical stretcher devices. A prototype was built using polydimethylsiloxane soft lithography. In a proof of premise experiment, we show that in the IFOS it is possible to deform cells of mammalian origin which have been treated with cytochalasin. All recorded successful experiments took place in less than 2 s each, as opposed to 10-20 s in state-of-the-art optical stretcher devices. Although other microfluidic rheology devices achieve significantly higher throughput rates, they operate in different frequency regimes and probe different mechanical responses. The IFOS still captures viscoelastic properties and active responses of cells while aiming to maximize the throughput at creep times on the order of seconds. It can be assumed that an automatic IFOS reaches a throughput an order of magnitude higher than current devices that are based on optical stretching for cell rheology. DOI: 10.1088/2057-1739/aa6eb1  Journal Homepage

Convergent Science Physical Oncology, Volume 3, Number 2, 024004  (2017)

 

Frieda Kage, Moritz Winterhoff, Vanessa Dimchev, Jan Mueller, Tobias Thalheim, Anika Freise, Stefan Brühmann, Jana Kollasser, Jennifer Block, Georgi Dimchev, Matthias Geyer, Hans-Joachim Schnittler, Cord Brakebusch, Theresia E. B. Stradal, Marie-France Carlier, Michael Sixt, Josef Käs, Jan Faix, Klemens Rottner:

FMNL formins boost lamellipodial force generation

Abstract: Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching. DOI: 10.1038/ncomms14832  Journal Homepage

Nature Communications, Volume 8, 14832 (2017)

 

Sonja Kallendrusch, Felicitas Merz, Ingo Bechmann, Stefan G. Mayr, Mareike Zink:

Long-Term Tissue Culture of Adult Brain and Spleen Slices on Nanostructured Scaffolds

Abstract: Long-term tissue culture of adult mammalian organs is a highly promising approach to bridge the gap between single cell cultures and animal experiments, and bears the potential to reduce in vivo studies. Novel biomimetic materials open up new possibilities to maintain the complex tissue structure in vitro; however, survival times of adult tissues ex vivo are still limited to a few days with established state-of-the-art techniques. Here, it is demonstrated that TiO2 nanotube scaffolds with specific tissue-tailored characteristics can serve as superior substrates for long-term adult brain and spleen tissue culture. High viability of the explants for at least two weeks is achieved and compared to tissues cultured on standard polytetrafluoroethylene (PTFE) membranes. Histological and immunohistochemical staining and live imaging are used to investigate tissue condition after 5 and 14 d in vitro, while environmental scanning electron microscopy qualifies the interaction with the underlying scaffold. In contrast to tissues cultured on PTFE membranes, enhanced tissue morphology is detected in spleen slices, as well as minor cell death in neuronal tissue, both cultured on nanotube scaffolds. This novel biomimetic tissue model will prove to be useful to address fundamental biological and medical questions from tissue regeneration up to tumor progression and therapeutic approaches. DOI: 10.1002/adhm.201601336 Journal Homepage

Advances Healthcare Materials, Volume 6, Issue 9, 1601336 (2017)

 

Benedikt Heyart, Astrid Weidt, Emilia I. Wisotzki, Mareike Zink, Stefan G. Mayr:

Micropatterning of reagent-free high energy crosslinked gelatin hydrogels for bioapplications

Abstract: Hydrogels are crosslinked polymeric gels of great interest in the field of tissue engineering, particularly as biocompatible cell or drug carriers. Reagent-free electron irradiated gelatin is simple to manufacture, inexpensive and biocompatible. Here, the potential to micropattern gelatin hydrogel surfaces during electron irradiation crosslinking was demonstrated as a promising microfabrication technique to produce thermally stable structures on highly relevant length scales for bioapplications. In the present work, grooves of 3.75 to 170 µm width and several hundred nanometers depth were transferred onto gelatin hydrogels during electron irradiation and characterized by 3D confocal microscopy after exposure to ambient and physiological conditions. The survival and influence of these microstructures on cellular growth was further characterized using NIH 3T3 fibroblasts. Topographical modifications produced surface structures on which the cultured fibroblasts attached and responded by adapting their morphologies. This developed technique allows for simple and effective structuring of gelatin and opens up new possibilities for irradiation crosslinked hydrogels in biomedical applications in which cell attachment and contact guidance are favored. DOI: 10.1002/jbm.b.33849  Journal Homepage

Journal of Biomedical Materials Research B: Applied Biomaterials, published online before print (2017)

2016
 

Carsten Schuldt, Jörg Schnauß, Tina Händler, Martin Glaser, Jessica Lorenz, Tom Golde, J. A. Käs, David M. Smith:

Tuning synthetic semiflexible networks by bending stiffness

Abstract: The mechanics of complex soft matter often cannot be understood in the classical physical frame of flexible polymers or rigid rods. The underlying constituents are semiflexible polymers, whose finite bending stiffness (k) leads to nontrivial mechanical responses. A natural model for such polymers is the protein actin. Experimental studies of actin networks, however, are limited since the persistence length (lp ∝ k) cannot be tuned. Here, we experimentally characterize this parameter for the first time in entangled networks formed by synthetically produced, structurally tunable DNA nanotubes. This material enabled the validation of characteristics inherent to semiflexible polymers and networks thereof, i.e., persistence length, inextensibility, reptation, and mesh size scaling. While the scaling of the elastic plateau modulus with concentration G0 ∝ c7/5 is consistent with previous measurements and established theories, the emerging persistence length scaling G0 ∝ lp opposes predominant theoretical predictions. DOI: 10.1103/PhysRevLett.117.197801 Journal Homepage

Physical Review Letters, Volume 117, Issue 19, 197801 (2016)

 

Jörg Schnauß, Tina Händler, Josef A. Käs:

Semiflexible biopolymers in bundled arrangements

Abstract: Bundles and networks of semiflexible biopolymers are key elements in cells, lending them mechanical integrity while also enabling dynamic functions. Networks have been the subject of many studies, revealing a variety of fundamental characteristics often determined via bulk measurements. Although bundles are equally important in biological systems, they have garnered much less scientific attention since they have to be probed on the mesoscopic scale. Here, we review theoretical as well as experimental approaches, which mainly employ the naturally occurring biopolymer actin, to highlight the principles behind these structures on the single bundle level. DOI: 10.3390/polym8080274 Journal Homepage

Polymers, Volume 8, Issue 8, 274 (2016)

 

Martin Glaser, Jörg Schnauß, Teresa Tschirner, B. U. Sebastian Schmidt, Maximilian Moebius-Winkler, Josef A. Käs, David M. Smith:

Self-assembly of hierarchically ordered structures in DNA nanotube systems

Abstract: The self-assembly of molecular and macromolecular building blocks into organized patterns is a complex process found in diverse systems over a wide range of size and time scales. The formation of star- or aster-like configurations, for example, is a common characteristic in solutions of polymers or other molecules containing multi-scaled, hierarchical assembly processes. This is a recurring phenomenon in numerous pattern-forming systems ranging from cellular constructs to solutions of ferromagnetic colloids or synthetic plastics. To date, however, it has not been possible to systematically parameterize structural properties of the constituent components in order to study their influence on assembled states. Here, we circumvent this limitation by using DNA nanotubes with programmable mechanical properties as our basic building blocks. A small set of DNA oligonucleotides can be chosen to hybridize into micron-length DNA nanotubes with a well-defined circumference and stiffness. The self-assembly of these nanotubes to hierarchically ordered structures is driven by depletion forces caused by the presence of polyethylene glycol. This trait allowed us to investigate self-assembly effects while maintaining a complete decoupling of density, self-association or bundling strength, and stiffness of the nanotubes. Our findings show diverse ranges of emerging structures including heterogeneous networks, aster-like structures, and densely bundled needle-like structures, which compare to configurations found in many other systems. These show a strong dependence not only on concentration and bundling strength, but also on the underlying mechanical properties of the nanotubes. Similar network architectures to those caused by depletion forces in the low-density regime are obtained when an alternative hybridization-based bundling mechanism is employed to induce self-assembly in an isotropic network of pre-formed DNA nanotubes. This emphasizes the universal effect inevitable attractive forces in crowded environments have on systems of self-assembling soft matter, which should be considered for macromolecular structures applied in crowded systems such as cells. DOI: 10.1088/1367-2630/18/5/055001 Journal Homepage

New Journal of Physics, Volume 18, Issue 5, 055001 (2016)

 

Jörg Schnauß, Tom Golde, Carsten Schuldt, B. U. Sebastian Schmidt, Martin Glaser, Dan Strehle, Tina Händler, Claus Heussinger, Josef A. Käs:

Transition from a linear to a harmonic potential in collective dynamics of a multifilament actin bundle

Abstract: Attractive depletion forces between rodlike particles in highly crowded environments have been shown through recent modeling and experimental approaches to induce different structural and dynamic signatures depending on relative orientation between rods. For example, it has been demonstrated that the axial attraction between two parallel rods yields a linear energy potential corresponding to a constant contractile force of 0.1 pN. Here, we extend pairwise, depletion-induced interactions to a multifilament level with actin bundles, and find contractile forces up to 3 pN. Forces generated due to bundle relaxation were not constant, but displayed a harmonic potential and decayed exponentially with a mean decay time of 3.4 s. Through an analytical model, we explain these different fundamental dynamics as an emergent, collective phenomenon stemming from the additive, pairwise interactions of filaments within a bundle. DOI: 10.1103/PhysRevLett.116.108102 Journal Homepage

Physical Review Letters, Volume 116, Issue 10, 108102 (2016)

 

Emilia I. Wisotzki, Ralf P. Friedrich, Astrid Weidt, Christoph Alexiou, Stefan G. Mayr, Mareike Zink:

Cellular response to reagent-free electron-irradiated gelatin hydrogels

Abstract: As a biomaterial, it is well established that gelatin exhibits low cytotoxicity and can promote cellular growth. However, to circumvent the potential toxicity of chemical crosslinkers, reagent-free crosslinking methods such as electron irradiation are highly desirable. While high energy irradiation has been shown to exhibit precise control over the degree of crosslinking, these hydrogels have not been thoroughly investigated for biocompatibility and degradability. Here, NIH 3T3 murine fibroblasts are seeded onto irradiated gelatin hydrogels to examine the hydrogel's influence on cellular viability and morphology. The average projected area of cells seeded onto the hydrogels increases with irradiation dose, which correlates with an increase in the hydrogel's shear modulus up to 10 kPa. Cells on these hydrogels are highly viable and exhibits normal cell cycles, particularly when compared to those grown on glutaraldehyde crosslinked gelatin hydrogels. However, proliferation is reduced on both types of crosslinked samples. To mimic the response of the hydrogels in physiological conditions, degradability is monitored in simulated body fluid to reveal strongly dose-dependent degradation times. Overall, given the low cytotoxicity, influence on cellular morphology and variability in degradation times of the electron irradiated gelatin hydrogels, there is significant potential for application in areas ranging from regenerative medicine to mechanobiology.  DOI: 10.1002/mabi.201500408 Journal Homepage

Macromolecular Bioscience, Volume 16, Issue 6, Pages 914-924 (2016)

 

S. Mayazur Rahman, Andreas Reichenbach, Mareike Zink, Stefan G. Mayr:

Mechanical spectroscopy of retina explants at the protein level employing nanostructured scaffolds

Abstract: Development of neuronal tissue, such as folding of the brain, and formation of the fovea centralis in the human retina are intimately connected with the mechanical properties of the underlying cells and the extracellular matrix. In particular for neuronal tissue as complex as the vertebrate retina, mechanical properties are still a matter of debate due to their relation to numerous diseases as well as surgery, where the tension of the retina can result in tissue detachment during cutting. However, measuring the elasticity of adult retina wholemounts is difficult and until now only the mechanical properties at the surface have been characterized with micrometer resolution. Many processes, however, such as pathological changes prone to cause tissue rupture and detachment, respectively, are reflected in variations of retina elasticity at smaller length scales at the protein level. In the present work we demonstrate that freely oscillating cantilevers composed of nanostructured TiO2 scaffolds can be employed to study the frequency-dependent mechanical response of adult mammalian retina explants at the nanoscale. Constituting highly versatile scaffolds with strong tissue attachment for long-term organotypic culture atop, these scaffolds perform damped vibrations as fingerprints of the mechanical tissue properties that are derived using finite element calculations. Since the tissue adheres to the nanostructures via constitutive proteins on the photoreceptor side of the retina, the latter are stretched and compressed during vibration of the underlying scaffold. Probing mechanical response of individual proteins within the tissue, the proposed mechanical spectroscopy approach opens the way for studying tissue mechanics, diseases and the effect of drugs at the protein level. DOI: 10.1039/C6SM00293E Journal Homepage

Soft Matter, Volume 12, Issue 14, Pages 3431-3441 (2016)

2015
 

Jörg Schnauß, Martin Glaser, Carsten Schuldt, Tom Golde, Tina Händler, Sebastian Schmidt, Stefan Diez, Josef Käs:

Motor-free force generation in biological systems

Abstract: A central part of soft matter physics is the investigation of effects in an active environment. These systems are driven out of equilibrium by a constant energy consumption. In biological systems, for instance, energy is consumed in the dynamic polymerization process of cytoskeletal filaments or by motor-filament interactions. These active processes convert chemical energy into mechanical work and impede a trapping of cellular structures in thermodynamically frozen states. Thus, active soft matter is crucial for biological systems to fulfill a broad range of tasks. Inherent physical principles relying on entropy maximizing arguments, however, cannot be easily switched off even in active systems. Cells might even employ these principles to accomplish certain tasks without the need to arrange elaborate, energy dissipating structures. Within the presented studies we demonstrate possibilities how biological relevant forces can be generated in the absence of any active accessory proteins. The presented studies are based on the cytoskeletal key components actin and microtubules. We demonstrate different approaches ranging from light induced softening to cross-linker expansion, which realize entropy driven contractions of the according system. Publication Journal Homepage

diffusion-fundamentals.org, Volume 23, Issue 5, Pages 1-15 (2015)

 

Steve Pawlizak, Anatol W. Fritsch, Steffen Grosser, Dave Ahrens, Tobias Thalheim, Stefanie Riedel, Tobias R. Kießling, Linda Oswald, Mareike Zink, M. Lisa Manning, Josef A. Käs:

Testing the differential adhesion hypothesis across the epithelial-mesenchymal transition

Abstract: We analyze the mechanical properties of three epithelial/mesenchymal cell lines (MCF-10A, MDA-MB-231, MDA-MB-436) that exhibit a shift in E-, N- and P-cadherin levels characteristic of an epithelial-mesenchymal transition associated with processes such as metastasis, to quantify the role of cell cohesion in cell sorting and compartmentalization. We develop a unique set of methods to measure cell-cell adhesiveness, cell stiffness and cell shapes, and compare the results to predictions from cell sorting in mixtures of cell populations. We find that the final sorted state is extremely robust among all three cell lines independent of epithelial or mesenchymal state, suggesting that cell sorting may play an important role in organization and boundary formation in tumours. We find that surface densities of adhesive molecules do not correlate with measured cell-cell adhesion, but do correlate with cell shapes, cell stiffness and the rate at which cells sort, in accordance with an extended version of the differential adhesion hypothesis (DAH). Surprisingly, the DAH does not correctly predict the final sorted state. This suggests that these tissues are not behaving as immiscible fluids, and that dynamical effects such as directional motility, friction and jamming may play an important role in tissue compartmentalization across the epithelial-mesenchymal transition. DOI: 10.1088/1367-2630/17/8/083049 Journal Homepage

New Journal of Physics, Volume 17, Issue 8, 083049 (2015)

 

Paul Heine, Allen Ehrlicher, Josef Käs:

Neuronal and metastatic cancer cells: Unlike brothers

Abstract: During development neuronal cells traverse substantial distances across the developing tissue. In the mature organism, however, they are bound to the confines of the nervous system. Likewise metastatic cancer cells have the potential to establish auxiliary tumor sites in remote tissues or entirely different organs. The epithelial–mesenchymal transition is the transformation of proliferative cancer cells into a highly invasive state, which facilitates the crossing of tissue boundaries and migration across various environments. This review contributes a first look into the parallels and contrasts between physical aspects of neuronal and metastatic cancer cells. DOI: 10.1016/j.bbamcr.2015.06.011 Journal Homepage

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Volume 1853, Issue 11, Part B, Pages 3126-3131 (2015)

 

Chris Händel, Sebastian Schmidt, Jürgen Schiller, Undine Dietrich, Till Möhn, Tobias Kießling, Steve Pawlizak, Anatol Fritsch, Lars-Christian Horn, Susanne Briest, Michael Höckel, Mareike Zink, Josef Käs:

Cell membrane softening in human breast and cervical cancer cells

Abstract: Biomechanical properties are key to many cellular functions such as cell division or cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. Mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the  plasma membrane with the exception of red blood cells is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.  DOI: 10.1088/1367-2630/17/8/083008 Journal Homepage

New Journal of Physics, Volume 17, Issue 8, 083008 (2015)

 

Simone Braig, Sebastian U. Schmidt, Katharina Ferkaljuk, Chris Händel, Till Möhn, Oliver Werz, Rolf Müller, Stefan Zahler, Andreas Koeberle, Josef A. Käs, Angelika M. Vollmar:

Pharmacological targeting of membrane rigidity: Implications on cancer cell migration and invasion

Abstract: The invasive potential of cancer cells strongly depends on cellular stiffness, a physical quantity that is not only regulated by the mechanical impact of the cytoskeleton but also influenced by the membrane rigidity. To analyze the specific role of membrane rigidity in cancer progression, we treated cancer cells with the Acetyl-CoA carboxylase inhibitor Soraphen A and revealed an alteration of the phospholipidome via mass spectrometry. Migration, invasion, and cell death assays were employed to relate this alteration to functional consequences, and a decrease of migration and invasion without significant impact on cell death has been recorded. Fourier fluctuation analysis of giant plasma membrane vesicles showed that Soraphen A increases membrane rigidity of carcinoma cell membranes. Mechanical measurements of the creep deformation response of whole intact cells were performed using the optical stretcher. The increase in membrane rigidity was observed in one cell line without changing the creep deformation response indicating no restructuring of the cytoskeleton. These data indicate that the increase of membrane rigidity alone is sufficient to inhibit invasiveness of cancer cells, thus disclosing the eminent role of membrane rigidity in migratory processes. DOI: 10.1088/1367-2630/17/8/083007 Journal Homepage

New Journal of Physics, Volume 17, Issue 8, 083007 (2015)

 

Uta Allenstein, Susanne Selle, Meike Tadsen, Christian Patzig, Thomas Höche, Mareike Zink, Stefan G. Mayr:

Coupling of Metals and Biominerals: Characterizing the Interface between Ferromagnetic Shape-Memory Alloys and Hydroxyapatite

Abstract: Durable, mechanically robust osseointegration of metal implants poses one of the largest challenges in contemporary orthopedics. Application of biomimetic hydroxyapatite (HAp) coatings as mediator for enhanced mechanical coupling to natural bone constitutes a promising approach. Motivated by recent advances in the field of smart metals that might open the venue for alternate therapeutic concepts, we explore their mechanical coupling to sputter-deposited HAp layers in a combined experimental-theoretical study. While experimentally delamination tests and comprehensive structural characterization, including high resolution transmission electron microscopy, are utilized to establish structure-property relationships, density functional theory based total energy calculations unravel the underlying physics and chemistry of bonding and confirm the experimental findings. Experiments and modeling indicate, that sputter deposited hydroxyapatite coatings are strongly adherent to the exemplary ferromagnetic shape memory alloys, Ni-Mn-Ga and Fe-Pd, with delamination stresses and interface bonding strength exceeding the physiological scales by orders of magnitudes.  DOI: 10.1021/acsami.5b03189 Journal Homepage

ACS Applied Materials & Interfaces, Volume 7, Issue 28, Pages 15331-15338 (2015)

 

Sebastian B. Schmidt, Tobias R. Kießling, Enrico Warmt, Anatol W. Fritsch, Roland Stange, Josef Käs:

Complex thermorheology of living cells

Abstract: Temperature has a reliable and nearly instantaneous influence on mechanical responses of cells. As recently published, MCF-10A normal epithelial breast cells follow the time–temperature superposition (TTS) principle. Here, we measured thermorheological behaviour of eight common cell types within physiologically relevant temperatures and applied TTS to creep compliance curves. Our results showed that superposition is not universal and was seen in four of the eight investigated cell types. For the other cell types, transitions of thermorheological responses were observed at 36 °C. Activation energies (EA) were calculated for all cell types and ranged between 50 and 150 kJ mol?1. The scaling factors of the superposition of creep curves were used to group the cell lines into three categories. They were dependent on relaxation processes as well as structural composition of the cells in response to mechanical load and temperature increase. This study supports the view that temperature is a vital parameter for comparing cell rheological data and should be precisely controlled when designing experiments. DOI: 10.1088/1367-2630/17/7/073010 Journal Homepage

New Journal of Physics, Volume 17, 073010 (2015)

 

D. Michalski, W. Härtig, M. Krueger, C. Hobohm, J. A. Käs, T. Fuhs:

A novel approach for mechanical tissue characterization indicates decreased elastic strength in brain areas affected by experimental thromboembolic stroke

Abstract: As treatment of ischemic stroke remains a challenge with respect to the failure of numerous neuroprotective attempts, there is an ongoing need for better understanding of pathophysiological mechanisms causing tissue damage. Although ischemic outcomes have been studied extensively at the cellular and molecular level using histological and biochemical methods, properties of ischemia-affected brain tissue with respect to mechanical integrity have not been addressed so far. As a novel approach, this study used fluorescence-based detection of regions affected by experimental thromboembolic stroke in combination with scanning force microscopy to examine mechanical alterations in selected rat brain areas. Twenty-five hours after onset of ischemia, a decreased elastic strength in the striatum as the region primarily affected by ischemia was found compared with the contralateral nonaffected hemisphere. Additional intrahemispheric analyses showed decreased elastic strength in the ischemic border zone compared with the more severely affected striatum. In conclusion, these data strongly indicate a critical alteration in mechanical tissue integrity caused by focal cerebral ischemia. Further, on the basis of data that have been obtained in relation to the ischemic border zone, a shell-like pattern of mechanical tissue damage was found in good accordance with the penumbra concept. These findings might enable the development of specific therapeutic interventions to protect affected areas from critical loss of mechanical integrity.  DOI: 10.1097/WNR.0000000000000395 Journal Homepage

Neuroreport, Volume 26, Issue 10, Pages 583-587 (2015)

 

U. Allenstein, S. G. Mayr, M. Zink:

Contractile cell forces deform macroscopic cantilevers and quantify biomaterial performance

Abstract: Cells require adhesion to survive, proliferate and migrate, as well as for wound healing and many other functions. The strength of contractile cell forces on an underlying surface is a highly relevant quantity to measure the affinity of cells to a rigid surface with and without coating. Here we show with experimental and theoretical studies that these forces create surface stresses that are sufficient to induce measurable bending of macroscopic cantilevers. Since contractile forces are linked to the formation of focal contacts, results give information on adhesion promoting qualities and allow a comparison of very diverse materials. In exemplary studies, in vitro fibroblast adhesion on the magnetic shape memory alloy Fe–Pd and on the L-lysine derived plasma-functionalized polymer PPLL was determined. We show that cells on Fe–Pd are able to induce surface stresses three times as high as on pure titanium cantilevers. A further increase was observed for PPLL, where the contractile forces are four times higher than on the titanium reference. In addition, we performed finite element simulations on the beam bending to back up the calculation of contractile forces from cantilever bending under non-homogenous surface stress. Our findings consolidate the role of contractile forces as a meaningful measure of biomaterial performance. DOI: 10.1039/C5SM01212K  Journal Homepage

Soft Matter, Volume 11, Issue 25, Pages 5053-5059 (2015)

 

Florian Huber, Dan Strehle, Jörg Schnauß, Josef Käs:

Formation of regularly spaced networks as a general feature of actin bundle condensation by entropic forces

Abstract: Biopolymer networks contribute mechanical integrity as well as functional organization to living cells. One of their major constituents, the protein actin, is present in a large variety of different network architectures, ranging from extensive networks to densely packed bundles. The shape of the network is directly linked to its mechanical properties and essential physiological functions. However, a profound understanding of architecture-determining mechanisms and their physical constraints remains elusive. We use experimental bottom-up systems to study the formation of confined actin networks by entropic forces. Experiments based on molecular crowding as well as counterion condensation reveal a generic tendency of homogeneous filament solutions to aggregate into regular actin bundle networks connected by aster-like centers. The network architecture is found to critically rely on network formation history. Starting from identical biochemical compositions, we observe drastic changes in network architecture as a consequence of initially biased filament orientation or mixing-induced perturbations. Our experiments suggest that the tendency to form regularly spaced bundle networks is a rather general feature of isotropic, homogeneous filament solutions subject to uniform attractive interactions. Due to the fundamental nature of the considered interactions, we expect that the investigated type of network formation further implies severe physical constraints for cytoskeleton self-organization on the more complex level of living cells. DOI: 10.1088/1367-2630/17/4/043029 Journal Homepage

New Journal of Physics, Volume 17, 043029 (2015)

 

Alba C. De Luca, Mareike Zink, Astrid Weidt, Stefan G. Mayr, Athina E. Markaki:

Effect of microgrooved surface topography on osteoblast maturation and protein adsorption

Abstract:  Microgrooved surfaces have been used extensively to influence cell contact guidance. Guiding cell growth, extracellular matrix deposition, and mineralization is important for bone implant longevity. In this study, we investigated the osteoblast response to microgrooved metallic surfaces in serum-supplemented medium. Groove spacing was comparable with the spread osteoblast size. Focal adhesions were observed to confine to the intervening ridge/groove boundaries. Osteoblasts bridged over the grooves and were unable to conform to the concave shape of the underlying grooves. Microgrooved surfaces induced higher osteoblast proliferation and metabolic activity after 14 days in osteogenic medium compared with as-received surfaces, resulting in higher mineralization and alignment of cell-secreted collagen after 28 days. To establish whether preferential cell attach- ment at the ridge/groove boundaries was influenced by the adhesion proteins contained in the serum-supplemented media, fluorescently labeled fibronectin was adsorbed onto the microgrooved substrates at low concentrations, mimicking the concentrations found in blood serum. Fibronectin was found to selectively adsorb onto the ridge/groove boundaries, the osteoblast focal adhesion sites, suggesting that protein adsorption may have influenced the cell attachment pattern. DOI: 10.1002/jbm.a.35407 Journal Homepage

Journal of Biomedical Materials Research Part A, Volume 103, Issue 8, 2689-2700 (2015)

 

Steffen Grosser, Anatol W. Fritsch, Tobias R. Kießling, Roland Stange, Josef A. Käs:

The lensing effect of trapped particles in a dual-beam optical trap

Abstract: In dual-beam optical traps, two counterpropagating, divergent laser beams emitted from opposing laser fibers trap and manipulate dielectric particles. We investigate the lensing effect that trapped particles have on the beams. Our approach makes use of the intrinsic coupling of a beam to the opposing fiber after having passed the trapped particle. We present measurements of this coupling signal for PDMS particles, as well as a model for its dependence on size and refractive index of the trapped particle. As a more complex sample, the coupling of inhomogeneous biological cells is measured and discussed. We show that the lensing effect is well captured by the simple ray optics approximation. The measurements reveal intricate details, such as the thermal lens effect of the beam propagation in a dual-beam trap. For a particle of known size, the model further allows to infer its refractive index simply from the coupling signal. DOI: 10.1364/OE.23.005221 Journal Homepage

Optics Express, Volume 23, Issue 4, Pages 5221-5235 (2015)

2014
 

U. Allenstein, F. Szillat, A. Weidt, M. Zink, S. G. Mayr:

Interfacing hard and living matter: plasma-assembled proteins on inorganic functional materials for enhanced coupling to cells and tissue

Abstract: When bringing functional hard materials to biomedical applications, control of interfaces with cells and tissue poses one of the largest challenges. Assembly of protein monomers within an inert-gas-plasma constitutes a novel approach to synthesize strongly adherent bioactive coatings that dramatically enhance coupling to living matter. As proof of concept this is demonstrated for a Fe-Pd ferromagnetic shape memory transducer that is functionalized with the amino-acid, L-lysine, by plasma-treatment, resulting in flexible, yet ultra-durable coatings. Containing high amounts of NH2 functional groups, they fulfill all requirements for strong adhesion of cells and tissues. This is confirmed by cell tests with living NIH/3T3 embryonic murine fibroblasts that demonstrate excellent biocompatibility, exceeding conventional poly-L-lysine coatings in terms of cells focal adhesion density. The physics and chemistry behind these scenarios are unraveled by employing ab initio computer calculations. This combined approach opens the way for plasma-assisted functionalization strategies for a broad class of metals and semiconductors with polypeptides. DOI: 10.1039/C4TB01061B Journal Homepage

Journal of Materials Chemistry B, Volume 2, Issue 44, Pages 7739-7746 (2014)

 

Mareike Zink, S. G. Mayr:

Ferromagnetic shape memory alloys: synthesis, characterisation and biocompatibility of Fe-Pd for mechanical coupling to cells

Abstract: Yielding magnetically switchable strains of several percentage, ferromagnetic shape memory alloys (FSMAs) constitute a highly attractive materials class for engineering and biomedical applications. The Fe-Pd alloy is considered as a promising candidate to fulfil this goal, yet synthesis in the desirable martensite phase poses a major challenge, particularly when miniaturised as thin films. The present contribution reviews recent progress in synthesis via the splat-quenching and molecular beam epitaxy routes and reports on the physical picture that emerges from systematic characterisation. Moreover, since no cell damaging temperature variations are required to induce shape changes of FSMAs, Fe-Pd bears great potential for medical devices if good biocompatibility is ensured. Here we review studies on cellular response in contact with Fe-Pd, as well as morphological changes on roughness graded surfaces. We end with a summary how polymer coatings can enhance bioactive properties of Fe-Pd to promote cell adhesion and viability for future applications. DOI: 10.1179/1743284714Y.0000000592 Journal Homepage

Materials Science and Technology, Volume 30, Issue 13a, Pages 1579-1589 (2014)

 

Enrico Warmt, Tobias R. Kießling, Roland Stange, Anatol W. Fritsch, Mareike Zink, Josef A Käs:

Thermal instability of cell nuclei

Abstract: DNA is known to be a mechanically and thermally stable structure. In its double stranded form it is densely packed within the cell nucleus and is thermo-resistant up to 70 °C. In contrast, we found a sudden loss of cell nuclei integrity at relatively moderate temperatures ranging from 45 to 55 °C. In our study, suspended cells held in an optical double beam trap were heated under controlled conditions while monitoring the nuclear shape. At specific critical temperatures, an irreversible sudden shape transition of the nuclei was observed. These temperature induced transitions differ in abundance and intensity for various normal and cancerous epithelial breast cells, which clearly characterizes different cell types. Our results show that temperatures slightly higher than physiological conditions are able to induce instabilities of nuclear structures, eventually leading to cell death. This is a surprising finding since recent thermorheological cell studies have shown that cells have a lower viscosity and are thus more deformable upon temperature increase. Since the nucleus is tightly coupled to the outer cell shape via the cytoskeleton, the force propagation of nuclear reshaping to the cell membrane was investigated in combination with the application of cytoskeletal drugs. DOI: 10.1088/1367-2630/16/7/073009  Journal Homepage

New Journal of Physics, Volume 16, Issue 7, 073009 (2014)

 

J. Lippoldt, C. Händel, U. Dietrich, J. A. Käs:

Dynamic membrane structure induces temporal pattern formation

Abstract: The understanding of temporal pattern formation in biological systems is essential for insights into regulatory processes of cells. Concerning this problem, the present work introduces a model to explain the attachment/detachment cycle of MARCKS and PKC at the cell membrane, which is crucial for signal transduction processes. Our model is novel with regard to its driving mechanism: Structural changes within the membrane fuel an activator–inhibitor based global density oscillation of membrane related proteins. Based on simulated results of our model, phase diagrams were generated to illustrate the interplay of MARCKS and PKC. They predict the oscillatory behavior in the form of the number of peaks, the periodic time, and the damping constant depending on the amounts of MARCKS and PKC, respectively. The investigation of the phase space also revealed an unexpected intermediate state prior to the oscillations for high amounts of MARCKS in the system. The validation of the obtained results was carried out by stability analysis, which also accounts for further enhanced understanding of the studied system. It was shown, that the occurrence of the oscillating behavior is independent of the diffusion and the consumption of the reactants. The diffusion terms in the used reaction–diffusion equations only act as modulating terms and are not required for the oscillation. The hypothesis of our work suggests a new mechanism of temporal pattern formation in biological systems. This mechanism includes a classical activator–inhibitor system, but is based on the modifications of the membrane structure, rather than a reaction–diffusion system. DOI: 10.1016/j.bbamem.2014.05.018 Journal Homepage

Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1838, Issue 10, Pages 2380-2390 (2014)

 

Emilia I. Wisotzki, Marcel Hennes, Carsten Schuldt, Florian Engert, Wolfgang Knolle, Ulrich Decker, Josef A. Käs, Mareike Zink, Stefan G. Mayr:

Tailoring the material properties of gelatin hydrogels by high energy electron irradiation

Abstract: Natural hydrogels such as gelatin are highly desirable biomaterials for application in drug delivery, biosensors, bioactuators and extracellular matrix components due to strong biocompatibility and biodegradability. Typically, chemical crosslinkers are used to optimize material properties, often introducing toxic byproducts into the material. In this present work, electron irradiation is employed as a reagent-free crosslinking technique to precisely tailor the viscoelasticity, swelling behavior, thermal stability and structure of gelatin. With increasing electron dose, changes in swelling behavior and rheology indicate increasing amounts of random coils and dangling ends as opposed to helical content, a result confirmed through Fourier transform infrared spectroscopy. Gel fraction, rheology and swelling measurements at 37 °C were used to verify thermal stability in biological conditions. Scanning electron microscopy images of dried gelatin samples support these conclusions by revealing a loss of free volume and apparent order in the fracture patterns. The degree of crosslinking and mesh size is quantified by rubber elasticity theory and the Flory–Rehner equation. Overall, precise control of material properties is demonstrated through the interplay of concentration and irradiation dose, while providing an extensive parameter–property database suitable for optimized synthesis. DOI: 10.1039/c4tb00429a Journal Homepage

Journal of Materials Chemistry B, Volume 2, Issue 27, Pages 4297-4309 (2014)

 

Daniel E. Minner, Philipp Rauch, Josef Käs, Christoph A. Naumann:

Polymer-tethered lipid multi-bilayers: a biomembrane-mimicking cell substrate to probe cellular mechano-sensing

Abstract: Cells tiptoe through their environment forming highly localized and dynamic focal contacts. Experiments on polymeric gels of adjustable elasticity have shown that cells probe the viscoelasticity of their environment through an adaptive process of focal contact assembly/disassembly that critically affects cell adhesion, morphology, and motility. However, the specific mechanisms of this process have not yet been fully revealed. Here we report, for the first time, that fibroblast adhesion, morphology, and migration can also be controlled by altering the number of bilayers in a stack of multiple polymer-tethered lipid bilayers stabilized via maleimide–sulfhydral coupling chemistry. The observed changes in cell morphology, migration, and cytoskeletal organization in response to bilayer stacking correspond well with those previously observed on polymeric substrates of different polymer crosslinking density suggesting that variations in bilayer stacking are associated with changes in substrate viscoelasticity. This is in conceptual agreement with the existing knowledge about the structural, dynamic, and mechanical properties of polymer–lipid composite materials. Several distinct features, such as the lateral mobility of individual cell linkers and the immobilization of linker clusters, make the described substrates highly attractive tools for the study of dynamic, mechano-regulated cell linkages and cellular mechano-sensing. DOI: 10.1039/C3SM52298A Journal Homepage

Soft Matter, Volume 10, Issue 8, 1189-1198 (2014)

 

Markus Gyger, Roland Stange, Tobias Kießling, Anatol Fritsch, Katja B. Kostelnik, Annette G. Beck-Sickinger, Mareike Zink, Josef A. Käs:

Active contractions in single suspended epithelial cells

Abstract: Investigations of active contractions in tissue cells to date have been focused on cells that exert forces via adhesion sites to substrates or to other cells. In this study we show that also suspended epithelial cells exhibit contractility, revealing that contractions can occur independently of focal adhesions. We employ the Optical Stretcher to measure adhesion-independent mechanical properties of an epithelial cell line transfected with a heat-sensitive cation channel. During stretching the heat transferred to the ion channel causes a pronounced Ca2+ influx through the plasma membrane that can be blocked by adequate drugs. This way the contractile forces in suspended cells are shown to be partially triggered by Ca2+ signaling. A phenomenological mathematical model is presented, incorporating a term accounting for the active stress exerted by the cell, which is both necessary and sufficient to describe the observed increase in strain when the Ca2+ influx is blocked. The median and the shape of the strain distributions depend on the activity of the cells. Hence, it is unlikely that they can be described by a simple Gaussian or log normal distribution, but depend on specific cellular properties such as active contractions. Our results underline the importance of considering activity when measuring cellular mechanical properties even in the absence of measurable contractions. Thus, the presented method to quantify active contractions of suspended cells offers new perspectives for a better understanding of cellular force generation with possible implications for medical diagnosis and therapy. DOI: 10.1007/s00249-013-0935-8 Journal Homepage

European Biophysics Journal, Volume 43, Issue 1, Pages 11-23 (2014)

 

Thomas Fuhs, Michael Gögler, Claudia A. Brunner, Charles W. Wolgemuth, Josef A. Käs:

Causes of retrograde flow in fish keratocytes

Abstract: Confronting motile cells with obstacles doubling as force sensors we tested the limits of the driving actin-and-myosin-machinery. We could directly measure the force necessary to stop actin polymerization as well as the force present in the retrograde actin flow. Combined with detailed measurements of the retrograde flow velocity and specific manipulation of actin and myosin we found that actin polymerization and myosin contractility are not enough to explain the cells behavior. We show that ever-present depolymerization forces, a direct entropic consequence of actin filament recycling, are sufficient to fill this gap, even under heavy loads. DOI: 10.1002/cm.21151 Journal Homepage

Cytoskeleton, Volume 71, Issue 1, Pages 24-35 (2014)

 

Janine Runge, Torsten E. Reichert, Anatol Fritsch, Josef Käs, Julia Bertolini, Torsten W. Remmerbach:

Evaluation of single cell biomechanics as potential marker for oral squamous cell carcinomas: a pilot study

Abstract: Objectives: Early detection of oral cancer is a major health issue. The objective of this pilot study was to analyze the deformability of healthy and cancer cells using a microfluidic optical stretcher (OS). Material and Methods: Different cancer cell lines, primary oral cancer cells and their healthy counterparts were cultivated and characterized respectively. A measurable deformation of the cells along the optical axis was detected, caused by surface stress, which is optically induced by the laser power. Results: All cells revealed a viscoelastic extension behavior and showed a characteristic deformation response under laser light exposure. The CAL-27/-33 cells exhibited the highest relative deformation. All other cells achieved similar values, but on a lower level. The cytoskeleton reacts sensitively of changing environmental conditions, which may be influenced by growth behavior of the cancer specimens. Nevertheless, the statistical analysis showed significant differences between healthy and cancer cells. Conclusion: Generally malignant and benign cells showed significantly different mechanical behavior. Cancer related changes influence the composition of the cytoskeleton and thus affect the deformability, but this effect may be superimposed by cell cultivation conditions, or cell doubling time. These influences had to be substituted by brush biopsies to minimize confounders in pursuing investigations. DOI: 10.1111/odi.12171 Journal Homepage

Oral Diseases, Volume 20, Issue 3, e120-e127 (2014)

2013
 

Tom Golde, Carsten Schuldt, Jörg Schnauß, Dan Strehle, Martin Glaser, Josef A. Käs:

Fluorescent Beads Disintegrate Actin Networks

Abstract: We studied the influence of fluorescent polystyrene beads on both entangled and cross-linked actin networks. Thermal bead fluctuations were observed via video particle tracking and analyzed with one-point microrheology. Illumination of fluorescent beads with their appropriate excitation wavelength leads to a drastic softening of actin gels. Other wavelengths and bright field microscopy do not increase thermal bead fluctuations. This effect cannot be significantly reduced by adding common oxygen scavengers. We conclude that the usage of fluorescent beads impairs results when studying the microrheology of actin networks. DOI: 10.1103/PhysRevE.88.044601 Journal Homepage

Physical Review E, Volume 88, Issue 4, 044601 (2013)

 

Kristin Seltmann, Anatol W. Fritsch, Josef A. Käs, Thomas M. Magin:

Keratins significantly contribute to cell stiffness and impact invasive behavior

Abstract: Cell motility and cell shape adaptations are crucial during wound healing, inflammation and malignant progression. These processes require the remodeling of the keratin cytoskeleton, to facilitate cell-cell and matrix adhesion. However, the role of keratins for biomechanical properties and invasion of epithelial cells are only partially understood. Here, we address this issue in murine keratinocytes lacking all keratins upon genome engineering. In contrast to prediction, keratin-free cells show about 60% higher cell deformability even for small deformations. This is compared to less pronounced softening effects for actin depolymerization induced via latrunculin A. To relate these findings with functional consequences, we use invasion and three-dimensional growth assays. These reveal higher invasiveness of keratin-free cells. Re-expression of a small amount of the keratin pair K5/K14 in keratin-free cells reverses the above phenotype for the invasion but does not with respect to cell deformability. Our data shows a novel role of keratins as major player of cell stiffness influencing invasion with implications for epidermal homeostasis and pathogenesis. This study supports the view that downregulation of keratins observed during epithelial-mesenchymal transition directly contributes to the migratory and invasive behavior of tumor cells. DOI: 10.1073/pnas.1310493110  Journal Homepage

PNAS, Volume 110, No. 46, 18507-18512 (2013)

 

Kenechukwu David Nnetu, Melanie Knorr, Steve Pawlizak, Thomas Fuhs, Josef A. Käs:

Slow and anomalous dynamics of an MCF-10A epithelial cell monolayer

Abstract: Understanding the mechanics and dynamics of active matter at high density is indispensable to a range of physical and biological processes such as swarm dynamics, tissue formation and cancer metastasis. Here, we study the dynamics and mechanics of an MCF-10A epithelial cell monolayer on the multi-cellular and single-cell scales and over a wide density range. We show that the dynamics and Young’s modulus of the monolayer are spatially heterogeneous on the multi-cellular scale. With increasing cell density, the monolayer approached kinetic arrest and the Young’s modulus scaled critically. On the single-cell scale, as the cell density increased, cells were intermittently trapped in cages formed by their neighbors and their motion evolved from a ballistic motion to a sub-diffusive motion. Furthermore, the relaxation time and inverse self-diffusivity increased exponentially with the cell density. These findings provide a mechanism for long-ranged mechanical stress propagation, tissue remodeling and patterning at very high cell densities. DOI: 10.1039/C3SM50806D  Journal Homepage

Soft Matter, Volume 9, 9335-9341 (2013)

 

Tobias R. Kießling, Roland Stange, Josef A. Käs, Anatol W. Fritsch:

Thermorheology of living cells - impact of temperature variations on cell mechanics

Abstract: Upon temperature changes, we observe a systematic shift of creep compliance curves J(t) for single living breast epithelial cells. We use a dual-beam laser trap (optical stretcher) to induce temperature jumps within milliseconds, while simultaneously measuring the mechanical response of whole cells to optical force. The cellular mechanical response was found to differ between sudden temperature changes compared to slow, long-term changes implying adaptation of cytoskeletal structure. Interpreting optically induced cell deformation as a thermorheological experiment allows us to consistently explain data on the basis of time-temperature superposition, well known from classical polymer physics. Measured time shift factors give access to the activation energy of the viscous flow of MCF-10A breast cells, which was determined to be ~80 kJ/mol. The presented measurements highlight the fundamental role that temperature plays for the deformability of cellular matter. We propose thermorheology as a powerful concept to assess the inherent material properties of living cells and to investigate cell regulatory responses upon environmental changes. DOI: 10.1088/1367-2630/15/4/045026 Journal Homepage

New Journal of Physics, Volume 15, Issue 4 (2013)

 

T. R. Kießling, M. Herrera, K. D. Nnetu, E. M. Balzer, M. Girvan, A. W. Fritsch, S. S. Martin, J. A. Käs, W. Losert:

Analysis of multiple physical parameters for mechanical phenotyping of living cells

Abstract: Since the cytoskeleton is known to regulate many cell functions, an increasing amount of effort to characterize cells by their mechanical properties has occured. Despite the structural complexity and dynamics of the multicomponent cytoskeleton, mechanical measurements on single cells are often fit to simple models with two to three parameters, and those parameters are recorded and reported. However, different simple models are likely needed to capture the distinct mechanical cell states, and additional parameters may be needed to capture the ability of cells to actively deform. Our new approach is to capture a much larger set of possibly redundant parameters from cells’ mechanical measurement using multiple rheological models as well as dynamic deformation and image data. Principal component analysis and network-based approaches are used to group parameters to reduce redundancies and develop robust biomechanical phenotyping. Network representation of parameters allows for visual exploration of cells’ complex mechanical system, and highlights unexpected connections between parameters. To demonstrate that our biomechanical phenotyping approach can detect subtle mechanical differences, we used a Microfluidic Optical Cell Stretcher to mechanically stretch circulating human breast tumor cells bearing genetically-engineered alterations in c-src tyrosine kinase activation, which is known to influence reattachment and invasion during metastasis. DOI: 10.1007/s00249-013-0888-y Journal Homepage

European Biophysics Journal, Volume 42, Issue 5, 383-394 (2013)

 

Florian Huber, Jörg Schnauß, Susanne Rönicke, Philipp Rauch, Karla Müller, Claus Fütterer, Josef Käs:

Emergent complexity of the cytoskeleton: from single filaments to tissue

Abstract: Despite their overwhelming complexity, living cells display a high degree of internal mechanical and functional organization which can largely be attributed to the intracellular biopolymer scaffold, the cytoskeleton. Being a very complex system far from thermodynamic equilibrium, the cytoskeleton’s ability to organize is at the same time challenging and fascinating. The extensive amounts of frequently interacting cellular building blocks and their inherent multifunctionality permits highly adaptive behavior and obstructs a purely reductionist approach. Nevertheless (and despite the field’s relative novelty), the physics approach has already proved to be extremely successful in revealing very fundamental concepts of cytoskeleton organization and behavior. This review aims at introducing the physics of the cytoskeleton ranging from single biopolymer filaments to multicellular organisms. Throughout this wide range of phenomena the focus is set on the intertwined nature of the different physical scales (levels of complexity) that give rise to numerous emergent properties by means of selforganization or self-assembly. DOI: 10.1080/00018732.2013.771509  Journal Homepage

Advances in Physics, Volume 62, Issue 1 (2013)

 

Lydia Woiterski, Thomas Claudepierre, Robert Luxenhofer, Rainer Jordan, Josef A. Käs:

Stages of neuronal network formation

Abstract: Graph theoretical approaches have become a powerful tool to investigate the architecture and dynamics of complex networks. The topology of network graphs revealed small-world properties for very different real systems among these neuronal networks. In this study we observed the early development of mouse retinal ganglion cell (RGC) networks in vitro using time-lapse video microscopy. By means of a time-resolved graph theoretical analysis of the connectivity, shortest path length and edge length, we were able to discover different stages during the network formation. Starting from single cells, at the first stage neurons connected to each other ending up in a network with maximum complexity. In the further course, we observed a simplification of the network which manifested in a change of relevant networks parameters such as the minimization of the path length. Moreover, we found that RGC networks self-organized as small-world networks at both stages, however, the optimization occurred only in the second stage.  DOI: 10.1088/1367-2630/15/2/025029  Journal Homepage

New Journal of Physics, Volume 15, 025029 (2013)

 

Uta Allenstein, Yanhong Ma, Ariyan Arabi-Hashemi, Mareike Zink, Stefan G. Mayr:

Fe-Pd based ferromagnetic shape memory actuators for medical applications: Biocompatibility, effect of surface roughness and protein coatings

Abstract: Ferromagnetic shape memory (FMSM) alloys constitute an exciting new class of smart materials that can yield magnetically switchable strains of several per cent at constant temperatures and frequencies from quasi-static up to some kilohertz. In addition to their FMSM properties, these alloys can still be operated as conventional shape memory materials and also exhibit related superelasticity, important features for medical devices. In this study, extensive in vitro assessments demonstrate for the first time that vapor-deposited single crystalline Fe70Pd30 thin films and roughness graded polycrystalline splats of the same stoichiometry exhibit excellent biocompatibility and even bioactivity features in contact with different cell types—a prerequisite for medical applications. The study shows that fibroblast and epithelial cell lines, as well as primary osteoblast cells, proliferate well on Fe–Pd. The number of focal contacts important for strong tissue bonding can be improved with different binding agents from the extracellular matrix. However, even without coating, there is clear evidence that cells on Fe–Pd substrates behave similarly to control experiments. Additionally, cytotoxic effects of polycrystalline surfaces with various roughness profiles can be excluded, giving another tunable parameter for applying Fe–Pd in magnetically switchable membranes, e.g., stents and valves. DOI: 10.1016/j.actbio.2012.10.040 Journal Homepage

Acta Biomaterialia, Volume 9, Issue 3, 5845-5853 (2013)

 

Michael Krahe, Iris Wenzel, Kao-Nung Lin, Julia Fischer, Joseph Goldmann, Markus Kästner, Claus Fütterer:

Fluctuations and differential contraction during regeneration of Hydra vulgaris tissue toroids

Abstract: We studied regenerating bilayered tissue toroids dissected from Hydra vulgaris polyps and relate our macroscopic observations to the dynamics of force-generating mesoscopic cytoskeletal structures. Tissue fragments undergo a specific toroid–spheroid folding process leading to complete regeneration towards a new organism. The time scale of folding is too fast for biochemical signalling or morphogenetic gradients, which forced us to assume purely mechanical self-organization. The initial pattern selection dynamics was studied by embedding toroids into hydro-gels, allowing us to observe the deformation modes over longer periods of time. We found increasing mechanical fluctuations which break the toroidal symmetry, and discuss the evolution of their power spectra for various gel stiffnesses. Our observations are related to single-cell studies which explain the mechanical feasibility of the folding process. In addition, we observed switching of cells from a tissue bound to a migrating state after folding failure as well as in tissue injury. We found a supra-cellular actin ring assembled along the toroid's inner edge. Its contraction can lead to the observed folding dynamics as we could confirm by finite element simulations. This actin ring in the inner cell layer is assembled by myosin-driven length fluctuations of supra-cellular F-actin bundles (myonemes) in the outer cell layer. DOI: 10.1088/1367-2630/15/3/035004 Journal Homepage

New Journal of Physics, Volume 15, 035004 (2013)

 

Philipp Rauch, Paul Heine, Barbara Goettgens, Josef A Käs:

Forces from the rear: deformed microtubules in neuronal growth cones influence retrograde flow and advancement

Abstract: The directed motility of growth cones at the tip of neuronal processes is a key function in neuronal path-finding and relies on a complex system of interacting cytoskeletal components. Despite intensive research in this field, many aspects of the mechanical roles of actin structures and, in particular, of microtubules throughout this process remain unclear. Mostly, force generation is ascribed to actin–myosin-based structures such as filopodia bundles and the dynamic polymer gel within the lamellipodium. Our analysis of microtubule buckling and deformation in motile growth cones reveals that extending microtubule filaments significantly contributes to the overall protrusion force. In this study, we establish a relationship of the local variations in stored bending energy and deformation characteristics to growth cone morphology and retrograde actin flow. This implies the relevance of microtubule pushing and deformation for general neurite advancement as well as steering processes. DOI: 10.1088/1367-2630/15/1/015007 Journal Homepage

New Journal of Physics, Volume 15, 015007 (2013)

 

Mareike Zink, Florian Szillat, Uta Allenstein, Stefan G. Mayr:

Interaction of Ferromagnetic Shape Memory Alloys and RGD Peptides for Mechanical Coupling to Cells: from Ab Initio Calculations to Cell Studies

Abstract: Due to their magneto-mechanical coupling and biocompatibility, Fe-Pd based ferromagnetic shape memory alloys are a highly promising materials class for application as contact-less magneto-mechanical transducers in biomedical environments. For use in cell and tissue actuators or strain sensors, suffi cient adhesion to mediate strains clearly constitutes a prerequisite. As the RGD sequence is the most important binding motif for mammalian cells, which they express to facilitate adhesion, the potential of RGD coatings to achieve this goal is explored. Employing large-scale density functional theory calculations the physics of bonding between RGD and Fe-Pd surfaces, which is characterized by coordinate bonds of O and N atoms to Fe, accompanied by electrostatic contributions, is clarifi ed. Theoretical predictions on adhesion, that are confi rmed experimentally, suggest RGD as suitable strain mediator to Fe-Pd surfaces. On the cell side, favorable adhesion properties of RGDcoated Fe-Pd are manifested in cell morphology and spreading behavior. Demonstrating that the adhesion forces between RGD and Fe-Pd exceed those exerted by cells to the RGD coating, as well as traction forces acting onto integrin bonds, the fi ndings pave the way for novel type of applications as cell and tissue actuator or sensor within the areas of tissue engineering and regenerative medicine. DOI: 10.1002/adfm.201201789 Journal Homepage

Advanced Functional Materials, Volume 23, Issue 11, 1383-1391 (2013)

2012
 

Karim El-Laithy, Melanie Knorr, Josef Käs, Martin Bogdan:

Digital detection and analysis of branching and cell contacts in neural cell cultures

Abstract: Changes in human/animal behaviour and the involved neural functions are characterized by structural alterations in the brain circuitry. These changes comprise the formation of new synapses and the elimination of existing synapses aside from the modulation of connecting properties within other ones. The mechanisms of neuronal branching and cell contacting regulate and prepare for the processes of synaptic formation. In this study, we present a set of methods to detect, describe and analyse the dynamics attributed to the process of cell contacting in cell cultures in vitro. This involves the dynamics of branching and seeking for synaptic partners. The proposed technique formally distinguishes between the actual formed synapses and the potential synaptic sites, i.e. where cell contacts are likely. The study investigates the dynamic behaviour of these potential synaptic sites within the process of seeking for contacts. The introduced tools use morphological image processing algorithms to automatically detect the sites of interest. Results indicate that the introduced tools can reliably describe experimentally observed branching and seeking for contacts dynamics. Being straightforward in terms of implementation and analysis, our framework represents a solid method for studying the neural preparation phases of synaptic formation via cell contacting in random networks using standard phase contrast microscopy. DOI: 10.1016/j.jneumeth.2012.07.007 Journal Homepage

Journal Neuroscience Methods, Volume 210, Issue 2, 206-219 (2012)

 

Christian Kappel, Nicole Dölker, Rajendra Kumar, Mareike Zink, Ulrich Zachariae, Helmut Grubmüller:

Universal Relaxation Governs the Nonequilibrium Elasticity of Biomolecules

Abstract: Experimental and computational dynamic force spectroscopy is widely used to determine the mechanical properties of single biomolecules. Whereas so far the focus has mainly been on rupture or unfolding forces, recent force-probe molecular dynamics simulations have revealed a strong loading rate dependence of biomolecular elasticities, which cannot be explained by the established one-dimensional transition-state treatments. We show that this nonequilibrium behavior can be explained by a theory that includes relaxation effects. For three structurally and mechanically quite diverse systems, a single relaxation mode suffices to quantitatively describe their loading-rate-dependent elastic behavior. Atomistic simulations of these systems revealed the microscopic nature of the respective relaxation modes. This result suggests a new type of "elasticity spectroscopy" experiment, which should render nonequilibrium properties of structured macromolecules accessible to single-molecule force spectroscopy. DOI: 10.1103/PhysRevLett.109.118304 Journal Homepage

Physical Review Letters, Volume 109, 118304 (2012)

 

Thomas Fuhs, Lydia Reuter, Iris Vonderhaid, Thomas Claudepierre, Josef A. Käs:

Inherently slow and weak forward forces of neuronal growth cones measured by a drift-stabilized atomic force microscope

Abstract: Previous results have shown that glial cells provide a soft environment for the neurons of the mammalian central nervous system . This raises the question whether neurons are confined to the CNS and cannot wander off into more rigid tissues, such as brain capillary walls. We investigated the mechanical properties and force generation of extending mouse retinal ganglion cells and NG108-15 growth cones using different AFM based methods. For the first time, to our knowledge, we were able to measure the forward pushing forces at the leading edge of outgrowing neuronal growth cones with our driftstabilized AFM. Our results demonstrate that these growth cones have neither the required stability nor the ability to produce forces necessary to penetrate tissues that are at least an order of magnitude stiffer. DOI: 10.1002/cm.21080 Journal Homepage

Cytoskeleton, Volume 70, Issue 1, 44-53 (2012)

 

Kenechukwu David Nnetu, Melanie Knorr, Josef A. Käs, Mareike Zink:

The impact of jamming on boundaries of collectively moving weak-interacting cells

Abstract: Collective cell migration is an important feature of wound healing, embryonic and tumor development. The origin of collective cell migration is mainly intercellular interaction through effects such as a line tension preventing cells from detaching from the boundary. In contrast, in this study, we show for the first time that the formation of a constant cell front of a monolayer can also be maintained by the dynamics of the underlying migrating cells. Ballistic motion enables the maintenance of the integrity of the sheet, while a slowed down dynamics and glass-like behavior cause jamming of cells at the front when two monolayers - even of the same cell type - meet. By employing a velocity autocorrelation function to investigate the cell dynamics in detail, we found a compressed exponential decay as described by the Kohlrausch-William-Watts function of the form C(dx)_t ~ exp(-(x/x0(t))^beta(t)), with 1.5 <= beta(t) <= 1.8. This clearly shows that although migrating cells are an active, non-equilibrium system, the cell monolayer behaves glass-like which requires jamming as a part of intercellular interactions. Since it is the dynamics which determine the integrity of the cell sheet and its front for weakly interacting cells, it becomes evident why changes of the migratory behavior during epithelial to mesenchymal transition can result in the escape of single cells and metastasis. DOI: 10.1088/1367-2630/14/11/115012 Journal Homepage

New Journal of Physics, Volume 14, 115012 (2012)

 

Kenechukwu David Nnetu, Melanie Knorr, Dan Strehle, Mareike Zink, Josef A. Käs:

Directed persistent motions maintain sheet integrity during multi-cellular spreading and migration

Abstract: Multi-cellular migration plays an important role in physiological processes such as embryogenesis, cancer metastasis and tissue repair. Collective cell migration involves specific single cell motility behaviour that maintains the integrity of the monolayer and the fluid-like behavior of the sheet on long time scales. By studying the dynamics of MCF-10A, MDA-MB-231 epithelial cell monolayers and that of a NIH-3t3 fibroblast monolayer, we show that for the MCF-10A cells, in the case where cell-cell interactions are so weak that single cells can detach from the monolayer, a collective cell front can be maintained by the interplay between the directed persistent motion of the monolayer and the random motion of escaping single cells. The dynamics of the MCF-10A monolayer is contrasted with that of the MDA-MB-231 monolayer where single cells did not detach, non-interacting NIH-3t3 fibroblast cells which are always random walkers and that of a MCF-10A monolayer treated with a calcium chalating agent which reduces intercellular interactions. DOI: 10.1039/C2SM07208D Journal Homepage

Soft Matter, Soft Matter, Volume 8, Issue 26, 2913-2921 (2012)

 

Mireille Martin, Karla Müller, Cristina Cadenas, Matthias Hermes, Mareike Zink, Jan G. Hengstler, Josef A. Käs:

ERBB2 overexpression triggers transient high mechanoactivity of breast tumor cells

Abstract: Biomechanical properties of tumor cells play an important role for the metastatic capacity of cancer. Cellular changes of viscoelastic features are prerequisite for cancer progression since they are essential for proliferation and metastasis. However, only little is known about the way how expression of oncogenes influences these biomechanical properties. To address this aspect we used a breast cancer cell line with inducible expression of an oncogenic version of ERBB2. ERBB2 is known to be correlated with bad prognosis in breast cancer. Cell elasticity was determined by the Optical Stretcher, where suspended cells are deformed by two slightly divergent laser beams. We found that induction of ERBB2 caused remarkable biomechanical alterations of the MCF-7 cells after 24 h: the cells actively contracted in response to mechanical stimuli, a phenomenon known as mechanoactivation. After this period, as the cells became senescent, the mechanoactivity returned to control levels. Time-resolved gene array analysis revealed that mechanoactivation was accompanied by temporal upregulation of 46 cytoskeletal genes. A possible role of these genes in tumor progression was investigated by expression analyses of 766 breast cancer patients. This showed an association of 12 out of these 46 genes with increased risk of metastasis. Our results demonstrate that overexpression of ERBB2 causes mechanoactivation of tumor cells, which may enhance tumor cell motility fostering distant metastasis. DOI: 10.1002/cm.21023 Journal Homepage

Cytoskeleton, Volume 69, Issue 5, 267-277 (2012)

 

Valentina Dallacasagrande, Mareike Zink, Steven Huth, Alexander Jakob, Marcus Müller, Andreas Reichenbach, Josef A. Käs, S. G. Mayr:

Tailoring Substrates for Long-Term Organotypic Culture of Adult Neuronal Tissue

Abstract: Organotypic tissue cultures are highly promising for performing in vivo type studies in vitro. Currently, however, very limited survival times of only a few days for adult tissue often severely limit their application. Here, superhydrophilic nanostructured substrates with ideal material properties ensure tissue adhesion, essential for organotypic culture, while migration of single cells out of the tissue is hampered. Tuning substrate properties, for the first time, adult neuronal tissue could be cultured for 14 days with no indications of degeneration. DOI: 10.1002/adma.201200816 Journal Homepage

Advanced Materials, Volume 24, Issue 18, 2399-2403 (2012)

 

Hans Kubitschke, Claus Fütterer:

Dynamics of pore synthesis and degradation in protocells

Abstract: Liposomes have found countless applications as microreactors or for studying the evolution of protocells. However, to keep reactions ongoing, exchange with the environment is required. Based on experiments with nanopores expressed by an enclosed gene expression system, we developed a model describing the observed growth dynamics quantitatively. The model depends on one parameter only and allowed estimations of hitherto unknown parameters: the diffusion coefficient of amino acids through a single pore and the initial amino acid concentration. The long-term consequences of different degradation mechanisms are also discussed: we found a surprisingly sharp threshold deciding on the question of survival of the protocell. DOI: 10.1088/1367-2630/14/10/103008 Journal Homepage

New Journal of Physics, Volume 14, 103008 (2012)

 

Lydia Woiterski, David W. Britt, Josef A. Käs, Carsten Selle:

Oriented Confined Water Induced by Cationic Lipids

Abstract: We report on attenuated total reflection Fourier-transform infrared (ATR FTIR) spectroscopic measurements on oriented lipid multilayers of N,N-dimethyl-N,N-dioctadecyl-ammonium halides (DODAX, X = F, Cl, Br, I). The main goal of this study is the investigation of the structure and spectroscopic properties of water absorbed to these model membranes. Intensities of the water stretch absorptions were used to determine the amount of bound water. At high water activity, DODAF membranes bind ~11 water molecules/lipid while DODAC and DODAB adsorb 1-2 water/lipid and DODAI was hydrophobic. By adjustment of DODAF hydration to ~2 water molecules, stretching absorptions from water of the first hydration shell were accessible for the fluoride, chloride, and bromide analogs. The polarized measurements demonstrate highly confined and oriented water with infrared (IR) order parameters ranging from 0.2 to -0.4. Resolved IR water band components are attributed to different hydrogen-bonded populations. Complementary molecular dynamics simulations of DODAB strongly support the existence of differently hydrogen-bonded and oriented water within DODAB multilayers. A combination of both techniques was used for an assignment of water stretch band components to structures. The described cationic lipid systems are a prototype for a bottom-up approach to understand the IR spectroscopy of structured water at biological interfaces since they permit a defined increase of hydrophilic water-anionic interactions leading to extended water networks at membranes. DOI: 10.1021/la205043x Journal Homepage

Langmuir, Volume 28, Issue 10, 4712-4722 (2012)

 

Aldo Leal-Egaña, Anatol Fritsch, Felicia Heidebrecht, Aránzazu Díaz-Cuenca, Marcin Nowicki, Augustinus Bader, Josef Käs:

Tuning liver stiffness against tumours: An in vitro study using entrapped cells in tumour-like microcapsules

Abstract: Liver fibrosis is a reversible pathology characterized by the up-regulated secretion and deposition of ECM proteins and inhibitors of metalloproteinases, which increase the stiffness and viscosity of this organ. Due to recent studies have shown that fibrosis preceded the apparition of hepatocellular carcinomas, we hypothesize that liver fibrosis could play a role as a mechanism for restricting uncontrolled cell proliferation, inducing the mortality of cancer cells and subsequent development of primary tumours. DOI: 10.1016/j.jmbbm.2012.01.013 Journal Homepage

Journal of the Mechanical Behavior of Biomedical Materials, Volume 9, 113-121 (2012)

 

Juliane Zimmermann, Claudia Brunner, Mihaela Enculescu, Michael Goegler, Allen Ehrlicher, Josef Käs, Martin Falcke:

Actin Filament Elasticity and Retrograde Flow Shape the Force-Velocity Relation of Motile Cells

Abstract: Cells migrate through a crowded environment during processes such as metastasis or wound healing, and must generate and withstand substantial forces. The cellular motility responses to environmental forces are represented by their force-velocity relation, which has been measured for fish keratocytes but remains unexplained. Even pN opposing forces slow down lamellipodium motion by three orders of magnitude. At larger opposing forces, the retrograde flow of the actin network accelerates until it compensates for polymerization, and cell motion stalls. Subsequently, the lamellipodium adapts to the stalled state. We present a mechanism quantitatively explaining the cell's force-velocity relation and its changes upon application of drugs that hinder actin polymerization or actomyosin-based contractility. Elastic properties of filaments, close to the lamellipodium leading edge, and retrograde flow shape the force-velocity relation. To our knowledge, our results shed new light on how these migratory responses are regulated, and on the mechanics and structure of the lamellipodium. DOI: 10.1016/j.bpj.2011.12.023  Journal Homepage

Biophysical Journal, Volume 102, Issue 2, 287-295 (2012)

 

Florian Huber, Dan Strehle, Josef Käs:

Counterion-induced formation of regular actin bundle networks

Abstract: Dominating the cytoskeletal contribution to cell mechanics and migration, actin and actin-crosslinker systems have attained much attention with regard to rheology and network architecture. In contrast to protein crosslinkers or molecular motors -which self-assemble and self-organize actin into networks, bundles or asters- simple multivalent ions rely on the polyelectrolyte nature of actin and should not imply any particular binding geometry. Using controlled counterion condensation as a model linker at comparably high F-actin densities we report an entirely novel state displaying regularly spaced networks of actin bundles connected by aster-like clusters. Liquid crystalline effects at high densities of long filaments were found to substantially alter the bundle network structures directly reflecting the pre-existing order of the actin filaments. DOI: 10.1039/C1SM06019H  Journal Homepage

Soft Matter, Volume 8, Issue 4, 931-936 (2012)

2011
 

Silke Agte, Stephan Junek, Sabrina Matthias, Elke Ulbricht, Ines Erdmann, Antje Wurm, Detlev Schild, Josef A. Käs, Andreas Reichenbach:

Müller glial cell-provided cellular light guidance through the vital guinea-pig retina

Abstract: In vertebrate eyes, images are projected onto an inverted retina where light passes all retinal layers on its way to the photoreceptor cells. Light scattering within this tissue should impair vision. We show that radial glial (Müller) cells in the living retina minimize intraretinal light scatter and conserve the diameter of a beam that hits a single Müller cell endfoot. Thus, light arrives at individual photoreceptors with high intensity. This leads to an optimized signal-to-noise ratio, which increases visual sensitivity and contrast. Moreover, we show that the ratio between Müller cells and cones - responsible for acute vision - is roughly one. This suggests that high spatiotemporal resolution may be achieved by each cone receiving its part of the image via its 'individual' Müller cell-light guide. DOI:10.1016/j.bpj.2011.09.062 Journal Homepage

Biophysical Journal, Volume 101, Issue 11, 2611-2619 (2011)

 

Oliver Jonas, Claudia Mierke, Josef Käs:

Invasive cancer cell lines exhibit biomechanical properties that are distinct from their noninvasive counterparts

Abstract: Identifying the properties that distinguish invasive cells from noninvasive cells is of prime interest in understanding metastasis formation during cancer. We have taken pairs of cells from five organ types, each consisting of a highly invasive cell line and its noninvasive counterpart, and characterized their active and passive biomechanical properties in assays that mimic the mechanical perturbations likely experienced by in-vivo metastasizing tumor cells. Our setup applies compressive pressure to the entire cell via a flat SFM cantilever while directly measuring the cell’s deformation during loading, as well as its relaxation and plasticity during unloading. Simultaneously, the basal membrane is imaged via TIRF, yielding insights into the mechanism of force transduction during loading and force generation during relaxation. Four of the five noninvasive cell lines tested appear stiffer under whole cell compression than the corresponding invasive cell line. After force is lifted and cells are allowed to relax, all invasive cell lines exhibit greater recovery behavior as evidenced by higher retraction and lower plasticity. This retraction is observed to be an active process that is mostly dependent on the acto-myosin contractile apparatus and on actin remodeling. Combined with observed consistent distinctions in force transduction of invasive cells, the results hint at a direct connection between invasiveness and active and passive biomechanical properties of cancer cells, and as such may be relevant for understanding how some tumor cells adapt to the mechanical requirements of metastasis formation. DOI:10.1039/C1SM05532A  Journal Homepage

Soft Matter, Volume 7, Issue 24, 11488-11495 (2011)

 

Timo Betz, Daniel Koch, Yun-Bi Lu, Kristian Franze, Josef A. Käs

Growth cones as soft and weak force generators

Abstract: Many biochemical processes in the growth cone finally target its biomechanical properties, such as stiffness and force generation, and thus permit and control growth cone movement. Despite the immense progress in our understanding of biochemical processes regulating neuronal growth, growth cone biomechanics remains poorly understood. Here, we combine different experimental approaches to measure the structural and mechanical properties of a growth cone and to simultaneously determine its actin dynamics and traction force generation. Using fundamental physical relations, we exploited these measurements to determine the internal forces generated by the actin cytoskeleton in the lamellipodium. We found that, at timescales longer than the viscoelastic relaxation time of t = 8.5 ± 0.5 sec, growth cones show liquid-like characteristics, whereas at shorter time scales they behaved elastically with a surprisingly low elastic modulus of E = 106 ± 21 Pa. Considering the growth cone’s mechanical properties and retrograde actin flow, we determined the internal stress to be on the order of 30 pN per µm². Traction force measurements confirmed these values. Hence, our results indicate that growth cones are particularly soft and weak structures that may be very sensitive to the mechanical properties of their environment.  DOI:10.1073/pnas.1106145108  Journal Homepage

PNAS, Volume 108, Issue 33, 13420-13425 (2011)

 

Sebastian Koth, Michael Krahe, Claus Fütterer:

Fluctuations and symmetries in biology and physics

Abstract: Fluctuations can be found in physical as well as biological systems. Are they related? They also appear to be important for symmetry breaking. We compare common aspects of structural transitions in biological and physical systems: Hydra vulgaris tissue fragments always reconstitute a symmetric spherical shape prior to symmetry-breaking regeneration. Breaking this spherical symmetry is accompanied by strong specific fluctuations. Cells also show distinct lamellipod fluctuations. Structural change in such different phenomena as magnetism, liquid freezing and evaporation, cytoskeletal rearrangement and morphogenesis of tissues shows symmetry breaking scenarios accompanied by fluctuations. We are questioning their common purpose and consequences. Townes & Holtfreter wrote in 1955: “One of the most striking features of early vertebrate development is the transformation of a spherical egg into a body of about equal size in which groups of cells have shifted into specific arrangements.” Journal Homepage

DGZ Cell News, Volume 4/2011, 40-45 (2011)

 

Markus Gyger, Daniel Rose, Roland Stange, Tobias Kießling, Mareike Zink, Ben Fabry, Josef A. Käs:

Calcium imaging in the optical stretcher

Abstract: The Microfluidic Optical Stretcher has previously been shown to be a versatile tool to measure mechanical properties of single suspended cells. In this study we combine optical stretching and fluorescent calcium imaging. A cell line transfected with a heat sensitive cation channel was used as a model system to show the versatility of the setup. The cells were loaded with the Ca2+ dye Fluo-4 and imaged with confocal laser scanning microscopy while being stretched. During optical stretching heat is transferred to the cell causing a pronounced Ca2+ influx through the cation channel. The technique opens new perspectives for investigating the role of Ca2+ in regulating cell mechanical behavior. DOI:10.1364/OE.19.019212 Journal Homepage

Optics Express, Volume 19, Issue 20, 19212-19222 (2011)

 

Undine Dietrich, Peter Krüger, Josef A. Käs:

Structural investigation on the adsorption of the MARCKS peptide on anionic lipid monolayers - effects beyond electrostatic

Abstract: The presence of charged lipids in the cell membrane constitutes the background for the interaction with numerous membrane proteins. As a result, the valence of the lipids plays an important role concerning their lateral organization in the membrane and therefore the very manner of this interaction. This present study examines this aspect, particularly regarding to the interaction of the anionic lipid DPPS with the highly basic charged effector domain of the MARCKS protein, examined in monolayer model systems. Film balance, fluorescence microscopy and X-ray reflection/diffraction measurements were used to studythe behavior of DPPS in a mixture with DPPC for its dependance on the presence of MARCKS (151–175). In the mixed monolayer, both lipids are completely miscible therefore DPPS is incorporated in the ordered crystalline DPPC domains as well. The interaction of MARCKS peptide with the mixed monolayer leads to the formation of lipid/peptide clusters causing an elongation of the serine group of the DPPS up to 7Å in direction to surface normal into the subphase. The large cationic charge of the peptide pulls out the serine group of the interface which simultaneously causes an elongation of the phosphodiester group of the lipid fraction too. The obtained results were used to compare the interaction of MARCKS peptide with the polyvalent PIP2 in mixed monolayers. On this way we surprisingly find out, that the relative small charge difference of the anionic lipids causes a significant different interaction with MARCKS (151–175). The lateral arrangement of the anionic lipids depends on their charge values and determines the diffusion of the electrostatic binding clusters within the membrane. DOI:10.1016/j.chemphyslip.2011.02.004 Journal Homepage

Chemistry and Physics of Lipids, Volume 164, Issue 4, 266-275 (2011)

 

Franziska Wetzel, Susanne Rönicke, Karla Müller, Markus Gyger, Daniel Rose, Mareike Zink, Josef Käs:

Single cell viability and impact of heating by laser absorption

Abstract: Optical traps such as tweezers and stretchers are widely used to probe the mechanical properties of cells. Beyond their large range of applications, the use of infrared laser light in optical traps causes significant heating effects in the cell. This study investigated the effect of laser-induced heating on cell viability. Common viability assays are not very sensitive to damages caused in short periods of time or are not practicable for single cell analysis. We used cell spreading, a vital ability of cells, as a new sensitive viability marker. The optical stretcher, a two beam laser trap, was used to simulate heat shocks that cells typically experience during measurements in optical traps. The results show that about 60% of the cells survived heat shocks without vital damage at temperatures of up to 58 ± 2°C for 0.5 s. By varying the duration of the heat shocks, it was shown that 60% of the cells stayed viable when exposed to 48 ± 2°C for 5 s. DOI:10.1007/s00249-011-0723-2 Journal Homepage

European Biophysics Journal, Volume 40, Issue 9, 1109-1114 (2011)

 

Florian Huber, Josef Käs:

Self-regulative organization of the cytoskeleton

Abstract: Despite its impressive complexity the cytoskeleton succeeds to persistently organize itself and thus the cells' interior. In contrast to classical man-made machines, much of the cellular organization originates from inherent self-assembly and self-organization allowing a high degree of autonomy for various functional units. Recent experimental and theoretical studies revealed numerous examples of cytoskeleton components that arrange and organize in a self-regulative way. In the present review we want to shortly summarize some of the principle mechanisms that are able to inherently trigger and regulate the cytoskeleton organization. Although taken individually most of these regulative principles are rather simple with intuitively predictable consequences, combinations of two or more of these mechanisms can quickly give rise to very complex, unexpected behavior and might even be able to explain the formation of different functional units out of a common pool of available building blocks. DOI:10.1002/cm.20509 Journal Homepage

Cytoskeleton, Volume 68, Issue 5, 259-265 (2011)

 

Melanie Knorr, Daniel Koch, Thomas Fuhs, Ulrich Behn, Josef A. Käs:

Stochastic Actin Dynamics in Lamellipodia Reveal Parameter Space for Cell Type Classification

Abstract: The lamellipodium, a thin veil-like structure at the leading edge of motile cells, is fundamental for cell migration and growth. Orchestrated activities of membrane components and an underlying biopolymer film result in a controlled movement of the whole system. Dynamics in two-dimensional cell motility are primarily driven by the actively moving protein film in the lamellipodium. Polymerization of actin filaments at the leading edge, back-transport of the actin network due to myosin motor activity, depolymerization in the back, and diffusive transport of actin monomers to the front control these dynamics. The same molecular prerequisites for lamellipodial motion are found in most eukaryotic cells and can function independently of the cell body. Here we show that lamellipodial dynamics differ strongly in different cell types according to their function. Path finding neuronal growth cones display strong stochastic fluctuations, wound healing fibroblasts that locally migrate in tissues exhibit reduced fluctuations while fish keratocytes move highly persistently. Nevertheless, experimental analysis and computer simulations show that changes in the parameters for actin polymerization and retrograde actin transport alone are sufficient for the cell to utilize the same, highly adaptive machinery to display this rich variety of behaviors. DOI:10.1039/c0sm01028f Journal Homepage

Soft Matter, Volume 7, 3192-3203 (2011)

 

Sergio Alonso, Undine Dietrich, Chris Händel, Josef A. Käs, Markus Bär:

Oscillations in the Lateral Pressure of Lipid Monolayers Induced by Nonlinear Chemical Dynamics of the Second Messengers MARCKS and Protein Kinase C

Abstract: The binding of the MARCKS peptide to the lipid monolayer containing PIP2 increases the lateral pressure of the monolayer. The unbinding dynamics modulated by protein kinase C leads to oscillations in lateral pressure of lipid monolayers. These periodic dynamics can be attributed to changes in the crystalline lipid domain size. We have developed a mathematical model to explain these observations based on the changes in the physical structure of the monolayer by the translocation of MARCKS peptide. The model indicates that changes in lipid domain size drives these oscillations. The model is extended to an open system that sustains chemical oscillations. DOI:10.1016/j.bpj.2010.12.3702 Journal Homepage

Biophysical Journal, Volume 100, Issue 4, 939-947 (2011)

 

Björn Stuhrmann, Florian Huber, Josef A. Käs:

Robust organization principles of protrusive biopolymer networks in migrating living cells

Abstract: Cell migration is associated with the dynamic protrusion of a thin actin-based cytoskeletal extension at the cell front, which has been shown to consist of two different substructures, the leading lamellipodium and the subsequent lamellum. While the formation of the lamellipodium is increasingly well understood, organizational principles underlying the emergence of the lamellum are just beginning to be unraveled. We report here on a 1D mathematical model which describes the reactiondiffusion processes of a polarized actin network in steady state, and reproduces essential characteristics of the lamellipodium-lamellum system. We observe a steep gradient in filament lengths at the protruding edge, a local depolymerization maximum a few microns behind the edge, as well as a differential dominance of the network destabilizer ADF/cofilin and the stabilizer tropomyosin. We identify simple and robust organizational principles giving rise to the derived network characteristics, uncoupled from the specifics of any molecular implementation, and thus plausibly valid across cell types. An analysis of network length dependence on physico-chemical system parameters implies that to limit array treadmilling to cellular dimensions, network growth has to be truncated by mechanisms other than aging-induced depolymerization, e.g., by myosin-associated network dissociation at the transition to the cell body. Our work contributes to the analytical understanding of the cytoskeletal extension’s bisection into lamellipodium and lamellum and sheds light on how cells organize their molecular machinery to achieve motility. DOI:10.1371/journal.pone.0014471 Journal Homepage

PLoS ONE, Volume 6, Issue 1, e14471 (2011)

 

Yun-Bi Lu, Ianors Iandiev, Margrit Hollborn, Nicole Körber, Elke Ulbricht, Petra G. Hirrlinger, Thomas Pannicke, Er-Qing Wei, Andreas Bringmann, Hartwig Wolburg, Ulrika Wilhelmsson, Milos Pekny, Peter Wiedemann, Andreas Reichenbach, Josef A. Käs:

Reactive glial cells: increased stiffness correlates with increased intermediate filament expression

Abstract: Increased stiffness of reactive glial cells may impede neurite growth and contribute to the poor regenerative capabilities of the mammalian central nervous system. We induced reactive gliosis in rodent retina by ischemia-reperfusion and assessed intermediate filament (IF) expression and the viscoelastic properties of dissociated single glial cells in wild-type mice, mice lacking glial fibrillary acidic protein and vimentin (GFAP-/-Vim-/-) in which glial cells are consequently devoid of IFs, and normal Long-Evans rats. In response to ischemia-reperfusion, glial cells stiffened significantly in wild-type mice and rats but were unchanged in GFAP-/-Vim-/- mice. Cell stiffness (elastic modulus) correlated with the density of IFs. These results support the hypothesis that rigid glial scars impair nerve regeneration and that IFs are important determinants of cellular viscoelasticity in reactive glia. Thus, therapeutic suppression of IF up-regulation in reactive glial cells may facilitate neuroregeneration. DOI:10.1096/fj.10-163790  Journal Homepage

The FASEB Journal, Volume 25, Issue 2, 624-631 (2011)

 

Dan Strehle, Jörg Schnauß, Claus Heussinger, José Alvarado, Mark Bathe, Josef Käs, Brian Gentry:

Transiently crosslinked F-actin bundles

Abstract: F-actin bundles are prominent cytoskeletal structures in eukaryotes. They provide mechanical stability in stereocilia, microvilli, filopodia, stress fibers and the sperm acrosome. Bundles are typically stabilized by a wide range of specific crosslinking proteins, most of which exhibit off-rates on the order of 1s^-1. Yet F-actin bundles exhibit structural and mechanical integrity on time scales that are orders of magnitude longer. By applying large deformations to reconstituted F-actin bundles using optical tweezers, we provide direct evidence of their differential mechanical response in vitro: bundles exhibit fully reversible, elastic response on short time scales and irreversible, elasto-plastic response on time scales that are long compared to the characteristic crosslink dissociation time. Our measurements show a broad range of characteristic relaxation times for reconstituted F-actin bundles. This can be reconciled by considering that bundle relaxation behavior is also modulated by the number of filaments, crosslinking type and occupation number as well as the consideration of defects due to filament ends.  DOI:10.1007/s00249-010-0621-z Journal Homepage

European Biophysical Journal, Volume 40, Number 1, 93-101 (2011)

2010
 

Mareike Zink, Anatol Fritsch, Franziska Wetzel, K. David Nnetu, Tobias Kießling, Josef A. Käs:

Probing the physics of tumor cells from mechanical perspectives

Abstract: - Journal Homepage

Cell News - Newsletter of the German Society for Cell Biology, Volume 36, 17-21 (4/2010)

 

J. Galle, A. Bader, P. Hepp, W. Grill, B. Fuchs, J.A. Käs, A. Krinner, B. MarquaB, K. Muller, J. Schiller, R.M. Schulz, M. von Buttlar, E. von der Burg, M. Zscharnack, M. Loffler:

Mesenchymal Stem Cells in Cartilage Repair: State of the Art and Methods to monitor Cell Growth, Differentiation and Cartilage Regeneration

Abstract: Degenerative joint diseases caused by rheumatism, joint dysplasia or traumata are particularly widespread in countries with high life expectation. Although there is no absolutely convincing cure available so far, hyaline cartilage and bone defects resulting from joint destruction can be treated today by appropriate transplantations. Recently, procedures were developed based on autologous chondrocytes from intact joint areas. The chondrocytes are expanded in cell culture and subsequently transplanted into the defect areas of the affected joints. However, these autologous chondrocytes are characterized by low expansion capacity and the synthesis of extracellular matrix of poor functionality and quality. An alternative approach is the use of adult mesenchymal stem cells (MSCs). These cells effectively expand in 2D culture and have the potential to differentiate into various cell types, including chondrocytes. Furthermore, they have the ability to synthesize extracellular matrix with properties mimicking closely the healthy hyaline joint cartilage. Beside a more general survey of the architecture of hyaline cartilage, its composition and the pathological processes of joint diseases, we will describe here which advances were achieved recently regarding the development of closed, aseptic bioreactors for the production of autologous grafts for cartilage regeneration based on MSCs. Additionally, a novel mathematical model will be presented that supports the understanding of the growth and differentiation of MSCs. It will be particularly emphasized that such models are helpful to explain the well-known fact that MSCs exhibit improved growth properties under reduced oxygen pressure and limited supply with nutrients. Finally, it will be comprehensively shown how different analytical methods can be used to characterize MSCs on different levels. Besides discussing methods for non-invasive monitoring and tracking of the cells and the determination of their elastic properties, mass spectrometric methods to evaluate the lipid compositions of cells will be highlighted.  DOI:10.2174/092986710791331095 Journal Homepage

Current Medicinal Chemistry, Volume 17, Issue 21, 2274-2291 (2010)

 

Christian Schulze, Franziska Wetzel, Thomas Kueper, Anke Malsen, Gesa Muhr, Soeren Jaspers, Thomas Blatt, Klaus-Peter Wittern, Horst Wenck, Josef A. Käs:

Stiffening of Human Skin Fibroblasts with Age

Abstract: Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ?60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix. DOI:10.1016/j.bpj.2010.08.026 Journal Homepage

Biophysical Journal, Volume 99, Issue 8, 2434-2442 (2010)

 

Anatol Fritsch, Michael Höckel, Tobias Kiessling, Kenechukwu David Nnetu, Franziska Wetzel, Mareike Zink, Josef A. Käs:

Are biomechanical changes necessary for tumour progression?

Abstract: Cell biophysics sheds some new light on cancer by approaching this complex problem from a materials science perspective. DOI:10.1038/nphys1800 Journal Homepage

Nature Physics, Volume 6, Issue 10, 730-732 (2010)

 

Björn Kemper, Patrik Langehanenberg, Alexander Höink, Gert von Bally, Falk Wottowah, Stefan Schinkinger, Jochen Guck, Josef Käs, Ilona Bredebusch, Jürgen Schnekenburger, Karin Schütze:

Monitoring of Laser Micromanipulated Optically Trapped Cells by Digital Holographic Microscopy

Abstract: For a precise manipulation of particles and cells with laser light as well as for the understanding and the control of the underlying processes it is important to visualize and quantify the response of the specimens. Thus, we investigated if digital holographic microscopy (DHM) can be used in combination with microfluidics to observe optically trapped living cells in a minimally invasive fashion during laser micromanipulation. The obtained results demonstrate that DHM multi-focus phase contrast provides label-free quantitative monitoring of optical manipulation with a temporal resolution of a few milliseconds. DOI:10.1002/jbio.201000035 Journal Homepage

Journal of Biophotonics, Volume 3, Issue 7, 425-431 (2010)

 

Y. Ma, M. Zink, S. G. Mayr:

Biocompatibility of single crystalline Fe70Pd30 ferromagnetic shape memory films

Abstract: Controllable by an external magnetic field, ferromagnetic shape memory materials reveal a high potential for actuators in biomedical applications. Simulated body fluid (SBF) and cell tests were performed to assess the biocompatibility of Fe70Pd30  ferromagnetic shape memory thin films as grown on MgO substrates. Calcium-phosphate aggregates were detected on the film surface after soaking in SBF. Biocompatibility tests with NIH 3T3 fibroblasts revealed adhesion and proliferation on the film surface but morphological modifications with a reduced cell size became evident as well as changes in cell viability for continuous and noncontinuous FePd films. The results are compared to FePd on SiO2. DOI:10.1063/1.3435260  Journal Homepage

Applied Physics Letters, Volume 96, Issue 21, 213703 (2010)

2009
 

Norio Kikuchi, Allen Ehrlicher, Daniel Koch, Josef A. Käs, Sriram Ramaswamy, Madan Rao:

Buckling, stiffening, and negative dissipation in the dynamics of a biopolymer in an active medium

Abstract: We present a generic theory for the dynamics of a stiff filament under tension, in an active medium with orientational correlations, such as a microtubule in contractile actin. In sharp contrast to the case of a passive medium, we find the filament can stiffen, and possibly oscillate or buckle, depending on both the contractile or tensile nature of the activity and the filament-medium anchoring interaction. We also demonstrate a strong violation of the fluctuation-dissipation (FD) relation in the effective dynamics of the filament, including a negative FD ratio. Our approach is also of relevance to the dynamics of axons, and our model equations bear a remarkable formal similarity to those in recent work [Martin P, Hudspeth AJ, Juelicher F (2001) Proc Natl Acad Sci USA 98:14380-14385] on auditory hair cells. Detailed tests of our predictions can be made by using a single filament in actomyosin extracts or bacterial suspensions. DOI:10.1073/pnas.0900451106 Journal Homepage

PNAS, Volume 106, No. 47, 19776-19779 (2009)

 

Kristian Franze, Jens Gerdelmann, Michael Weick, Timo Betz, Steve Pawlizak, Melike Lakadamyali, Johannes Bayer, Katja Rillich, Michael Gögler, Yun-Bi Lu, Andreas Reichenbach, Paul Janmey, Josef Käs:

Neurite Branch Retraction Is Caused by a Threshold-Dependent Mechanical Impact

Abstract: Recent results indicate that, in addition to chemical cues, mechanical stimuli may also impact neuronal growth. For instance, unlike most other cell types, neurons prefer soft substrates. However, the mechanisms responsible for the neuronal affinity for soft substrates have not yet been identified. Here we show that, in vitro, neurons continuously probe their mechanical environment. Growth cones visibly deform substrates with a compliance commensurate with their own. To understand the growth cones’ sensing of stiff substrates, we investigated their precise temporal response to well-defined mechanical stress. When the applied stress exceeded a threshold of 274 ± 41 pN/µm², neurons retracted and re-extended their processes, thereby enabling exploration of alternative directions. A calcium influx through stretch-activated ion channels (SACs) and the detachment of adhesion sites were prerequisites for this retraction. Our data illustrate how growing neurons may detect and avoid stiff substrates - as a mechanism involved in axonal branch pruning - and provide novel support of the idea that mechanics may act as guidance cue for neuronal growth. DOI:10.1016/j.bpj.2009.07.033 Journal Homepage

Biophysical Journal, Volume 97, Issue 7, 1883-1890 (2009)

 

Timo Betz, Daniel Koch, Daryl Lim, Josef A. Käs:

Stochastic Actin Polymerization and Steady Retrograde Flow Determine Growth Cone Advancement

Abstract: Neuronal growth is an extremely complex yet reliable process that is directed by a dynamic lamellipodial structure at the tip of every growing neurite, called the growth cone. Lamellipodial edge fluctuations are controlled by the interplay between actin polymerization pushing the edge forward and molecular motor driven retrograde actin flow retracting the actin network. The leading edge switches randomly between extension and retraction processes. We identify switching of "on/off" states in actin polymerization as the main determinant of lamellipodial advancement. Our analysis of motility statistics allows for a prediction of growth direction. This was used in simulations explaining the amazing signal detection capabilities of neuronal growth by the experimentally found biased stochastic processes. Our measurements show that the intensity of stochastic fluctuations depend on changes in the underlying active intracellular processes and we find a power law eta = a*x^alpha with exponent alpha = 2.63 ± 0.12 between noise intensity eta and growth cone activity x, defined as the sum of protrusion and retraction velocity. Differences in the lamellipodial dynamics between primary neurons and a neuronal cell line further suggests that active processes tune the observed stochastic fluctuations. This hints at a possible role of noise intensity in determining signal detection sensitivity. DOI:10.1016/j.bpj.2009.03.045 Journal Homepage

Biophysical Journal, Volume 96, Issue 12, 5130-5138 (2009)

 

Undine Dietrich, Peter Krüger, Thomas Gutberlet, Josef A. Käs:

Interaction of the MARCKS Peptide with PIP2 in Phospholipid Monolayers

Abstract: In this present work we have studied the effect of MARCKS (151–175) peptide on a mixed DPPC/PIP2 monolayer. By means of film balance, fluorescence microscopy, x-ray reflection/diffraction and neutron reflection measurements we detected changes in the lateral organization of the monolayer and changes in the perpendicular orientation of the PIP2 molecules depending on the presence of MARCKS (151–175) peptide in the subphase. In the mixed monolayer, the PIP2 molecules are distributed uniformly in the disordered phase of the monolayer, whereas the PI(4,5) groups elongate up to 10 Å below the phosphodiester groups. This elongation forms the precondition for the electrostatic interaction of the MARCKS peptide with the PIP2 molecules. Due to the enrichment of PIP2 in the disordered phase, the interaction with the peptide occurs primarily in this phase, causing the PI(4,5) groups to tilt toward the monolayer interface. DOI:10.1016/j.bbamem.2009.04.001 Journal Homepage

Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1788, Issue 7, 1474-1481 (2009)

 

David Smith, Brian Gentry, Björn Stuhrmann, Florian Huber, Dan Strehle, Claudia Brunner, Daniel Koch, Matthias Steinbeck, Timo Betz, Josef A. Käs:

The Cytoskeleton: An Active Polymer-based Scaffold

Abstract: The motility of cells is a multifaceted and complicated cytoskeletal process. Significant inroads can be made into gaining a more detailed understanding, however, by focusing on the smaller, more simple subunits of the motile system in an effort to isolate the essential protein components necessary to perform a certain task. Identification of such functional modules has proven to be an effective means of working towards a comprehensive understanding of complex, interacting systems. By following a bottom-up approach in studying minimal actin-related sub-systems for keratocyte motility, we revealed several fundamentally important effects ranging from an estimation of the force generated by the polymerization of a single actin filament, to assembly dynamics and the production of force and tension of composite actin networks, to the contraction of actin networks or smaller bundled structures by the motor myosin II. While even motile keratocyte fragments represent a far more complex situation than the simple reconstituted systems presented here, clear parallels can be seen between in vivo cell motility and the idealized in vitro functional modules presented here, giving more weight to their continued focus. DOI:10.1142/S1793048009000983 Journal Homepage

Biophysical Reviews and Letters (BRL), Volume 4, Issues 1-2, 179-208 (2009)

 

Brian Gentry, David Smith, Josef Käs:

Buckling-induced zebra stripe patterns in nematic F-actin

Abstract: Rather than forming a simple and uniform nematic liquid crystal, concentrated solutions of semiflexible polymers, such as F-actin, have been observed to display a spatially periodic switching of the nematic director. When observed with polarization microscopy, these patterns appear as alternating light and dark bands, often referred to as zebra stripe patterns. Zebra stripe patterns, although not fully characterized, are due to periodic orientation distortions in the nematic order. We characterize such patterns by using a combination of two techniques. Using polarization microscopy, we quantify the periodic orientation distortions and show that the magnitude of the order parameter also varies periodically in the striped domains. When using fluorescently labeled filaments as markers, filaments spanning the striped domains are seen to undergo large angle bends. With fluorescence, clear density differences between adjacent stripes are also observed with domains of lesser density corresponding to strongly bent filaments. By directly comparing patterned areas with both polarization and fluorescence techniques, we show that periodic variation in the orientation, order parameter, filament bending, and density are correlated. We propose that these effects originate from the coupling of orientation and density that occurs for highly concentrated solutions of long semiflexible polymers subject to shear flows, as previously proposed [P. de Gennes, Mol. Cryst. Liq. Cryst. (Phila. Pa.) 34, 177 (1977)]. After cessation of shearing, strong interfilament interactions and high compressibility can lead to periodic buckling from the relaxation of filaments stretched during flows. The characterization of zebra stripe patterns presented here provides evidence that buckling in confined F-actin nematics produces strong periodic bending that is responsible for the observed features. DOI:10.1103/PhysRevE.79.031916 Journal Homepage

Physical Review E, Volume 79, Issue 3, 031916 (2009)

 

Claudia Brunner, Axel Niendorf, Josef A. Käs:

Passive and active single-cell biomechanics: a new perspective in cancer diagnosis

Abstract: The relationship between biological cells' mechanical behavior and their underlying molecular structures and cellular dynamics opens new perspectives for polymer physics. Intracellular organization and cellular stability rely on an active polymer scaffold, the cytoskeleton, which is unparalleled in the synthetic world. Here, we highlight micro- and nanotechnology-based methods that have been used to study the active and passive biomechanical properties of living cells. Correlating viscoelastic properties with the metastatic potential of the cells could produce new insights into the processes of cell migration and metastasis. DOI:10.1039/b807545j Journal Homepage

Soft Matter, Volume 5, 2171-2178 (2009)

 

Torsten W. Remmerbach, Falk Wottawah, Julia Dietrich, Bryan Lincoln, Christian Wittekind, Jochen Guck:

Oral Cancer Diagnosis by Mechanical Phenotyping

Abstract: Oral squamous cell carcinomas are among the 10 most common cancers and have a 50% lethality rate after 5 years. Despite easy access to the oral cavity for cancer screening, the main limitations to successful treatment are uncertain prognostic criteria for (pre-)malignant lesions. Identifying a functional cellular marker may represent a significant improvement for diagnosis and treatment. Toward this goal, mechanical phenotyping of individual cells is a novel approach to detect cytoskeletal changes, which are diagnostic for malignant change. The compliance of cells from cell lines and primary samples of healthy donors and cancer patients was measured using a microfluidic optical stretcher. Cancer cells showed significantly different mechanical behavior, with a higher mean deformability and increased variance. Cancer cells (n {approx} 30 cells measured from each patient) were on average 3.5 times more compliant than those of healthy donors [Dnormal = (4.43 ± 0.68) 10–3 Pa–1; Dcancer = (15.8 ± 1.5) 10–3 Pa–1; P < 0.01]. The diagnosis results of the patient samples were confirmed by standard histopathology. The generality of these findings was supported by measurements of two normal and four cancer oral epithelial cell lines. Our results indicate that mechanical phenotyping is a sensible, label-free approach for classifying cancer cells to enable broad screening of suspicious lesions in the oral cavity. It could in principle be applied to any cancer to aid conventional diagnostic procedures. DOI:10.1158/0008-5472.CAN-08-4073 Journal Homepage

Cancer Research, Volume 69, Issue 5, 1728-1732 (2009)

 

Christian Schulze, Karla Müller, Josef A. Käs, Jens C. Gerdelmann:

Compaction of cell shape occurs before decrease of elasticity in CHO-K1 cells treated with actin cytoskeleton disrupting drug cytochalasin D

Abstract: The actin filaments of the cytoskeleton form a highly dynamic polymer scaffold which is actively involved in many essential mechanisms such as cell migration, transport, mitosis, and mechanosensitivity. We treated CHO-K1 cells with different concentrations of the actin cytoskeleton disrupting drug cytochalasin D. Then investigating the cells' elastic behaviour by scanning force microscopy-based rheology we confirmed for high cytochalasin D concentrations (> or =1.5 µM) a significant decrease of mechanical stability. At lower concentrations we measured no significant softening, but flattening and a horizontal contraction was observable even at low concentrations (> or =0.3 µM) of cytochalasin D. The observed changes in cell shape resulted in a lower cell volume, showing that there is compensation by volume for small decreases in cytoskeletal strength resulting from reduced numbers or lengths of actin filaments. These results suggest that the characteristic functions defining a cell's mechanical stability such as mechanosensitivity can be maintained via small changes in cell volume in order to counter fluctuations in cytoskeletal composition. DOI:10.1002/cm.20341 Journal Homepage

Cell Motility and the Cytoskeleton, Volume 66, Issue 4, 193-201 (2009)

2008
 

Moritz K. Kreysing, Tobias Kießling, Anatol Fritsch, Christian Dietrich, Jochen R. Guck, Josef A. Käs:

The optical cell rotator

Abstract: The optical cell rotator (OCR) is a modified dual-beam laser trap for the holding and controlled rotation of suspended dielectric microparticles, such as cells. In contrast to optical tweezers, OCR uses two counter-propagating divergent laser beams, which are shaped and delivered by optical fibers. The rotation of a trapped specimen is carried out by the rotation of a dual-mode fiber, emitting an asymmetric laser beam. Experiments were performed on human erythrocytes, promyelocytic leukemia cells (HL60), and cell clusters (MCF-7). Since OCR permits the rotation of cells around an axis perpendicular to the optical axis of any microscope and is fully decoupled from imaging optics, it could be a suitable and expedient tool for tomographic microscopy.  DOI:10.1364/OE.16.016984 Journal Homepage

Optics Express, Volume 16, Issue 21, 16984-16992 (2008)

 

Markus Gyger, Florian Rückerl, Josef A. Käs, Jaime Ruiz-García:

Errors in two particle tracking at close distances

Abstract: Tracking single and multiple particles is of great importance for many physical investigations in a variety of different areas. It is essential to find and eliminate sources of systematic errors in the particle position determination (PPD) and to determine the limits of its applicability to a given problem. Particularly when measuring the interactions between colloids at close distances, artifacts in the image taking process pose a great problem. By means of a simulation technique, we investigated the accuracy of the PPD using two-dimensional Gaussian and Gaussian-like fitting functions. For the distance between the two colloidal particles this revealed a systematic overestimation of the inter-particle distance of up to 1.9 % of the particle diameter for the Gaussian fitting function. This deviation can be explained by the differences between the intensity distribution of the overlap of the simulated particles and the linear superposition of the Gaussian functions. Modifications of the fitting functions can reduce the systematic error significantly. DOI:10.1016/j.jcis.2008.06.046 Journal Homepage

Journal of Colloid and Interface Science, Volume 326, Issue 2, 382-386 (2008)

 

Max Semmling, Oliver Kreft, Almdena Munoz Javier, Gleb B. Sukhorukov, Josef Käs, Wolfgang J. Parak:

A Novel Flow-Cytometry-Based Assay for Cellular Uptake Studies of Polyelectrolyte Microcapsules

Abstract: A flow-cytometry-based assay is presented with which the uptake of polyelectrolyte capsules can be quantified. The cavity of the capsules is loaded with the pH-sensitive dye SNARF, which emits in the red and green in alkaline and acidic environments, respectively. By recording the fluorescence intensities in the red and green channels, the localization of capsules associated with cells can be determined. Capsules adherent to the outer cell membrane fluoresce in the red due to the alkaline pH of the cell medium, whereas capsules internalized by cells fluoresce in the green due to the acidic pH in the endosomal/lysosomal/phagosomal compartments in which incorporated capsules are located. Adding the SNARF readout to the scattering signal typically derived with flow cytometry analysis allows for a more detailed quantitative analysis of particle uptake, which can also distinguish between adherent and ingested particles. DOI:10.1002/smll.200800596 Journal Homepage

Small, Volume 4, Issue 10, 1763-1768 (2008)

 

Florian Huber, Josef Käs, Björn Stuhrmann:

Growing Actin Networks Form Lamellipodium and Lamellum by Self-Assembly

Abstract: Many different cell types are able to migrate by formation of a thin actin-based cytoskeletal extension. Recently, it became evident that this extension consists of two distinct substructures, designated lamellipodium and lamellum, which differ significantly in their kinetic and kinematic properties as well as their biochemical composition. We developed a stochastic two-dimensional computer simulation that includes chemical reaction kinetics, G-actin diffusion, and filament transport to investigate the formation of growing actin networks in migrating cells. Model parameters were chosen based on experimental data or theoretical considerations. In this work, we demonstrate the system's ability to form two distinct networks by self-organization. We found a characteristic transition in mean filament length as well as a distinct maximum in depolymerization flux, both within the first 1-2 µm. The separation into two distinct substructures was found to be extremely robust with respect to initial conditions and variation of model parameters. We quantitatively investigated the complex interplay between ADF/cofilin and tropomyosin and propose a plausible mechanism that leads to spatial separation of, respectively, ADF/cofilin- or tropomyosin-dominated compartments. Tropomyosin was found to play an important role in stabilizing the lamellar actin network. Furthermore, the influence of filament severing and annealing on the network properties is explored, and simulation data are compared to existing experimental data. DOI:10.1529/biophysj.108.134817 Journal Homepage

Biophysical Journal, Volume 95, Issue 12, 5508-5523 (2008)

 

Martin B. Forstner, Douglas S. Martin, Florian Rückerl, Josef Käs, Carsten Selle:

Attractive membrane domains control lateral diffusion

Abstract: Lipid membranes play a fundamental role in vital cellular functions such as signal transduction. Many of these processes rely on lateral diffusion within the membrane, generally a complex fluid containing ordered microdomains. However, little attention has been paid to the alterations in transport dynamics of a diffusing species caused by long-range interactions with membrane domains. In this paper, we address the effect of such interactions on diffusive transport by studying lateral diffusion in a phase-separated Langmuir phospholipid monolayer via single-particle tracking. We find that attractive dipole-dipole interactions between condensed phase domains and diffusing probe beads lead to transient confinement at the phase boundaries, causing a transition from two- to one-dimensional diffusion. Using Brownian dynamics simulations, the long-term diffusion constant for such a system is found to have a sensitive, Boltzmann-like, dependence on the interaction strength. In addition, this interaction strength is shown to be a strong function of the ratio of domain to particle size. As similar interactions are expected in biological membranes, the modulation of diffusive transport dynamics by varying interaction strength and/or domain size may offer cells selective spatial and temporal control over signaling processes. DOI:10.1103/PhysRevE.77.051906 Journal Homepage

Physical Review E, Volume 77, Issue 5, 051906 (2008)

 

Florian Rückerl, Josef Käs, Carsten Selle:

Diffusion of Nanoparticles in Monolayers is Modulated by Domain Size

Abstract: Langmuir monolayers are often used as simple models for biological membranes. The possibility to change their composition and phase state in a very controlled manner as well as access to a large observation area makes them a versatile tool for the investigation of membrane-related interactions. Inspired by experiments in our group, we investigate the interaction of single, partially charged nanoparticles with lipid microdomains by Monte Carlo simulations. Condensed domains in inhomogeneous Langmuir monolayers exhibit an electric dipole field interacting attractively with the nanoparticle's dipole moment. With increasing domain size, the resulting electric field changes from single dipole to semi-infinite domain characteristics, significantly influencing the motion of the particle. Small immobile domains (R = 1 µm) confine the movement of the tracer to the boundary of the domain whereas for large domains (R > or = 10 µm) its motion is only temporarily hindered. This suggests a powerful mechanism for controlling diffusive transport in lipid membranes. DOI:10.1021/la703140b Journal Homepage

Langmuir, Volume 24, Issue 7, 3365-3369 (2008)

2007
 

Timo Betz, Daniel Koch, Björn Stuhrmann, Allen Ehrlicher, Josef Käs:

Statistical analysis of neuronal growth: edge dynamics and the effect of a focused laser on growth cone motility

Abstract: The neuronal growth cone is a small dynamic structure at the tip of neuronal extensions that guides each neurite extension to its correct partner cell. To reach the designated target, the growth cone integrates chemical signals with high accuracy and reliability. This signal detection operates close to the thermal noise limit and is, therefore of high interest not only to understand neuronal growth, but also to investigate the biological mechanisms of signalling and information processing under the influence of noise. To further investigate neuronal growth, a focused laser positioned at the leading edge of the growth cone is used to bias growth direction, however, the mechanisms of this influence are still unclear. We present a detailed measurement and analysis of the leading edge dynamics of laser treated and control growth cones. Based on the edge motility measurements, we can consistently describe neuronal growth with a stochastic model that allows a bistable potential and the noise intensity of the stochastic process to be extracted. The investigation of control growth cones that were not influenced by the laser reveals a nonlinear dependence of the noise on the overall activity of the growth cones. The presented analysis further quantifies the edge dynamics in growth cones that are manipulated by a laser. Growth cones that actively follow the laser show a tilt of the bistable potential in the direction of the laser to favour protrusions, but no significant changes in the leading edge growth velocity. This is in contrast to the potential changes observed in stationary growth cones that were influenced by the laser. Here, the laser does not tilt the potential shape, but increases the edge velocities, probably by an increase in actin polymerization velocity. These measurements provide new quantitative insight into the dynamics underlying growth cone protrusion and movement. DOI:10.1088/1367-2630/9/11/426 Journal Homepage

New Journal of Physics, Volume 9, 426 (2007)

 

Frank Fleischer, Revathi Ananthakrishnan, Stefanie Eckel, Hendrik Schmidt, Josef Käs, Tatyana Svitkina, Volker Schmidt, Michael Beil:

Actin network architecture and elasticity in lamellipodia of melanoma cells

Abstract: Cell migration is an essential element in the immune response on the one hand and in cancer metastasis on the other hand. The architecture of the actin network in lamellipodia determines the elasticity of the leading edge and contributes to the regulation of migration. We have implemented a new method for the analysis of actin network morphology in the lamellipodia of B16F1 mouse melanoma cells. This method is based on fitting multi-layer geometrical models to electron microscopy images of lamellipodial actin networks. The chosen model and F-actin concentrations are thereby deterministic parameters. Using this approach, we identified distinct structural features of actin networks in lamellipodia. The mesh size which defines the elasticity of the lamellipodium was determined as 34 and 78 nm for a two-layer network at a total actin concentration of 9.6 mg/ml. These data lead to estimates of the low frequency elastic shear moduli which differ by more than a magnitude between the two layers. These findings indicate an anisotropic shear modulus of the lamellipodium with the stiffer layer being the dominant structure against deformations in the lamellipodial plane and the softer layer contributing significantly at lower indentations perpendicular to the lamellipodial plane. This combination creates a material that is optimal for pushing forward as well as squeezing through narrow spaces. DOI:10.1088/1367-2630/9/11/420 Journal Homepage

New Journal of Physics, Volume 9, 420 (2007)

 

Revathi Ananthakrishnan, Allen Ehrlicher:

The Forces Behind Cell Movement

Abstract: Cell movement is a complex phenomenon primarily driven by the actin network beneath the cell membrane, and can be divided into three general components: protrusion of the leading edge of the cell, adhesion of the leading edge and deadhesion at the cell body and rear, and cytoskeletal contraction to pull the cell forward. Each of these steps is driven by physical forces generated by unique segments of the cytoskeleton. This review examines the specific physics underlying these phases of cell movement and the origins of the forces that drive locomotion. Article Online (HTML) Journal Homepage

International Journal of Biological Sciences Volume 3, 303-317 (2007)

 

Susanne Ebert, Kort Travis, Bryan Lincoln, Jochen Guck:

Fluorescence ratio thermometry in a microfluidic dual-beam laser trap

Abstract: The dual-beam laser trap is a versatile tool with many possible applications. In order to characterize its thermal properties in a microfluidic trap geometry we have developed a non-intrusive fluorescence ratio technique using the temperature sensitive dye Rhodamine B and the temperature independent reference dye Rhodamine 110. We measured temperature distribution profiles in the trap with submicron spatial resolution on a confocal laser-scanning microscope. The maximum heating in the center of the trap amounts to (13 ± 2) °C/W for a wavelength of lambda = 1064 nm and scales linearly with the applied power. The measurements correspond well with simulated temperature distributions. DOI:10.1364/OE.15.015493 Journal Homepage

Optics Express, Volume 15, Issue 23, 15493-15499 (2007)

 

Bryan Lincoln, Stefan Schinkinger, Kort Travis, Falk Wottawah, Susanne Ebert, Frank Sauer, Jochen Guck:

Reconfigurable microfluidic integration of a dual-beam laser trap with biomedical applications

Abstract: A dual-beam fiber laser trap, termed the optical stretcher when used to deform objects, has been combined with a capillary-based microfluidic system in order to serially trap and deform biological cells. The design allows for control over the size and position of the trap relative to the flow channel. Data is recorded using video phase contrast microscopy and is subsequently analyzed using a custom edge fitting routine. This setup has been regularly used with measuring rates of 50-100 cells/h. One such experiment is presented to compare the distribution of deformability found within a normal epithelial cell line to that of a cancerous one. In general, this microfluidic optical stretcher can be used for the characterization of cells by their viscoelastic signature. Possible applications include the cytological diagnosis of cancer and the gentle and marker-free sorting of stem cells from heterogeneous populations for therapeutic cell-based approaches in regenerative medicine. DOI:10.1007/s10544-007-9079-x Journal Homepage

Biomedical Microdevices, Volume 9, Number 5, 703-710 (2007)

 

Jan Fuhrmann, Josef Käs, Angela Stevens:

Initiation of cytoskeletal asymmetry for cell polarization and movement

Abstract: A variety of mathematical models for the actin driven motility of eucaryotic cells have been discussed over the last decades. However, most of them do model polarized cells which are already in motion or at least have established lamellipods. Here we investigate the stimulus induced transition from a symmetric resting state of a cell to a polarized one. Our goal is to find a minimal scenario for this rearrangement of the cytoskeleton and to figure out which of the manifold proteins and processes associated with actin dynamics are essential for the initiation of movement. DOI:10.1016/j.jtbi.2007.08.013 Journal Homepage

Journal of Theoretical Biology, Volume 249, Issue 2, 278-288 (2007)

 

David Smith, Falko Ziebert, David Humphrey, Cynthia Duggan, Matthias Steinbeck, Walter Zimmerman, Josef Käs:

Molecular Motor-Induced Instabilities and Cross Linkers Determine Biopolymer Organization

Abstract: All eukaryotic cells rely on the active self-organization of protein filaments to form a responsive intracellular cytoskeleton. The necessity of motility and reaction to stimuli additionally requires pathways that quickly and reversibly change cytoskeletal organization. While thermally driven order-disorder transitions are, from the viewpoint of physics, the most obvious method for controlling states of organization, the timescales necessary for effective cellular dynamics would require temperatures exceeding the physiologically viable temperature range. We report a mechanism whereby the molecular motor myosin II can cause near-instantaneous order-disorder transitions in reconstituted cytoskeletal actin solutions. When motor-induced filament sliding diminishes, the actin network structure rapidly and reversibly self-organizes into various assemblies. Addition of stable cross linkers was found to alter the architectures of ordered assemblies. These isothermal transitions between dynamic disorder and self-assembled ordered states illustrate that the interplay between passive crosslinking and molecular motor activity plays a substantial role in dynamic cellular organization. DOI:10.1529/biophysj.106.095919 Journal Homepage

Biophysical Journal, Volume 93, Issue 12, 4445-4452 (2007)

 

Kristian Franze, Jens Grosche, Serguei N. Skatchkov, Stefan Schinkinger, Christian Foja, Detlev Schild, Ortrud Uckermann, Kort Travis, Andreas Reichenbach, Jochen Guck:

Müller cells are living optical fibers in the vertebrate retina

Abstract: Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact retinal tissue and individual Müller cells, which are radial glial cells spanning the entire retinal thickness. Müller cells have an extended funnel shape, a higher refractive index than their surrounding tissue, and are oriented along the direction of light propagation. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo showed that these cells provide a low-scattering passage for light from the retinal surface to the photoreceptor cells. Using a modified dual-beam laser trap we could also demonstrate that individual Müller cells act as optical fibers. Furthermore, their parallel array in the retina is reminiscent of fiberoptic plates used for low-distortion image transfer. Thus, Müller cells seem to mediate the image transfer through the vertebrate retina with minimal distortion and low loss. This finding elucidates a fundamental feature of the inverted retina as an optical system and ascribes a new function to glial cells. DOI:10.1073/pnas.0611180104 Journal Homepage

PNAS, Volume 104, No. 20, 8287-8292 (2007)

 

Michael Gögler, Timo Betz, Josef Käs:

Simultaneous manipulation and detection of living cell membrane dynamics

Abstract: We report a novel optical-tweezers-based method to study the membrane motion at the leading edge of biological cells with nanometer spatial and microsecond temporal resolution. A diffraction-limited laser spot was positioned at the leading edge of a cell, and the forward scattered light was imaged on a quadrant photodiode that served as a position sensitive device. The universality of this technique is demonstrated with different cell types. We investigated the membrane motion at the leading edge of red blood cells in detail and showed that this technique can achieve simultaneous manipulation and detection of cellular edge dynamics with unprecedented precision. DOI:10.1364/OL.32.001893 Journal Homepage

Optics Letters, Volume 32, Issue 13, 1893-1895 (2007)

2006
 

Mireille Martin, Karla Mueller, Falk Wottawah, Stefan Schinkinger, Bryan Lincoln, Maren Romeyke, Josef A. Käs:

Feeling with light for cancer

Abstract: Even minute alterations in a cell's intracellular scaffolds, i.e. the cytoskeleton, which organize a cell, result in significant changes in a cell's elastic strength since the cytoskeletal mechanics nonlinearly amplify these alterations.  Light has been used to observe cells since Leeuwenhoek's times and novel techniques in optical microscopy are frequently developed in biological physics. In contrast, with the optical stretcher we use the forces caused by light described by Maxwell's surface tensor to feel cells. Thus, the stretcher exemplifies the other type of biophotonic devices that do not image but manipulate cells. The optical stretcher uses optical surface forces to stretch cells between two opposing laser beams, while optical gradient forces, which are used in optical tweezers, play a minor role and only contribute to a stable trapping configuration. The combination of the optical stretcher's sensitivity and high throughput capacity make a cell's "optical stretchiness" an extremely precise parameter to distinguish different cell types. This avoids the use of expensive, often unspecific molecular cell markers. This technique applies particularly well to cells with dissimilar degrees of differentiation, as a cell's maturation correlates with an increase in cytoskeletal strength. Because malignant cells gradually dedifferentiate during the progression of cancer, the optical stretcher should allow, the direct staging from early dysplasia to metastasis of a tumor sample obtained by MRI-guided fine needle aspirations or cytobrushes. With two prototypes of a microfluidic optical stretcher at our hands, we prepare preclinical trials to study its potential in resolving breast tumors' progression towards metastasis. Since the optical stretcher represents a basic technology for cell recognition and sorting, an abundance of further biomedical applications can be envisioned. DOI:10.1117/12.637899 Journal Homepage

Proceedings of SPIE, Volume 6080, 126-135 (2006)

 

Yun-Bi Lu, Kristian Franze, Gerald Seifert, Christian Steinhäuser, Frank Kirchhoff, Hartwig Wolburg, Jochen Guck, Paul Janmey, Er-Qing Wei, Josef Käs, Andreas Reichenbach:

Viscoelastic properties of individual glial cells and neurons in the CNS

Abstract: One hundred fifty years ago glial cells were discovered as a second, non-neuronal, cell type in the central nervous system. To ascribe a function to these new, enigmatic cells, it was suggested that they either glue the neurons together (the Greek word “glia” means “glue”) or provide a robust scaffold for them (“support cells”). Although both speculations are still widely accepted, they would actually require quite different mechanical cell properties, and neither one has ever been confirmed experimentally. We investigated the biomechanics of CNS tissue and acutely isolated individual neurons and glial cells from mammalian brain (hippocampus) and retina. Scanning force microscopy, bulk rheology, and optically induced deformation were used to determine their viscoelastic characteristics. We found that (i) in all CNS cells the elastic behavior dominates over the viscous behavior, (ii) in distinct cell compartments, such as soma and cell processes, the mechanical properties differ, most likely because of the unequal local distribution of cell organelles, (iii) in comparison to most other eukaryotic cells, both neurons and glial cells are very soft (“rubber elastic”), and (iv) intriguingly, glial cells are even softer than their neighboring neurons. Our results indicate that glial cells can neither serve as structural support cells (as they are too soft) nor as glue (because restoring forces are dominant) for neurons. Nevertheless, from a structural perspective they might act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as a soft substrate required for neurite growth and facilitating neuronal plasticity.  DOI:10.1073/pnas.0606150103 Journal Homepage

PNAS, Volume 103, No. 47, 17759-17764 (2006)

 

Claudia A. Brunner, Allen Ehrlicher, Bernd Kohlstrunk, Detlef Knebel, Josef A. Käs, Michael Goegler:

Cell migration through small gaps

Abstract: Cell motility is a fundamental process associated with many phenomena in nature, such as immune response, wound healing, and cancer metastasis. In these processes, cells must squeeze through cell layers, and we characterize this ability to actively produce forces and simultaneously adapt their shapes. We have measured forward forces up to 15 nN that a migrating keratocyte was able to generate, in order to adjust its shape and successfully force its way under and past an obstacle. We also observed that 34 nN was capable of stalling the cell’s forward motion. Furthermore, we measured that under compression stresses up to 1,165 pN/µm² (1,165 Pa), cell morphology, and velocity remained unchanged. Additionally, we found that keratocytes were able to compress themselves up to 80% vertically in order to squeeze through a gap as small as 500 nm. DOI:10.1007/s00249-006-0079-1 Journal Homepage

European Biophysics Journal, Volume 35, Number 8, 713-719 (2006)

 

Frank Müller, Andreas Houben, Peter E. Barker, Yan Xiao, Josef A. Käs, Michael Melzer:

Quantum dots - a versatile tool in plant science?

Abstract: An optically stable, novel class of fluorophores (quantum dots) for in situ hybridisation analysis was tested to investigate their signal stability and intensity in plant chromosome analyses. Detection of hybridisation sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide. Comparison of quantum dots (QDs) with conventional detection systems (Alexa 488) in immunolabeling experiments demonstrated greater sensitivity than the conventional system. In contrast, detection of QDs in in situ hybridisation of several plant chromosomes, using several high-copy sequences, was less sensitve than Alexa 488. Thus, semiconductor nanocrystal fluorophores are more suitable for immunostaining but not for in situ hybridisation of plant chromosomes. DOI:10.1186/1477-3155-4-5 Journal Homepage

Journal of Nanobiotechnology, Volume 4, 5 (2006)

 

B. Stuhrmann, H.-G. Jahnke, M. Schmidt, K. Jähn, T. Betz, K. Müller, A. Rothermel, J. Käs, A. A. Robitzki:

Versatile optical manipulation system for inspection, laser processing, and isolation of individual living cells

Abstract: Isolation of individual cells from a heterogeneous cell population is an invaluable step in the analysis of single cell properties. The demands in molecular and cellular biology as well as molecular medicine are the selection, isolation, and monitoring of single cells and cell clusters of biopsy material. Of particular interest are methods which complement a passive optical or spectroscopic selection with a variety of active single cell processing techniques such as mechanical, biochemical, or genetic manipulation prior to isolation. Sophisticated laser-based cell processing systems are available which can perform single cell processing in a contact-free and sterile manner. Until now, however, these multipurpose turnkey systems offer only basic micromanipulation and are not easily modified or upgraded, whereas laboratory situations often demand simple but versatile and adaptable solutions. We built a flexible laser micromanipulation platform combining contact-free microdissection and catapulting capabilities using a pulsed ultraviolet (337 nm) laser with simultaneous generation of optical tweezing forces using a continuous wave infrared (1064 nm) laser. The potential of our platform is exemplified with techniques such as local laser-induced injection of biomolecules into individual living cells, laser surgery, isolation of single cells by laser catapulting, and control of neuronal growth using optical gradient forces. Arbitrary dynamic optical force patterns can be created by fast laser scanning with acousto-optical deflectors and galvanometer mirrors, allowing multibeam contact-free micromanipulation, a prerequisite for reliable handling of material in laboratory-on-a-chip applications. All common microscopy techniques can be used simultaneously with the offered palette of micromanipulation methods. Taken together, we show that advanced optical micromanipulation systems can be designed which combine quality, cost efficiency, and adaptability. DOI:10.1063/1.2214961 Journal Homepage

Review of Scientific Instruments, Volume 77, Issue 6, 063116 (2006)

 

Timo Betz, Daryl Lim, Josef A. Käs:

Neuronal Growth: A Bistable Stochastic Process

Abstract: The fundamentally stochastic nature of neuronal growth has hardly been addressed in neuroscience. We report on the stochastic fluctuations of a neuronal growth cone's leading edge movement, the basic step in neuronal growth. Describing the edge movement as a stochastic bistable process leads to an isotropic noise parameter that is successfully used to test the model. An analysis of growth cone motility confirms the model, and predicts that linear changes of the bistable potential, as known from stochastic filtering, result in directed growth cone translocation. DOI:10.1103/PhysRevLett.96.098103 Journal Homepage

Physical Review Letters, Volume 96, Issue 9, 098103 (2006)

 

Kort Travis, Jochen Guck:

Scattering from Single Nanoparticles: Mie Theory Revisited

Abstract: Recent intense interest in nanoparticle materials and nanoparticle-based contrast enhancement agents for biophysical applications gives new relevance to Mie scattering theory in its original context of application. The Mie theory still provides the most exact treatment of scattering from single nanoparticles of the noble metals. When recast in terms of modern electrodynamic formalism, the theory provides a concise closed-form representation for the scattered fields and also serves as a vehicle to elaborate the formal electrodynamic technique. The behavior of the Debye truncation condition for the multipole expansion is illustrated with numerical examples, clearly showing the features of the transition between the Rayleigh, dipole and higher order multipole approximations for the scattered fields. The classical Mie theory is an approximation in that only the transverse field components are included in the calculation. Extensions to the classical theory which include the effects of longitudinal fields are discussed and illustrated numerically. The example of scattering from multilayer composite particles is used to examine the feasibility of engineering spectral features of the scattering cross-section to target the requirements of specific applications. DOI:10.1142/S1793048006000136 Journal Homepage

Biophysical Reviews and Letters (BRL), Volume 1, Issue 2, 179-207 (2006)

 

Revathi Ananthakrishnan, Jochen Guck, Falk Wottawah, Stefan Schinkinger, Bryan Lincoln, Maren Romeyke, Tess Moon, Josef Käs:

Quantifying the contribution of actin networks to the elastic strength of fibroblasts

Abstract: The structural models created to understand the cytoskeletal mechanics of cells in suspension are described here. Suspended cells can be deformed by well-defined surface stresses in an Optical Stretcher [Guck, J., Ananthakrishnan, R., Mahmood, H., Moon, T.J., Cunningham, C.C., Käs, J., 2001. The optical stretcher: a novel laser tool to micromanipulate cells. Biophys. J. 81(2), 767–784], a two-beam optical trap designed for the contact-free deformation of cells. Suspended cells have a well-defined cytoskeleton, displaying a radially symmetric actin cortical network underlying the cell membrane with no actin stress fibers, and microtubules and intermediate filaments in the interior. Based on experimental data using suspended fibroblasts, we create two structural models: a thick shell actin cortex model that describes cell deformation for a localized stress distribution on these cells and a three-layered model that considers the entire cytoskeleton when a broad stress distribution is applied. Applying the models to data, we obtain a (actin) cortical shear moduli G of ~220 Pa for normal fibroblasts and ~185 Pa for malignantly transformed fibroblasts. Additionally, modeling the cortex as a transiently crosslinked isotropic actin network, we show that actin and its crosslinkers must be co-localized into a tight shell to achieve these cortical strengths. The similar moduli values and cortical actin and crosslinker densities but different deformabilities of the normal and cancerous cells suggest that a cell's structural strength is not solely determined by cytoskeletal composition but equally importantly by (actin) cytoskeletal architecture via differing cortical thicknesses. We also find that although the interior structural elements (microtubules, nucleus) contribute to the deformed cell's exact shape via their loose coupling to the cortex, it is the outer actin cortical shell (and its thickness) that mainly determines the cell's structural response. DOI:10.1016/j.jtbi.2006.03.021 Journal Homepage

Journal of Theoretical Biology, Volume 242, Issue 2, 502-516 (2006) Corrigendum to "Quantifying the contribution of actin networks to the elastic strength of fibroblasts" Journal of Theoretical Biology, Volume 255, Issue 1, 162 (2008)

 

F.-U. Gast, P. S. Dittrich, P. Schwille, M. Weigel, M. Mertig, J. Opitz, U. Queitsch, S. Diez, B. Lincoln, F. Wottawah, S. Schinkinger, J. Guck, J. Käs, J. Smolinski, K. Salchert, C. Werner, C. Duschl, M. S. Jäger, K. Uhlig, P. Geggier, S. Howitz:

The microscopy cell (MicCell), a versatile modular flowthrough system for cell biology, biomaterial research, and nanotechnology

Abstract: We describe a novel microfluidic perfusion system for high-resolution microscopes. Its modular design allows pre-coating of the coverslip surface with reagents, biomolecules, or cells. A poly(dimethylsiloxane) (PDMS) layer is cast in a special molding station, using masters made by photolithography and dry etching of silicon or by photoresist patterning on glass or silicon. This channel system can be reused while the coverslip is exchanged between experiments. As normal fluidic connectors are used, the link to external, computer-programmable syringe pumps is standardized and various fluidic channel networks can be used in the same setup. The system can house hydrogel microvalves and microelectrodes close to the imaging area to control the influx of reaction partners. We present a range of applications, including single-molecule analysis by fluorescence correlation spectroscopy (FCS), manipulation of single molecules for nanostructuring by hydrodynamic flow fields or the action of motor proteins, generation of concentration gradients, trapping and stretching of live cells using optical fibers precisely mounted in the PDMS layer, and the integration of microelectrodes for actuation and sensing. DOI:10.1007/s10404-005-0047-6 Journal Homepage

Microfluidics and Nanofluidics, Volume 2, Number 1, 21-36 (2006)

2005
 

B. Stuhrmann, M. Gögler, T. Betz, A. Ehrlicher, D. Koch, J. Käs:

Automated tracking and laser micromanipulation of motile cells

Abstract: Control over neuronal growth is a prerequisite for the creation of defined in vitro neuronal networks as assays for the elucidation of interneuronal communication. Neuronal growth has been directed by focusing a near-infrared laser beam at a nerve cell's leading edge [A. Ehrlicher, T. Betz, B. Stuhrmann, D. Koch, V. Milner, M. G. Raizen, and J. Käs, Proc. Natl. Acad. Sci. U.S.A. 99, 16024 (2002)]. The setup reported by Ehrlicher et al. was limited to local laser irradiation and relied on a great deal of subjective interaction since the laser beam could only be steered manually. To overcome the drawbacks of the reported setup, we developed and here present a fully automated low-contrast edge detection software package, which responds to detected cell morphological changes by rapidly actuating laser steering devices, such as acousto-optical deflectors or moving mirrors, thus enabling experiments with minimum human interference. The resulting radiation patterns can be arbitrary functions of space, time, and cell morphology, and are calculated by experiment specific feedback routines. Data processing is repeated on the order of 1  s allowing rapid reactions to morphological changes. The strengths of our program are the combination of real-time low contrast shape detection with complex feedback mechanisms, as well as easy adaptability due to a modular programming concept. In this article we demonstrate automated optical guidance; however, the software is easily adaptable to other problems requiring automated rapid responses of equipment to changes in the morphology of low contrast objects. DOI:10.1063/1.1863812  Journal Homepage

Review of Scientific Instruments, Volume 76, 035105 (2005)

 

Falk Wottawah, Stefan Schinkinger, Bryan Lincoln, Revathi Ananthakrishnan, Maren Romeyke, Jochen Guck, Josef Käs:

Optical Rheology of Biological Cells

Abstract: A step stress deforming suspended cells causes a passive relaxation, due to a transiently cross-linked isotropic actin cortex underlying the cellular membrane. The fluid-to-solid transition occurs at a relaxation time coinciding with unbinding times of actin cross-linking proteins. Elastic contributions from slowly relaxing entangled filaments are negligible. The symmetric geometry of suspended cells ensures minimal statistical variability in their viscoelastic properties in contrast with adherent cells and thus is defining for different cell types. Mechanical stimuli on time scales of minutes trigger active structural responses. DOI:10.1103/PhysRevLett.94.098103 Journal Homepage

Physical Review Letters, Volume 94, Issue 9, 98103 (2005)

 

Jochen Guck, Stefan Schinkinger, Bryan Lincoln, Falk Wottawah, Susanne Ebert, Maren Romeyke, Dominik Lenz, Harold M. Erickson, Revathi Ananthakrishnan, Daniel Mitchell, Josef Käs, Sydney Ulvick, Curt Bilby:

Optical Deformability as an Inherent Cell Marker for Testing Malignant Transformation and Metastatic Competence

Abstract: The relationship between the mechanical properties of cells and their molecular architecture has been the focus of extensive research for decades. The cytoskeleton, an internal polymer network, in particular determines a cell's mechanical strength and morphology. This cytoskeleton evolves during the normal differentiation of cells, is involved in many cellular functions, and is characteristically altered in many diseases, including cancer. Here we examine this hypothesized link between function and elasticity, enabling the distinction between different cells, by using a microfluidic optical stretcher, a two-beam laser trap optimized to serially deform single suspended cells by optically induced surface forces. In contrast to previous cell elasticity measurement techniques, statistically relevant numbers of single cells can be measured in rapid succession through microfluidic delivery, without any modification or contact. We find that optical deformability is sensitive enough to monitor the subtle changes during the progression of mouse fibroblasts and human breast epithelial cells from normal to cancerous and even metastatic state. The surprisingly low numbers of cells required for this distinction reflect the tight regulation of the cytoskeleton by the cell. This suggests using optical deformability as an inherent cell marker for basic cell biological investigation and diagnosis of disease. DOI:10.1529/biophysj.104.045476 Journal Homepage

Biophysical Journal, Volume 88, Issue 5, 3689-3698 (2005)

 

Revathi Ananthakrishnan, Jochen Guck, Falk Wottawah, Stefan Schinkinger, Bryan Lincoln, Maren Romeyke, Josef Käs:

Modelling the structural response of an eukaryotic cell in the optical stretcher

Abstract: The cytoskeleton of an eukaryotic cell is a composite polymer material with unique structural (mechanical) properties. To investigate the role of individual cytoskeletal polymers in the deformation response of a cell to an external force (stress), we created two structural models – a thick shell model for the actin cortex, and a three-layered model for the whole cell. These structural models for a cell are based on data obtained by deforming suspended cells, where each cell is stretched between two counter-propagating laser beams using an optical stretcher. Our models, with the data, suggest that the outer actin cortex is the main determinant of the structural response of the cell. Article (PDF) Journal Homepage

Current Science, Volume 88, Issue 9, 1434-1440 (2005)

 

S. Park, D. Koch, R. Cardenas, J. Käs, C. K. Shih:

Cell Motility and Local Viscoelasticity of Fibroblasts

Abstract: Viscoelastic changes of the lamellipodial actin cytoskeleton are a fundamental element of cell motility. Thus, the correlation between the local viscoelastic properties of the lamellipodium (including the transitional region to the cell body) and the speed of lamellipodial extension is studied for normal and malignantly transformed fibroblasts. Using our atomic force microscopy-based microrheology technique, we found different mechanical properties between the lamellipodia of malignantly transformed fibroblasts (H-ras transformed and SV-T2 fibroblasts) and normal fibroblasts (BALB 3T3 fibroblasts). The average elastic constants, K, in the leading edge of SV-T2 fibroblasts (0.48±0.51 kPa) and of H-ras transformed fibroblasts (0.42±0.35 kPa) are significantly lower than that of BALB 3T3 fibroblasts (1.01±0.40 kPa). The analysis of time-lapse phase contrast images shows that the decrease in the elastic constant, K, for malignantly transformed fibroblasts is correlated with the enhanced motility of the lamellipodium. The measured mean speeds are 6.1±4.5 µm/h for BALB 3T3 fibroblasts, 13.1±5.2 µm/h for SV-T2 fibroblasts, and 26.2±11.5 µm/h for H-ras fibroblasts. Furthermore, the elastic constant, K, increases toward the cell body in many instances which coincide with an increase in actin filament density toward the cell body. The correlation between the enhanced motility and the decrease in viscoelastic moduli supports the Elastic Brownian Ratchet model for driving lamellipodia extension. DOI:10.1529/biophysj.104.053462 Journal Homepage

Biophysical Journal, Volume 89, Issue 6, 4330-4342 (2005)

 

Timo Betz, Jörn Teipel, Daniel Koch, Wolfgang Härtig, Jochen Guck, Josef Käs, Harald Giessen:

Excitation beyond the monochromatic laser limit: simultaneous 3-D confocal and multiphoton microscopy with a tapered fiber as white-light laser source

Abstract: Confocal and multiphoton microscopy are essential tools in modern life sciences. They allow fast and highly resolved imaging of a steadily growing number of fluorescent markers, ranging from fluorescent proteins to quantum dots and other fluorophores, used for the localization of molecules and the quantitative detection of molecular properties within living cells and organisms. Up to now, only one physical limitation seemed to be unavoidable. Both confocal and multiphoton microscopy rely on lasers as excitation sources, and their monochromatic radiation allows only a limited number of simultaneously usable dyes, which depends on the specific number of laser lines available in the used microscope. We have overcome this limitation by successfully replacing all excitation lasers in a standard confocal microscope with pulsed white light ranging from 430 to 1300  nm generated in a tapered silica fiber. With this easily reproducible method, simultaneous confocal and multiphoton microscopy was demonstrated. By developing a coherent and intense laser source with spectral width comparable to a mercury lamp, we provide the flexibility to excite any desired fluorophore combination. DOI:10.1117/1.2114788 Journal Homepage

Journal of Biomedical Optics, Volume 10, Issue 5, 054009 (2005)

 

Falk Wottawah, Stefan Schinkinger, Bryan Lincoln, Susanne Ebert, Karla Müller, Frank Sauer, Kort Travis, Jochen Guck:

Characterizing single suspended cells by optorheology

Abstract: The measurement of the mechanical properties of individual cells has received much attention in recent years. In this paper we describe the application of optically induced forces with an optical stretcher to perform step-stress experiments on individual suspended fibroblasts. The conversion from creep-compliance to frequency-dependent complex shear modulus reveals characteristic viscoelastic signatures of the underlying cytoskeleton and its dynamic molecular properties. Both normal and cancerous fibroblasts display a single stress relaxation time in the observed time and frequency space that can be related to the transient binding of actin crosslinking proteins. In addition, shear modulus and steady-state viscosity of the shell-like actin cortex as the main module resisting small deformations are extracted. These values in combination with insight into the cells’ architecture are used to explain their different deformability. This difference can then be exploited to distinguish normal from cancerous cells. The nature of the optical stretcher as an optical trap allows easy incorporation in a microfluidic system with automatic trapping and alignment of the cells, and thus a high measurement throughput. This carries the potential for using the microfluidic optical stretcher to investigate cellular processes involving the cytoskeleton and to diagnose diseases related to cytoskeletal alterations. DOI:10.1016/j.actbio.2005.02.010  Journal Homepage

Acta Biomaterialia, Volume 1, Issue 3, 263-271 (2005)

2004
 

R. E. Mahaffy, S. Park, E. Gerde, J. Käs, C. K. Shih:

Quantitative Analysis of the Viscoelastic Properties of Thin Regions of Fibroblasts Using Atomic Force Microscopy

Abstract: Viscoelasticity of the leading edge, i.e., the lamellipodium, of a cell is the key property for a deeper understanding of the active extension of a cell's leading edge. The fact that the lamellipodium of a cell is very thin (<1000nm) imparts special challenges for accurate measurements of its viscoelastic behavior. It requires addressing strong substrate effects and comparatively high stresses (>1kPa) on thin samples. We present the method for an atomic force microscopy-based microrheology that allows us to fully quantify the viscoelastic constants (elastic storage modulus, viscous loss modulus, and the Poisson ratio) of thin areas of a cell (<1000nm) as well as those of thick areas. We account for substrate effects by applying two different models - a model for well-adhered regions (Chen model) and a model for nonadhered regions (Tu model). This method also provides detailed information about the adhered regions of a cell. The very thin regions relatively near the edge of NIH 3T3 fibroblasts can be identified by the Chen model as strongly adherent with an elastic strength of ~1.6±0.2kPa and with an experimentally determined Poisson ratio of ~0.4 to 0.5. Further from the edge of these cells, the adherence decreases, and the Tu model is effective in evaluating its elastic strength (~0.6±0.1kPa). Thus, our AFM-based microrheology allows us to correlate two key parameters of cell motility by relating elastic strength and the Poisson ratio to the adhesive state of a cell. This frequency-dependent measurement allows for the decomposition of the elastic modulus into loss and storage modulus. Applying this decomposition and Tu's and Chen's finite depth models allow us to obtain viscoelastic signatures in a frequency range from 50 to 300Hz, showing a rubber plateau-like behavior. DOI:10.1016/S0006-3495(04)74245-9 Journal Homepage

Biophysical Journal, Volume 86, Issue 3, 1777-1793 (2004)

 

Anna L. Lin, Bernward A. Mann, Gelsy Torres-Oviedo, Bryan Lincoln, Josef Käs, Harry L. Swinney:

Localization and Extinction of Bacterial Populations under Inhomogeneous Growth Conditions

Abstract: The transition from localized to systemic spreading of bacteria, viruses, and other agents is a fundamental problem that spans medicine, ecology, biology, and agriculture science. We have conducted experiments and simulations in a simple one-dimensional system to determine the spreading of bacterial populations that occurs for an inhomogeneous environment under the influence of external convection. Our system consists of a long channel with growth inhibited by uniform ultraviolet (UV) illumination except in a small “oasis”, which is shielded from the UV light. To mimic blood flow or other flow past a localized infection, the oasis is moved with a constant velocity through the UV-illuminated “desert”. The experiments are modeled with a convective reaction-diffusion equation. In both the experiment and model, localized or extinct populations are found to develop, depending on conditions, from an initially localized population. The model also yields states where the population grows everywhere. Further, the model reveals that the transitions between localized, extended, and extinct states are continuous and nonhysteretic. However, it does not capture the oscillations of the localized population that are observed in the experiment. DOI:10.1529/biophysj.103.034041 Journal Homepage

Biophysical Journal, Volume 87, Issue 1, 75-80 (2004)

 

Carsten Selle, Florian Rückerl, Douglas S. Martin, Martin B. Forstner, Josef A. Käs:

Measurement of diffusion in Langmuir monolayers by single-particle tracking

Abstract: There is a great amount of literature available indicating that membranes are inhomogeneous, complex fluids. For instance, observation of diffusion in cell membranes demonstrated confined motion of membrane constituents and even subdiffusion. In order to circumvent the small dimensions of cells leading to weak statistics when investigating the diffusion properties of single membrane components, a technique based on optical microscopy employing Langmuir monolayers as membrane model systems has been developed in our lab. In earlier work, the motion of labeled single lipids was visualized. These measurements with long observation times, thus far only possible with this method, were combined with respective Monte-Carlo simulations. We could conclude that noise can lead in general to the assumption of subdiffusion while interpreting the results of single-particle-tracking (SPT) experiments within membranes in general. Since the packing density of lipids within monolayers at the air/water interface can be changed easily, inhomogeneity with regard to the phase state can be achieved by isothermal compression to coexistence regions. Surface charged polystyrene latexes were used as model proteins diffusing in inhomogeneous monolayers as biomembrane mimics. Epifluorescence microscopy coupled to SPT revealed that domain associated, dimensionally reduced diffusion can occur in these kinds of model systems. This was caused by an attractive potential generated by condensed domains within monolayers. Monte-Carlo simulations supported this view point. Moreover, long-time simulations show that diffusion coefficients of respective particles were dependent on the strength of the attractive potential present: a behavior reflecting altered dimensionality of diffusion. The widths of those potentials were also found to be affected by the domain size of the more ordered lipid phase. In biological membrane systems, cells could utilize these physical mechanisms to adjust diffusion properties of membrane components. DOI:10.1039/b412680g Journal Homepage

Physical Chemistry Chemical Physics, Volume 6, Issue 24, 5535-5542 (2004)

 

Daniel Koch, Timo Betz, Allen Ehrlicher, Michael Gögler, Björn Stuhrmann, Josef Käs:

Optical control of neuronal growth

Abstract: Understanding and controlling neuronal growth are basic objectives in neuroscience, biology, biophysics, and biomedicine, and are vital for the formation of neural circuits in vitro, as well as for nerve regeneration in vivo.  All molecular stimuli for neuronal growth eventually address the polymeric cytoskeleton, which advances a neurite's leading edge also known as the growth cone. We have shown that optical forces of a highly focused infrared laser beam influence the motility of a growth cone by biasing the polymerization-driven intracellular machinery.  In actively extending growth cones, a laser spot placed at specific areas of the neurite's leading edge affects the growth speed, the direction taken by a growth cone, and the splitting of a growth cone. This novel optical tool manipulates a natural biological process, the cytoskeleton driven morphological changes in growth cones, with potential applications in the formation of neuronal networks and in understanding growth cone motility.  The current apparatus combines optical tweezers, phase contrast and fluorescence imaging, and real-time shape detection. Automated and dynamically readjusted irradiation of the growth cone is used to examine and to influence structural and morphological changes of neuronal growth. DOI:10.1117/12.559672 Journal Homepage

Proceedings of SPIE, Volume 5514 (Optical Trapping and Optical Micromanipulation), 428-436 (2004)

 

Bryan Lincoln, Harold M. Erickson, Stefan Schinkinger, Falk Wottawah, Daniel Mitchell, Sydney Ulvick, Curt Bilby, Jochen Guck:

Deformability-based flow cytometry

Abstract: Background: Elasticity of cells is determined by their cytoskeleton. Changes in cellular function are reflected in the amount of cytoskeletal proteins and their associated networks. Drastic examples are diseases such as cancer, in which the altered cytoskeleton is even diagnostic. This connection between cellular function and cytoskeletal mechanical properties suggests using the deformability of cells as a novel inherent cell marker. Methods: The optical stretcher is a new laser tool capable of measuring cellular deformability. A unique feature of this deformation technique is its potential for high throughput, with the incorporation of a microfluidic delivery of cells. Results: Rudimentary implementation of the microfluidic optical stretcher has been used to measure optical deformability of several normal and cancerous cell types. A drastic difference has been seen between the response of red blood cells and polymorphonuclear cells for a given optically induced stress. MCF-10, MCF-7, and modMCF-7 cells were also measured, showing that while cancer cells stretched significantly more (five times) than normal cells, optical deformability could even be used to distinguish metastatic cancer cells from nonmetastatic cancer cells. This trimodal distribution was apparent after measuring a mere 83 cells, which shows optical deformability to be a highly regulated cell marker. Conclusions: Preliminary work suggests a deformability-based cell sorter similar to current fluorescence-based flow cytometry without the need for specific labeling. This could be used for the diagnosis of all diseases, and the investigation of all cellular processes, that affect the cytoskeleton. DOI:10.1002/cyto.a.20050 Journal Homepage

Cytometry Part A, Volume 59A, Issue 2, 203-209 (2004)

 

Stefan Schinkinger, Falk Wottawah, Kort Travis, Bryan Lincoln, Jochen Guck:

Feeling for cells with light

Abstract: In an optical stretcher, infrared laser light is used to exert surface stress on biological cells, causing an elongation of the trapped cell body along the laser beam axis. These optically induced deformations characterize individual cells and cell lines. When integrated within a microfluidic chamber with high throughput, this enables diagnosis of diseases, on a cellular level, that are associated with cytoskeletal processes. Additionally, it allows sorting of cells with high accuracy in a non-contact manner. To determine the surface stress on the cell, ray optics calculations as well as the system transfer operator (T-matrix) approach with an appropriate incident field are used. The latter approach allows a more accurate modeling of the cell in the optical stretcher and reveals a more detailed stress profile acting on the cell surface. Analyzing the deformation behavior of normal and malignantly transformed fibroblasts, significant differences in axial elongation even for sample sizes as low as 30 cells are already measurable on a time scale of 0.1s. Here, malignant transformation of cells is discussed as an example of how any process that affects the cell's optical or mechanical properties allows classification with the optical stretcher. DOI:10.1117/12.556795 Journal Homepage

Proceedings of SPIE, Volume 5514 (Optical Trapping and Optical Micromanipulation), 170-178 (2004)

2003
 

Martin B. Forstner, Douglas S. Martin, Ann M. Navar, Josef A. Käs:

Simultaneous Single-Particle Tracking and Visualization of Domain Structure on Lipid Monolayers

Abstract: A new method is presented that combines dual fluorescence microscopy and single-particle tracking (SPT) to investigate the impact of spatial inhomogeneities in membranes on lateral diffusion within Langmuir monolayers. Incontrast to previous SPT experiments, random walks of individual fluorescent tracer particles and the structure of the membrane are simultaneously imaged. Monolayers of 1,2-dimyristoyl-sn-glycero-2-phosphoethanolamine (DMPE) in the liquid condensed/liquid expanded phase are used to model the inhomogeneous plasma membrane, while the fluorescent particles embedded in the membrane serve as model proteins. The interactions between tracers and the domain boundary and the resulting change in their random walks are directly observed. Since this technique permits systematic SPT studies of diffusion in inhomogeneous monolayers, its extensions to more biologically relevant monolayers are laid out as well. DOI:10.1021/la034152u Journal Homepage

Langmuir, Volume 19, Issue 12, 4876-4879 (2003)

2002
 

D. Humphrey, C. Duggan, D. Saha, D. Smith, J. Käs:

Active fluidization of polymer networks through molecular motors

Abstract: Entangled polymer solutions and melts exhibit elastic, solid-like resistance to quick deformations and a viscous, fluid-like response to slow deformations. This viscoelastic behaviour reflects the dynamics of individual polymer chains driven by brownian motion: since individual chains can only move in a snake-like fashion through the mesh of surrounding polymer molecules, their diffusive transport, described by reptation, is so slow that the relaxation of suddenly imposed stress is delayed. Entangled polymer solutions and melts therefore elastically resist deforming motions that occur faster than the stress relaxation time. Here we show that the protein myosin II permits active control over the viscoelastic behaviour of actin filament solutions. We find that when each actin filament in a polymerized actin solution interacts with at least one myosin minifilament, the stress relaxation time of the polymer solution is significantly shortened. We attribute this effect to myosin's action as a 'molecular motor', which allows it to interact with randomly oriented actin filaments and push them through the solution, thus enhancing longitudinal filament motion. By superseding reptation with sliding motion, the molecular motors thus overcome a fundamental principle of complex fluids: that only depolymerization makes an entangled, isotropic polymer solution fluid for quick deformations. DOI:10.1038/416413a Journal Homepage

Nature, Volume 416, 413-416 (2002)

 

Jochen Guck, Revathi Ananthakrishnan, Casey C. Cunningham, Josef Käs:

Stretching biological cells with light

Abstract: The radiation pressure of two counter-propagating laser beams traps and stretches individual biological cells. Using non-focused laser beams, cells stay viable when irradiated with up to 1.4 W of 780 nm Ti-sapphire laser light for several minutes. Fluorescence microscopy has demonstrated that the essential features of the cytoskeleton, excluding stress fibres, are maintained for stretched cells in suspension. The optical stretcher provides accurate measurements of whole cell elasticity and thus can distinguish between different cells by their cytoskeletal characteristics. A model has been derived for the forces on the surface of a spherical cell that explains the observed deformations. The peak stresses on the surface of cells are 1-150 Pa for light powers of 0.2-1.4 W and depending on the refractive index of the cell trapped. Precursors of rat nerve cells exhibit a homogeneous Young's modulus E of 500±25 Pa, whereas for osmotically inflated, spherical red blood cells (RBCs) the homogeneous Young's modulus is E = 11.0±0.5 Pa. Thus, PC12 cells are about 40-50 times more elastic than RBCs. DOI:10.1088/0953-8984/14/19/311 Journal Homepage

Journal of Physics: Condensed Matter, Volume 14, Issue 19, 4843-4856 (2002)

 

A. Ehrlicher, T. Betz, B. Stuhrmann, D. Koch, V. Milner, M.G. Raizen, J. Käs:

Guiding neuronal growth with light

Abstract: Control over neuronal growth is a fundamental objective in neuroscience, cell biology, developmental biology, biophysics, and biomedicine and is particularly important for the formation of neural circuits in vitro, as well as nerve regeneration in vivo [Zeck, G. & Fromherz, P. (2001) Proc. Natl. Acad. Sci. USA 98, 10457–10462]. We have shown experimentally that we can use weak optical forces to guide the direction taken by the leading edge, or growth cone, of a nerve cell. In actively extending growth cones, a laser spot is placed in front of a specific area of the nerve's leading edge, enhancing growth into the beam focus and resulting in guided neuronal turns as well as enhanced growth. The power of our laser is chosen so that the resulting gradient forces are sufficiently powerful to bias the actin polymerization-driven lamellipodia extension, but too weak to hold and move the growth cone. We are therefore using light to control a natural biological process, in sharp contrast to the established technique of optical tweezers [Ashkin, A. (1970) Phys. Rev. Lett. 24, 156–159; Ashkin, A. & Dziedzic, J. M. (1987) Science 235, 1517–1520], which uses large optical forces to manipulate entire structures. Our results therefore open an avenue to controlling neuronal growth in vitro and in vivo with a simple, noncontact technique.  DOI:10.1073/pnas.252631899 Journal Homepage

PNAS, Volume 99, No. 25, 16024-16028 (2002)

 

Douglas S. Martin, Martin B. Forstner, Josef A. Käs:

Apparent Subdiffusion Inherent to Single Particle Tracking

Abstract: Subdiffusion and its causes in both in vivo and in vitro lipid membranes have become the focus of recent research. We report apparent subdiffusion, observed via single particle tracking (SPT), in a homogeneous system that only allows normal diffusion (a DMPC monolayer in the fluid state). The apparent subdiffusion arises from slight errors in finding the actual particle position due to noise inherent in all experimental SPT systems. A model is presented that corrects this artifact, and predicts the time scales after which the effect becomes negligible. The techniques and results presented in this paper should be of use in all SPT experiments studying normal and anomalous diffusion. DOI:10.1016/S0006-3495(02)73971-4 Journal Homepage

Biophysical Journal, Volume 83, Issue 4, 2109-2117 (2002)

2001
 

Jochen Guck, Revathi Ananthakrishnan, Hamid Mahmood, Tess J. Moon, C. Casey Cunningham, Josef Käs:

The Optical Stretcher: A Novel Laser Tool to Micromanipulate Cells

Abstract: When a dielectric object is placed between two opposed, nonfocused laser beams, the total force acting on the object is zero but the surface forces are additive, thus leading to a stretching of the object along the axis of the beams. Using this principle, we have constructed a device, called an optical stretcher, that can be used to measure the viscoelastic properties of dielectric materials, including biologic materials such as cells, with the sensitivity necessary to distinguish even between different individual cytoskeletal phenotypes. We have successfully used the optical stretcher to deform human erythrocytes and mouse fibroblasts. In the optical stretcher, no focusing is required, thus radiation damage is minimized and the surface forces are not limited by the light power. The magnitude of the deforming forces in the optical stretcher thus bridges the gap between optical tweezers and atomic force microscopy for the study of biologic materials. DOI:10.1016/S0006-3495(01)75740-2 Journal Homepage

Biophysical Journal, Volume 81, Issue 2, 767-784 (2001)

 

Martin B. Forstner, Josef Käs, Douglas Martin:

Single Lipid Diffusion in Langmuir Monolayers

Abstract: Individual lipid movement in a monolayer is studied over long time intervals (500 s) by darkfield microscopy of single lipids labeled with gold colloids (30 or 100 nm in diameter). Dimyristoyl phosphatidylcholine in the fluid phase shows normal diffusion, with a diffusion coefficient of (1.1 ± 0.2) × 10-8 cm2/s. Since this is consistent with values derived from the diffusive transport of many lipids, the analysis of gold-tagged lipids in a monolayer provides a reliable picture of lipid diffusion on the level of single molecules. DOI:10.1021/la000795n Journal Homepage

Langmuir, Volume 17, Issue 3, 567-570 (2001)

 

Elizabeth J. Furnish, Wei Zhou, Casey C. Cunningham, Josef A. Kas, Christine E. Schmidt:

Gelsolin overexpression enhances neurite outgrowth in PC12 cells

Abstract: The rational design of therapies for treating nerve injuries requires an understanding of the mechanisms underlying neurite extension. Neurite motility is driven by actin polymerization; however, the mechanisms are not clearly understood. One actin accessory protein, gelsolin, is involved with remodeling the cytoskeleton, although its role in cell motility is unclear. We report a two-fold upregulation of gelsolin upon differentiation with nerve growth factor. Cells that were genetically modified to overexpress gelsolin have longer neurites and a greater neurite motility rate compared to controls. These data suggest that gelsolin plays an important role in neurite outgrowth. DOI:10.1016/S0014-5793(01)03078-2 Journal Homepage

FEBS Letters, Volume 508, Issue 2, 282-286 (2001)

 

Jay X. Tang, Josef A. Käs, Jagesh V. Shah, Paul A. Janmey:

Counterion-induced actin ring formation

Abstract: Actin filaments form rings and loops when > 20 mM divalent cations are added to very dilute solutions of phalloidin-stabilized filamentous actin (F-actin). Some rings consist of very long single actin filaments partially overlapping at their ends, and others are formed by small numbers of filaments associated laterally. In some cases, undulations of the rings are observed with amplitudes and dynamics similar to those of the thermal motions of single actin filaments. Lariat-shaped aggregates also co-exist with rings and rodlike bundles. These polyvalent cation-induced actin rings are analogous to the toroids of DNA formed by addition of polyvalent cations, but the much larger diameter of actin rings reflects the greater bending stiffness of F-actin. Actin rings can also be formed by addition of streptavidin to crosslink sparsely biotinylated F-actin at very low concentrations. The energy of bending in a ring, calculated from the persistence length of F-actin and the ring diameter, provides an estimate for the adhesion energy mediated by the multivalent counterions, or due to the streptavidin-biotin bonds, required to keep the ring closed. DOI:10.1007/s002490100178 Journal Homepage

European Biophysics Journal, Volume 30, Issue 7, 477-484 (2001)

2000
 

R. E. Mahaffy, C. K. Shih, F. C. MacKintosh, J. Käs:

Scanning Probe-Based Frequency-Dependent Microrheology of Polymer Gels and Biological Cells

Abstract: A new scanning probe-based microrheology approach is used to quantify the frequency-dependent viscoelastic behavior of both fibroblast cells and polymer gels. The scanning probe shape was modified using polystyrene beads for a defined surface area nondestructively deforming the sample. An extended Hertz model is introduced to measure the frequency-dependent storage and loss moduli even for thin cell samples. Control measurements of the polyacrylamide gels compare well with conventional rheological data. The cells show a viscoelastic signature similar to in vitro actin gels. DOI:10.1103/PhysRevLett.85.880 Journal Homepage

Physical Review Letters, Volume 85, Issue 4, 880-883 (2000)

 

Jochen Guck, Revathi Ananthakrishnan, Tess J. Moon, C. Casey Cunningham, Josef Käs:

Optical Deformability of Soft Biological Dielectrics

Abstract: Two counterpropagating laser beams were used to significantly stretch soft dielectrics such as cells. The deforming forces act on the surface between the object and the surrounding medium and are considerably higher than the trapping forces on the object. Radiation damage is avoided since a double-beam trap does not require focusing for stable trapping. Ray optics was used to describe the stress profile on the surface of the trapped object. Measuring the total forces and deformations of well-defined elastic objects validated this approach. DOI:10.1103/PhysRevLett.84.5451 Journal Homepage

Physical Review Letters, Volume 84, Issue 23, 5451-5454 (2000)