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

Branched and crosslinked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analysed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thereby 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 crosslinked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independently of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 seconds 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 assembled actin network. The expansion is sensitive to the amount of filamentous actin and its crosslinking. Our results suggest that the mechanism by which actin reshapes the membrane adapts to force requirements that vary during the progress of endocytosis.