Self-organizing actin networks drive sequential endocytic protein recruitment and vesicle release on synthetic lipid bilayers.

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, we show that supported lipid bilayers coated with purified yeast WASP, an endocytic actin assembly regulator, and incubated in cytoplasmic yeast extracts, recruit downstream endocytic proteins and assemble actin tails. Time-lapse imaging of WASP-coated bilayers revealed sequential recruitment of proteins from different endocytic modules, faithfully replicating Significance Statement: Actin filament assembly participates in many vesicle-forming processes. However, the underlying principles for how assembly is initiated and organized to effectively harness assembly forces remain elusive. To address this gap, we report a novel reconstitution of actin-driven vesicle release from supported lipid bilayers. Using real-time imaging, we observe sequential recruitment of endocytic proteins and, following a burst of actin assembly, vesicle release from bilayers. Given the absence of cargo or upstream endocytic regulatory proteins on the bilayers, and the participation of actin in many vesicle-forming processes, we posit that this mode of vesicle formation represents an early evolutionary precursor for multiple trafficking pathways. We expect that this assay will be of great use for future investigations of actin-mediated vesicle-forming processes.