An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy

DOI PDF 被引用文献19件
  • Andrew I. Shevchuk
    Division of Experimental Medicine, Department of Medicine, Imperial College London, London W12 0NN, England, UK 1
  • Pavel Novak
    Division of Experimental Medicine, Department of Medicine, Imperial College London, London W12 0NN, England, UK 1
  • Marcus Taylor
    Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, England, UK 4
  • Ivan A. Diakonov
    Department of Cardiovascular Sciences, National Heart and Lung Institute, Imperial College London, London SW3 6LY, England, UK 3
  • Azza Ziyadeh-Isleem
    Institut National de la Santé et de la Recherche Médicale, UMRS 956, Fondation ICAN, Paris, Cedex 13, France 5
  • Marc Bitoun
    UMR S974 Université Pierre et Marie Curie, U974 Institut National de la Santé et de la Recherche Médicale, UMR 7215 Centre National de la Recherche Scientifique, Institut de Myologie, 75013 Paris, France 7
  • Pascale Guicheney
    Institut National de la Santé et de la Recherche Médicale, UMRS 956, Fondation ICAN, Paris, Cedex 13, France 5
  • Max J. Lab
    Department of Cardiovascular Sciences, National Heart and Lung Institute, Imperial College London, London SW3 6LY, England, UK 3
  • Julia Gorelik
    Department of Cardiovascular Sciences, National Heart and Lung Institute, Imperial College London, London SW3 6LY, England, UK 3
  • Christien J. Merrifield
    Laboratoire d’Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France 8
  • David Klenerman
    Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, England, UK 9
  • Yuri E. Korchev
    Division of Experimental Medicine, Department of Medicine, Imperial College London, London W12 0NN, England, UK 1

説明

<jats:p>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.</jats:p>

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