An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy
0301 basic medicine
Dynamin II
Microscopy
03 medical and health sciences
COS Cells
Chlorocebus aethiops
Green Fluorescent Proteins
Animals
Coated Pits, Cell-Membrane
Research Articles
Clathrin
Endocytosis
DOI:
10.1083/jcb.201109130
Publication Date:
2012-05-08T06:51:53Z
AUTHORS (12)
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.
SUPPLEMENTAL MATERIAL
Coming soon ....
REFERENCES (40)
CITATIONS (76)
EXTERNAL LINKS
PlumX Metrics
RECOMMENDATIONS
FAIR ASSESSMENT
Coming soon ....
JUPYTER LAB
Coming soon ....