title: An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy.
abstract: Current knowledge of the structural changes taking place during clathrin-mediated endocytosis is largely based on electron microscopy images of fixed preparations and x-ray crystallography data of purified proteins. In this paper, we describe a study of clathrin-coated pit dynamics in living cells using ion conductance microscopy to directly image the changes in pit shape, combined with simultaneous confocal microscopy to follow molecule-specific fluorescence. We find that 70% of pits closed with the formation of a protrusion that grew on one side of the pit, covered the entire pit, and then disappeared together with pit-associated clathrin-enhanced green fluorescent protein (EGFP) and actin-binding protein-EGFP (Abp1-EGFP) fluorescence. This was in contrast to conventionally closing pits that closed and cleaved from flat membrane sheets and lacked accompanying Abp1-EGFP fluorescence. Scission of both types of pits was found to be dynamin-2 dependent. This technique now enables direct spatial and temporal correlation between functional molecule-specific fluorescence and structural information to follow key biological processes at cell surfaces.
authors.affiliation: Division of Experimental Medicine, Department of Medicine, Imperial College London, London W12 0NN, England, UK.
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Shevchuk
authors.forename: Pavel
authors.initials: P
authors.lastname: Novak
authors.forename: Marcus
authors.initials: M
authors.lastname: Taylor
authors.forename: Ivan A
authors.initials: IA
authors.lastname: Diakonov
authors.forename: Azza
authors.initials: A
authors.lastname: Ziyadeh-Isleem
authors.forename: Marc
authors.initials: M
authors.lastname: Bitoun
authors.forename: Pascale
authors.initials: P
authors.lastname: Guicheney
authors.forename: Max J
authors.initials: MJ
authors.lastname: Lab
authors.forename: Julia
authors.initials: J
authors.lastname: Gorelik
authors.forename: Christien J
authors.initials: CJ
authors.lastname: Merrifield
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
authors.forename: Yuri E
authors.initials: YE
authors.lastname: Korchev
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D034285:Dynamin II / Q000378:metabolism; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008853:Microscopy
chemical_list: D002966:Clathrin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D034285:Dynamin II