Interdependence of Filament Nucleation, Elongation and Crosslinking in Actin Bundle Assembly

Abstract

A network of actin-binding proteins tightly regulates each stage of actin assembly, including filament nucleation, elongation and crosslinking. These processes are integrated together to assemble higher-order actin structures specialized for specific cellular roles. Actin bundles are composed of unbranched filaments that are crosslinked in parallel or anti-parallel orientations by bundling proteins. These structures form the basis of a number of cytoskeletal assemblies, including the contractile ring, stress fibers, and filopodia. Aberrant regulation of actin bundles and their associated proteins is linked to diseases such as focal segmental glomerulosclerosis and metastatic cancers. Yet despite their importance, the dynamic properties of actin bundles are not well understood. To gain insight into the mechanism of bundle assembly, we investigated how the processes of nucleation, elongation, and crosslinking influence each other. We utilized formins, which function as both nucleators and actin polymerases, to elucidate the interdependence between nucleation and elongation. We found that, whereas filament nucleation necessarily precedes and promotes filament elongation, the dependence of both processes on actin monomer availability gives rise to competition that ultimately favors nucleation over elongation. To investigate the relationship between elongation and crosslinking, we generated pools of actin filaments of uniform lengths and analyzed their kinetics of crosslinking by the bundling protein fascin. We found that fascin crosslinks short filaments more slowly than long filaments, suggesting that filament elongation plays a role in regulating filament bundling. We propose that elongating filaments undergo changes in curvature and rotational diffusion that modulate the probability of filament alignment and ultimately dictate the efficiency of fascin-mediated crosslinking. This work suggests that upregulation of fascin might alter the mechanical properties of bundled actin structures by promoting the incorporation of short filaments.