Effects of solution crowding on actin polymerization reveal the energetic basis for nucleotide-dependent filament stability.
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 (C(c)) 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. Here, we 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 C(c) of ADP-actin in a concentration-dependent manner. The stabilities of ATP- and ADP-actin filaments are comparable in the presence of physiological amounts (approximately 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 osmotic stress theory implicates water as an allosteric regulator of actin activity and hydration as the molecular basis for nucleotide-dependent filament stability.
Frederick
Kendra B
KB
Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 208114, New Haven, CT 06520-8114, USA.
Sept
David
D
De La Cruz
Enrique M
EM
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D012996:Solutions; D013499:Surface Properties; D013816:Thermodynamics
D012996:Solutions; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate