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The contributions of the actin machinery to endocytic membrane bending and vesicle formation.

ABSTRACT: Branched and cross-linked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analyzed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thus temporally dissect the effects of different actin network perturbations, revealing distinct stages of actin-based membrane reshaping. Our data show that initiation of membrane bending requires the actin network to be physically linked to the plasma membrane and to be optimally cross-linked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independent of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 s before scission, when the filament nucleation rate drops. From that time point on, invagination growth and vesicle scission are driven by an expansion of the actin network without a proportional increase of net actin amounts. The expansion is sensitive to the amount of filamentous actin and its cross-linking. Our results suggest that the mechanism by which actin reshapes the membrane changes during the progress of endocytosis, possibly adapting to varying force requirements.

SUBMITTER: Picco A

PROVIDER: S-EPMC5994895 | biostudies-other | 2018 Jun

REPOSITORIES: biostudies-other

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The contributions of the actin machinery to endocytic membrane bending and vesicle formation.

Picco Andrea A Kukulski Wanda W Manenschijn Hetty E HE Specht Tanja T Briggs John A G JAG Kaksonen Marko M

Molecular biology of the cell 20180410 11

Branched and cross-linked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analyzed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thus temporally dissect the effects of different actin network perturbations, revealing distinct stages of actin-based membrane reshaping. Our d ...[more]

PMID: 29851558

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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

The contributions of the actin machinery to endocytic membrane bending and vesicle formation.

Publications

The contributions of the actin machinery to endocytic membrane bending and vesicle formation.

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