Deciphering Protein Dynamics during Endocytic Budding by Time Resolved Electron Microscopy

Trabajo presentado en el 28th Annual Symposium of The Protein Society, celebrado en San Diego, California, Estados Unidos, del 27 al 30 de julio de 2014Fluorescence 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 spatial resolution of these studies is insufficient to unveil subtle changes in the topography of the lipid bilayer coupled to the recruitment or reorganization of the endocytic machinery. In this study, we use quantitative immuno-electron microscopy to introduce the temporal dimension to the ultrastructural analysis of membrane budding. Our data define functional modules with unprecedented spatio-temporal resolution, frame the emergence of membrane curvature with respect to the recruitment of endocytic factors and demonstrate the constriction of the invaginations coupled to the translocation of membrane sculpting proteins. Further, we show that initial bending of the PM is independent of actin and clathrin polymerization and precedes building of a 70 nm actin cap branched by the Arp2/3 complex and crosslinked by fimbrin. Finally our data indicates that further elongation of the endocytic profiles and constriction of the invagination neck requires the mechanochemical activity of the myosins-I. Altogether, this work provides major insights into the molecular mechanisms driving membrane deformation in a cellular context.Peer Reviewe