Soft Matter Physics Division - Biophysics at the University of Leipzig University of Leipzig
IntroductionActin Dynamics and Kinetics
(Figure from Julie Hodgkinson, Imperial College School of Medicine, National Heart and Lung Institute)
Actin is a semi-flexible polymer found as a part of the cytoskeleton of cells in higher biological organisms. Its semi-flexible nature means that the typical lengths of the filaments in solution are on the order of its persistence length, LP, or the distance over which the angles of the ends become uncorrelated. In simple terms, this means the persistence length represents the distance over which bending at one end does not affect the other end. In vitro, persistence length of actin filaments is on the order of 15 µm.

Actin filaments consist of repeating subunits of actin monomers in a right-handed double helical structure. The individual units or G-actin (globular actin) are 43 kD in size, and have a diameter of approximately 5 nm. The monomers have several subunits, and an internal cleft which can bind ATP and Magnesium ions. Each monomer has an asymmetrical structure, giving polymerized actin filaments a "polarity", with the ends referred to as the "pointed" (or minus) and "barbed" (or plus) ends.

Actin monomers move freely throughout solution according to Brownian diffusion. At a critical concentration of 0.1 mg/ml, the monomers begin to form stable nuclei consisting of 3-4 individual units. At this point, the filaments begin their elongation phase, where free monomers add to both sides of the filament, hydrolyzing ATP and releasing inorganic phosphate in the process. While filaments add to both sides of the filaments, they do not add with equal rates – the barbed, or plus end is the fast-growing end, while the pointed, or minus end is the slow-growing end.
(Figures from Joyce J. Diwan, Rensselaer Polytechnic Institute)

(Figure from Professor Daniel L. Purich, Department of Biochemistry & Molecular Biology, University of Florida College of Medicine)


The polymerization rate depends largely on the concentration of the monomer pool, so a balance is eventually reached between these two factors, and the filaments enter a steady-state phase. In this state, the filaments undergo a process referred to as "treadmilling", whereby there is a constant polymerization and depolymerization of the filaments. On average, the filaments polymerize at their barbed ends and depolymerize at their pointed ends, so there is a net transport of monomers from the barbed to pointed ends. This process is a non-equilibrium effect as ATP is being constantly hydrolyzed and the system continually remains in a dissipative state.
(Treadmilling movie from Josef A. Käs, University of Leipzig.)

(Figure from Josef A. Käs, University of Leipzig)
Individual actin filaments are only approximately 6 nm in width, several orders of magnitude less than the minimal resolution of a light microscope (~200 nm). As a result, ordinary light microscopy methods are not used for visualization of actomyosin networks in solution. Techniques previously developed to visualize single actin filaments through fluorescent microscopy in F-actin networks (Käs, 1994) are frequently utilized to microscopically investigate actin-myosin solutions. This involves labeling each actin monomer with a rhodamine molecule bound to an antibody raised against rabbit skeletal muscle actin. The rhodamine is excited by green light and fluoresces brightly red. In this way, each monomer on the actin filament (approximately 4000 monomers per 10 µm filament) acts as a pinhole emitting light, thus making individual filaments visible through fluorescent microscopy.

Pattern Formation of Actin Filaments

Actin filaments pose an interesting and difficult challenge for contemporary polymer physics. They are semi-flexible, with a bending stiffness much greater than that of flexible biopolymers such as DNA, but significantly more flexible than rigid rod-like macromolecules such as Microtubules. Being relatively rigid, actin polymers can undergo strong steric interactions with one another, and concentration -dependent phase transitions occur in which the system partially enters a liquid crystalline phase. We have seen evidence of an even higher degree of ordering characterized by large scale spatial structuring. We are currently using a variety of microscopy techniques, including fluorescence, confocal, laser scanning microscopy and a novel polarization device to investigate the appearance of a concentration- dependent order parameter in the system. Our aim is to characterize both patterns and the parameter space in which they appear.
There is an additional interesting aspect to these actin systems known as treadmilling. This is a process due to actin's unique reaction kinetics whereby individual monomers are steadily cycled through a filament which maintains an overall constant length. This is an energy dissipative process and assures that the system cannot attain thermodynamic equilibrium. We are currently investigating the role that this nonequilibrium process might play in the formation of the spatial structures. Perhaps this will shed some light on the role of treadmilling in cells, which as of yet remains a mystery.

In the Presence of the Molecular Motor Myosin II

All eukaryotic cells depend upon mechanisms of protein filament self-assembly to form their cytoskeletons. The cell's need for motility and rapid response to stimuli additionally require the existence of pathways which serve to restructure and disassemble cytoskeletal elements. While temperature-driven increases in disorder are the most physically fundamental methods for breaking down complex structures, they would compromise the cell's viability. Molecular machinery provides a non-destructive means to accomplish the same goals. This method is utilized on the genetic level with the unfolding of DNA strands for replication and cell division. Molecular machinery unfolds the DNA strands without heat-induced damage to the cell, providing an alternative to temperature-driven methods (Lodish et al, 2000). We have observed experimental evidence of a similar mechanism functioning on actin cytoskeletal dynamics, involving collections of the actin-specific molecular motor Myosin II. Crosslink-driven bundling self-assembles complex actomyosin structures in the near-chemical-equilibrium state, including bundles, asters, and large aggregates. Activation of the motors, however, causes a rapid disassembly of all structures. Such a mechanism is not only harmless to cell function, but occurs on a very rapid timescale which is favorable for quick cytoskeletal dynamics.

Other Actin Dynamics Experiments

We are investigating the effect of cytoplasm contents on the bending stiffness of individual filaments. From these experiments we hope to gain a more accurate picture of the stiffness of actin filaments in vivo, as this property is crucial to the integrity of the actin cytoskeleton. We are also using torsion pendulum rheometry to explore bulk properties of actin networks under a variety of conditions, including in the presence of cross-linking proteins. We hope that these in vitro investigations will shed new light on the dynamic properties of the actin cytoskeleton.

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