title: Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit.
abstract: Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission.
authors.affiliation: Department of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115 Department of Pediatrics, Harvard Medical School, Boston, MA 02115.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Cocucci
authors.affiliation: Department of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115.
authors.forename: Raphaël
authors.initials: R
authors.lastname: Gaudin
authors.affiliation: Department of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115 Department of Pediatrics, Harvard Medical School, Boston, MA 02115 kirchhausen@crystal.harvard.edu.
authors.forename: Tom
authors.initials: T
authors.lastname: Kirchhausen
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism* / Q000648:ultrastructure; D034285:Dynamin II; D034281:Dynamins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004705:Endocytosis; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D002966:Clathrin; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C000593490:DNM2 protein, human; D034285:Dynamin II; D034281:Dynamins