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Lipid-mediated PX-BAR domain recruitment couples local membrane constriction to endocytic vesicle fission.

ABSTRACT: Clathrin-mediated endocytosis (CME) involves membrane-associated scaffolds of the bin-amphiphysin-rvs (BAR) domain protein family as well as the GTPase dynamin, and is accompanied and perhaps triggered by changes in local lipid composition. How protein recruitment, scaffold assembly and membrane deformation is spatiotemporally controlled and coupled to fission is poorly understood. We show by computational modelling and super-resolution imaging that phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂] synthesis within the clathrin-coated area of endocytic intermediates triggers selective recruitment of the PX-BAR domain protein SNX9, as a result of complex interactions of endocytic proteins competing for phospholipids. The specific architecture induces positioning of SNX9 at the invagination neck where its self-assembly regulates membrane constriction, thereby providing a template for dynamin fission. These data explain how lipid conversion at endocytic pits couples local membrane constriction to fission. Our work demonstrates how computational modelling and super-resolution imaging can be combined to unravel function and mechanisms of complex cellular processes.

SUBMITTER: Schoneberg J

PROVIDER: S-EPMC5481832 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Lipid-mediated PX-BAR domain recruitment couples local membrane constriction to endocytic vesicle fission.

Schöneberg Johannes J Lehmann Martin M Ullrich Alexander A Posor York Y Lo Wen-Ting WT Lichtner Gregor G Schmoranzer Jan J Haucke Volker V Noé Frank F

Nature communications 20170619

Clathrin-mediated endocytosis (CME) involves membrane-associated scaffolds of the bin-amphiphysin-rvs (BAR) domain protein family as well as the GTPase dynamin, and is accompanied and perhaps triggered by changes in local lipid composition. How protein recruitment, scaffold assembly and membrane deformation is spatiotemporally controlled and coupled to fission is poorly understood. We show by computational modelling and super-resolution imaging that phosphatidylinositol 3,4-bisphosphate [PI(3,4) ...[more]

PMID: 28627515

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Project description: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 actinâ€™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 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.Significance statementActin 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.

Dataset Information

Lipid-mediated PX-BAR domain recruitment couples local membrane constriction to endocytic vesicle fission.

Publications

Lipid-mediated PX-BAR domain recruitment couples local membrane constriction to endocytic vesicle fission.

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