An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy |
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Authors: | Shevchuk Andrew I Novak Pavel Taylor Marcus Diakonov Ivan A Ziyadeh-Isleem Azza Bitoun Marc Guicheney Pascale Lab Max J Gorelik Julia Merrifield Christien J Klenerman David Korchev Yuri E |
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Affiliation: | Division of Experimental Medicine, Department of Medicine, Imperial College London, London W12 0NN, England, UK. |
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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. |
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