Emergent Mechanics of Endocytic Actin Networks

Forces generated by actin assembly assist membrane invagination during clathrin-mediated endocytosis (CME). The sequential recruitment of core endocytic proteins and regulatory proteins, and assembly of the actin network, are well documented in live cells and are highly conserved from yeasts to humans. However, understanding of CME protein self-organization, as well as the biochemical and mechanical principles that underlie actin’s role in CME, is lacking. Here, I describe two studies revealing potential mechanistic explanations for how actin and associated proteins robustly organize for productive force generation during CME./nI first helped to construct and analyze an experimentally constrained multiscale model showing that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension. The model predicts that around 200 activated Arp2/3 complexes are required for robust internalization, which was confirmed by experiments in live cells. Simulations reveal that actin self-organizes into a radial branched array with growing ends oriented toward the base of the pit. Long actin filaments bend between attachment sites in the coat and the base of the pit. Elastic energy stored in bent filaments, whose presence was confirmed experimentally, contributes to endocytic internalization. Elevated membrane tension directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints./nI then developed an experimental system and analysis strategies to show that supported lipid bilayers coated with purified yeast Wiskott Aldrich Syndrome Protein (WASP), an endocytic actin assembly regulator, and incubated in cytoplasmic yeast extracts, recruit downstream endocytic proteins and assemble actin networks. Time-lapse imaging of WASP-coated bilayers reveal sequential recruitment of proteins from different endocytic modules, faithfully replicating in vivo behavior. Reconstituted actin networks assemble in a WASP-dependent manner and deform lipid bilayers, as seen by electron microscopy. Time-lapse imaging reveals that vesicles are released from the lipid bilayers with a burst of actin assembly. Actin networks pushing on membranes have previously been reconstituted; here, we have reconstituted a biologically important variation of these actin networks that self-organize on bilayers and produce pulling forces sufficient to bud off membrane vesicles. I propose that actin-driven vesicle generation may represent an ancient evolutionary precursor to diverse vesicle forming processes adapted for a wide array of cellular environments and applications./nThese studies align with the mission of the nascent field of emergent mechanics: to understand new mechanical properties that arise from collective interactions among a system’s building blocks. With these results I demonstrate pathways by which the building blocks of actin networks can self-organize into mechanically adaptive, higher-order structures to generate the forces necessary to carry out CME.

Advisor: Drubin, David