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Actin Growth Kinetics and Actin, one of the most abundant proteins in eukaryotic cells, readily assembles into long double stranded helical filaments. Cells continuously control the growth and shrinkage of actin filament networks in order to perform tasks crucial for their survival such as cell motion, cell division, and phagocytosis. These cellular processes are very complex and involve a cascade of convoluted biochemical and biophysical events whose result is the formation of specialized actin structures. In a systematic effort to deconvolute these cellular processes into their essential components, an enormous body of experimental work has focused on the properties of purified solutions of actin and other actin-binding proteins. Our goal is to theoretically interpret the results of these experiments in order to extract detailed mechanistic information from complex data. Our theoretical work also addresses the dynamic integration of these steps into coherent cellular tasks. A major objective is to unravel mechanisms underlying cell motion.
GROWTH RATES OF SINGLE FILAMENTS Each actin monomer binds ATP whose hydrolysis into ADP and phosphate (Pi) provides the free energy which maintains actin networks far from equilibrium in vivo. It is well established that in the presence of ATP actin monomers are added to filament ends as ATP-actin. Rapidly the ATP is then hydrolyzed, resulting in ADP-Pi-actin. After a long delay phosphate is released into solution generating ADP-actin which is less stable than the other species, i.e. its tendency to depolymerize is greater. These events generate a 3-species “cap” at each end of a filament. The cap dynamics lead to a peculiar dependence of the filament growth rate, j, on actin monomer concentration, c.
We have studied theoretically how the changing cap structure leads to distinct non-linear regimes in the j(c) curves and we compared our analytical and numerical results to measurements of growth rates. It is well known that detailed balance is invalid for actin growth due to irreversible ATP hydrolysis and thus the values of the critical concentration (where the growth rate vanishes) are different at the two filament ends. This difference between the two ends leads to treadmilling of single filaments in vitro. The precise values of the critical concentration depend on the structure of the cap. Our work leads to insights and testable conclusions as to why the critical concentration at the pointed end can be six times larger than that of the barbed end.
Fluctuations IN GROWTH and DYNAMIC INSTABILITY Beyond the average growth rates, fluctuations around the mean reveal important information on actin growth kinetics, unavailable from measurements of j(c). Fluctuations are measured by the length “diffusion” coefficient, D. Using total internal reflection microscopy, Fujiwara et al. (Nature Cell Biol. (2002) 4 666) observed unexpectedly large length fluctuations at steady state, provoking speculation that growth may be governed by oligomeric rather than the standard monomeric on-off events. Using the single monomer picture, our analysis of the full D(c) curve showed that fluctuations are large near and below the critical concentration due to cap loss events which lead to filaments alternating between growth and shrinkage. This is a much milder version of the dynamic instability of microtubule polymerization. Future measurements of the full D(c) curve will help to test conclusively the origin of large fluctuations observed experimentally.
Click the following image to see a movie of a Monte Carlo simulation of actin growth showing large length fluctuations.
References (click to download) Vavylonis, Yang, and O’Shaughnessy, Proc. Natl. Acad. Sci. USA, 102, 8543-8548 (2005). |
