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 and Applied Science > Optoelectronics Research Centre |
| Item ID: | 342982 |
| Date Deposited: | 19 Sep 2012 10:25 |
| Last Modified: | 26 Apr 2013 07:02 |
| Contributors: | Shevchuk, A. I. (Author) Novak, P. (Author) Taylor, M. (Author) Diakonov, I. A. (Author) Ziyadeh-Isleem, A. (Author) Bitoun, M. (Author) Guicheney, P. (Author) Lab, M. J. (Author) Gorelik, J. (Author) Merrifield, C. J. (Author) Klenerman, D. (Author) Korchev, Y. E. (Author) |
| Date: | May 2012 |
| Status: | Published |
| URI: | http://eprints.soton.ac.uk/id/eprint/342982 |
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