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

Shevchuk, A.I., Novak, P., Taylor, M., Diakonov, I.A., Ziyadeh-Isleem, A., Bitoun, M., Guicheney, P., Lab, M.J., Gorelik, J., Merrifield, C.J., Klenerman, D. and Korchev, Y.E. (2012) An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy PloS one, 197, (4), pp. 499-508. (doi:10.1083/jcb.201109130).

Record type: Article

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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Published date: May 2012
Organisations: Optoelectronics Research Centre, Centre for Biological Sciences

Identifiers

Local EPrints ID: 342982
URI: http://eprints.soton.ac.uk/id/eprint/342982
ISSN: 1932-6203
PURE UUID: 230e59f8-4ec2-4bc9-a455-eb903d977407

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Date deposited: 19 Sep 2012 10:25
Last modified: 18 Jul 2017 05:26

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Contributors

Author: A.I. Shevchuk
Author: P. Novak
Author: M. Taylor
Author: I.A. Diakonov
Author: A. Ziyadeh-Isleem
Author: M. Bitoun
Author: P. Guicheney
Author: M.J. Lab
Author: J. Gorelik
Author: C.J. Merrifield
Author: D. Klenerman
Author: Y.E. Korchev

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