Ultrastructural dynamics of proteins involved in endocytic budding

Fluorescence live-cell imaging has temporally resolved the conserved choreography ofmore 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 ofmembrane 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 ofmembrane-sculpting proteins. Furthermore,we showthat 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 themechanochemical activity of themyosins-I. Altogether, this work provides major insights into the molecularmechanisms driving membrane deformation in a cellular context.This work was supported by Grants MTM2009-10893 (to A.E.), and BFU2008-03500, BFU2011-30185, and CSD2009-00016 (to M.I.G.) from the Spanish Government.Peer Reviewe