Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3363.

Adviser: Enrique M. De La Cruz.

Actin polymerization is a fundamental cellular process involved in cell structure maintenance, force generation, and motility. Phosphate release from filament subunits following ATP hydrolysis destabilizes the filament lattice and increases the critical concentration (Cc) for assembly. The structural differences between ATP- and ADP-actin are still debated, as well as the energetic factors that underlie nucleotide-dependent filament stability, particularly under crowded intracellular conditions. I investigate the effect of crowding agents on ATP- and ADP-actin polymerization, and find that ATP-actin polymerization is largely unaffected by solution crowding, while crowding agents lower the Cc of ADP-actin in a concentration-dependent manner. The stabilities of ATP- and ADP-actin filaments are comparable in the presence of physiological amounts (∼30% w/v) and types (sorbitol) of low molecular weight crowding agents. Crowding agents act to stabilize ADP-F-actin by slowing subunit dissociation. These observations suggest that nucleotide hydrolysis and phosphate release per se do not introduce intrinsic differences in the in vivo filament stability. Rather, the preferential disassembly of ADP-actin filaments in cells is driven through interactions with regulatory proteins. Interpretation of the experimental data according to the binding-based osmotic stress theory implicates water as a regulator of actin activity and hydration as the molecular basis for nucleotide-dependent filament stability differences in dilute solution.

ADP-actin provides a model system with which to perform a comparison of crowding theories. Excluded volume theories consider only entropic effects from the reduced volume available to system components. Depletion attraction theory is unable to predict the effects of crowding, while scaled particle theory explains the trend but not the magnitude of the observed effects. Covolume theory performs well, suggesting that ADP-actin subunits do not undergo large conformational changes such as nucleotide cleft closure upon polymerization. Binding-based analyses consider enthalpic effects from "soft" interactions between solution components. Preferential interaction and osmotic stress theories invoke the interaction of specific residues with solvent as underlying the effect of crowding on ADP-actin polymerization.