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. The Journal of Cell Biology, 197, (4), 499-508. (doi:10.1083/jcb.201109130).

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Description/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.

Item Type: Article
ISSNs: 0021-9525 (print)
1540-8140 (electronic)
Related URLs:
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
Divisions: Faculty of Natural and Environmental Sciences > Biological Sciences
Faculty of Physical Sciences and Engineering > Optoelectronics Research Centre
ePrint ID: 342982
Date Deposited: 19 Sep 2012 10:25
Last Modified: 27 Mar 2014 20:25
URI: http://eprints.soton.ac.uk/id/eprint/342982

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