Interdependence of Nucleation, Elongation, and Bundling in the Assembly of Crosslinked Actin Structures

Abstract

Remodeling of the actin cytoskeleton drives essential cell processes like morphogenesis, motility, and cytokinesis. A network of actin-binding proteins tightly regulates each stage of actin dynamics, leading to the coordinated assembly of higher-order actin structures that are specialized for specific cellular roles. Many of these cytoskeletal assemblies are made from unbranched actin filaments that are crosslinked into actin bundles or meshworks. Cells generate unbranched filaments using proteins called formins, which control both actin filament nucleation and elongation. After filaments are formed, they are held together in specific geometries by crosslinkers like fascin. Within the dynamic cellular environment, the processes of nucleation, elongation, and crosslinking take place concurrently. Thus, to understand the assembly of actin structures, we need to know how these processes are integrated together. To investigate this interdependence, I developed TIRF microscopy-based assays and computational image analysis pipelines to visualize, quantify, and characterize reconstituted actin filaments and crosslinked actin structures. I first employed these tools to dissect the contributions of formin-mediated nucleation and elongation to actin filament assembly, finding that the dependence of filament length on the elongation rate is limited by formin’s nucleation activity. I next explored how elongation influences fascin-mediated bundling during the assembly of crosslinked actin structures. I found that bundling of filaments earlier in elongation prevents discrete bundles from merging together to form interconnected meshworks. These results indicate that uncoordinated filament elongation and crosslinking can alter the architecture of bundled actin networks. Taken together, these studies offer important insight into the integration of nucleation, elongation, and crosslinking and provide a framework for future investigations into the assembly of specialized actin structures.