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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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.
|Digital Object Identifier (DOI):||doi:10.1083/jcb.201109130|
|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
|Date Deposited:||19 Sep 2012 10:25|
|Last Modified:||12 Aug 2015 15:33|
|RDF:||RDF+N-Triples, RDF+N3, RDF+XML, Browse.|
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