Spatio-temporal properties of membrane-localized actin nucleating complexes

The actin cytoskeleton plays a vital role in various biological processes such as cell migration, morphogenesis, and intracellular trafficking. The polymerization of actin filaments at membranes provides the force for generating dynamic actin structures such as protrusions and invaginations that drive these processes. In filopodia, which are finger-like protrusions comprised of bundled actin filaments, actin regulatory proteins are believed to assemble a distal 'tip complex' which stimulates actin nucleation at the membrane. However how these regulators collectively behave in a macromolecular complex still remains poorly understood. To understand the macromolecular nature of these complexes, I investigated the dynamic properties and spatial organization of actin regulatory factors, using an in vitro reconstitution assay for filopodia-like structures (FLS) utilizing artificial lipid bilayers and Xenopus laevis egg extracts. FRAP analysis of seven actin regulatory factors (Toca-1, N-WASP, GTPase-binding domain, Ena, VASP, Diaph3, Fascin) revealed that the FLS tip complex has both dynamic and stable properties, with different proteins displaying distinct dynamics. Further analyses on the membrane-binding protein Toca-1 showed that its dynamic turnover is controlled by interactions with actin and exchange of molecules with solution. Single-molecule localization microscopy resolved the nanoscale organization of Toca-1, showing its arrangement into flat plaque-like and narrowly elevated tubular substructures. Plaque-like structures showed similarities to phase-transition patterns, while tubule-like structures closely resembled those previously found to decorate membrane tubules in vitro, which are thought to be involved in endocytic membrane remodeling. Endocytic accessory proteins such as SNX9 and Dynamin2 were also found to localize to FLS tips. This work provides new insights into the dynamics and organization of protein ensembles at actin nucleation sites, and proposes a novel link between endocytosis and filopodia formation, which is relevant to understanding how cells decide when and where to assemble actin at the membrane