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Publications Journal
Articles
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.
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| APL Bioengineering accepted (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.
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| 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.
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| 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.
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| Glycobiology, April 1;34(3) (2024) |
2023
| 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.
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| 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.
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| 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.
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| 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.
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| 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.
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| 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.
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| 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.
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| 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.
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| Nanoscale, Advance Article (2023) |
2022
| 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.
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| 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.
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| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| Polymers 2022, 14(3), 614 |
2021
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| Physical Review X, 11, 011033 (2021) |
2020
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| Jahrgang 2020, Seiten 53-57, ISSN: 2195-0334 |
2019
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| ACS Appl. Bio Mater., (2019) 2,3, 1066-1077 |
2018
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.”
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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: -
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| 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.
|
| Physical Review Letters, Volume 84, Issue 23, 5451-5454
(2000) |
|
|
