title: Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography.
abstract: Endocytosis, like many dynamic cellular processes, requires precise temporal and spatial orchestration of complex protein machinery to mediate membrane budding. To understand how this machinery works, we directly correlated fluorescence microscopy of key protein pairs with electron tomography. We systematically located 211 endocytic intermediates, assigned each to a specific time window in endocytosis, and reconstructed their ultrastructure in 3D. The resulting virtual ultrastructural movie defines the protein-mediated membrane shape changes during endocytosis in budding yeast. It reveals that clathrin is recruited to flat membranes and does not initiate curvature. Instead, membrane invagination begins upon actin network assembly followed by amphiphysin binding to parallel membrane segments, which promotes elongation of the invagination into a tubule. Scission occurs on average 9 s after initial bending when invaginations are ∼100 nm deep, releasing nonspherical vesicles with 6,400 nm2 mean surface area. Direct correlation of protein dynamics with ultrastructure provides a quantitative 4D resource.
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
authors.forename: Wanda
authors.initials: W
authors.lastname: Kukulski
authors.forename: Martin
authors.initials: M
authors.lastname: Schorb
authors.forename: Marko
authors.initials: M
authors.lastname: Kaksonen
authors.forename: John A G
authors.initials: JA
authors.lastname: Briggs
mesh_terms: D000199:Actins / Q000378:metabolism; D002462:Cell Membrane / Q000648:ultrastructure*; D055032:Electron Microscope Tomography; D004705:Endocytosis; D008954:Models, Biological; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000648:ultrastructure*; D022161:Transport Vesicles / Q000378:metabolism
chemical_list: D000199:Actins