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 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 revealed 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 revealed 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. We 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.

• PMID: 37216532
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Journal Article | Research Support, N.I.H., Extramural | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Stoops EH, Ferrin MA, Jorgens DM, Drubin DG

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