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.
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.
Pattern Formation of Actin Filaments
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.
In the Presence of the Molecular Motor Myosin II
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
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.