期刊
JOURNAL OF CELL BIOLOGY
卷 197, 期 4, 页码 499-508出版社
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.201109130
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资金
- Biotechnology and Biological Sciences Research Council [BBD0208751]
- Medical Research Council [G0700926]
- British Heart Foundation [JG-NH/10/3/28574]
- Biotechnology and Biological Sciences Research Council [BB/D020816/1, C20199, BB/D020875/1] Funding Source: researchfish
- British Heart Foundation [NH/10/3/28574] Funding Source: researchfish
- Medical Research Council [G0700926, MC_U105178789] Funding Source: researchfish
- BBSRC [BB/D020875/1, BB/D020816/1] Funding Source: UKRI
- MRC [MC_U105178789, G0700926] Funding Source: UKRI
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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