Abstract
Fluorescence live-cell imaging has temporally resolved the conserved choreography of more than 30 proteins involved in clathrin and actin-mediated endocytic budding from the plasma membrane. However, the resolution of these studies is insufficient to unveil how the endocytic machinery actually drives membrane deformation in vivo. In this study, we use quantitative immuno-EM to introduce the temporal dimension to the ultrastructural analysis of membrane budding and define changes in the topography of the lipid bilayer coupled to the dynamics of endocytic proteins with unprecedented spatiotemporal resolution. Using this approach, we frame the emergence of membrane curvature with respect to the recruitment of endocytic factors and show that constriction of the invaginations correlates with translocation of membrane-sculpting proteins. Furthermore, we show that initial bending of the plasma membrane is independent of actin and clathrin polymerization and precedes building of an actin cap branched by the Arp2/3 complex. Finally, our data indicate that constriction and additional elongation of the endocytic profiles require the mechanochemical activity of the myosins-I. Altogether, this work provides major insights into the molecular mechanisms driving membrane deformation in a cellular context.