A CORRELATIVE ANALYSIS OF ACTIN FILAMENT ASSEMBLY, STRUCTURE, AND DYNAMICS

The effect of the type of metal ion (i.e., Ca2+, Mg2+, or none) bound to the high-affinity divalent cation binding site (HAS) of actin on fi lament assembly, structure, and dynamics was investigated in the absen ce and presence of the mushroom toxin phalloidin. In agreement with ea rlier reports, we found the polymerization reaction of G-actin into F- actin filaments to be tightly controlled by the type of divalent catio n residing in its HAS. Moreover, novel polymerization data are present ed indicating that LD, a dimer unproductive by itself, does incorporat e into growing F-actin filaments. This observation suggests that durin g actin filament formation, in addition to the obligatory nucleation-c ondensation pathway involving UD, a productive filament dimer, a facul tative, LD-based pathway is implicated whose abundance strongly depend s on the exact polymerization conditions chosen. The ''ragged'' and '' branched'' filaments observed during the early stages of assembly repr esent a hallmark of LD incorporation and might be key to producing an actin meshwork capable of rapidly assembling and disassembling in high ly motile cells, Hence, LD incorporation into growing actin filaments might provide an additional level of regulation of actin cytoskeleton dynamics. Regarding the structure and mechanical properties of the F-a ctin filament at steady state, no significant correlation with the div alent cation residing in its HAS was found, However, compared to nativ e filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes, Together, our data indicate that whereas the G-actin conformation is tightly controlled by the di valent cation in its HAS, the F-actin conformation appears more robust than this variation, Hence, we conclude that the structure and dynami cs of the Mg-F-actin moiety within the thin filament are not significa ntly modulated by the cyclic Ca2+ release as it occurs in muscle contr action to regulate the actomyosin interaction via troponin.