Quantitative Analysis of Approaches to Measure Cooperative Phosphate Release in Polymerized Actin

Abstract

We use stochastic simulations that treat several experimental probes of actin dynamics to explore the extent to which phosphate dissociation in filamentous actin may be cooperative. Phosphate time-courses from polymerization and copolymerization experiments of ATP- and ADP-actin are studied, including the effects of variations in filament-number concentration as well as single-filament depolymerization time-courses. We find that highly cooperative models are consistent with the treated experimental data. We also find that some types of experiments that are believed to provide strong constraints on the cooperativity of actin hydrolysis models do not provide such constraints.

Response to “On Phosphate Release in Actin Filaments”

“…Jégou et al disagree with some of the findings of our article (1). They argue that 1), a different value of the release rate r d can improve the fit to depolymerization dynamics obtained by the random release model; 2), plotting depolymerization dynamics of single filaments would be more appropriate than comparing an average to a single filament; and 3), data in their article (2) for the dependence of the phosphate time courses on initial actin concentration (during growth) supports the random release model.…”

Insights into the Cooperative Nature of ATP Hydrolysis in Actin Filaments

“…3), in which the rate of P i release at the interface is approximately the same as the average rate of P i release in the filament, characterized by a variation within 53% relative to the average rate. This is in contrast with the prediction of a strong cooperativity and the resulting implication of an enhancement in rate of P i release at the interface by a factor of 10 6 -10 8 relative to the average rate made in (44), although their analysis ignored any multibody effects beyond a simpler nearestneighbor cooperativity (44,48,49). Clearly, the nature of our predicted cooperativity does not show any signs in support of a purely vectorial ATP hydrolysis or P i release.…”

“…Fitting the predictions of a cooperative model to the time course of polymerization and ATP hydrolysis measured in these experiments eliminates the possibility of the strict vectorial model being accurate in all cases and suggests that the rate of hydrolysis at the ATP-ADP interface must be less than 100 times faster than the rate of hydrolysis away from the interface, although the predictions of a random hydrolysis mechanism were also shown to be able to explain the observed experimental data (42,46). By using the nearest-neighbor cooperativity in such a model as a parameter to fit experimentally measured P i release profiles, it was shown that the possibility of a high degree of cooperativity in P i release could not be completely excluded, however, with some ambiguity in the data analysis involved (41,44,48,49). The random and vectorial models are based on two simple microscopic physical hypotheses that are able to accurately explain the experimental observations.…”

“…In the strictest version of the vectorial model (35,43), at the most two interfaces (exactly two if the filament is growing from both its ends) can exist in a filament because hydrolysis is assumed to occur exclusively at the ATP/ADP interface and the interface simply moves toward the growing end of the filament as time progresses. The more realistic version of the vectorial model (cooperative model) assigns a relatively small nonzero hydrolysis rate for ATP subunits that are not present at the interface (8,44).…”

Theoretical Analysis of Microtubule Dynamics at All Times

“…1. 15,21 The hydrolysis mechanism for the cytoskeleton filament is still under discussion, [21][22][23][24][25][26][27][28][29][30] and we assume here that all T-subunits on microtubules can be hydrolyzed with the same rate r as indicated in Fig.1. The hydrolyzed D-subunits can detach from the "plus" end of the microtubules with a rate W D (see Fig.…”