biostudies-literatureShevchuk AIBritish Heart FoundationMedical Research CouncilBiotechnology and Biological Sciences Research Council499-508https://www.ebi.ac.uk/biostudies/studies/S-EPMC3352948biostudies-literatureUnknown197(4)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.The Journal of cell biologyAn alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy.PMC3352948MC_U105178789JG-NH/10/3/28574NH/10/3/28574BB/D020816/1BBD0208751G0700926C20199BB/D020875/1Ziyadeh-Isleem AMerrifield CJNovak PLab MJKorchev YEDiakonov IABitoun MGorelik JShevchuk AIGuicheney PTaylor MKlenerman DfalseAn alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy.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.2012-01-01T00:00:00Z2012 May2022-02-09T10:44:19.458Z2019-03-27T00:53:31ZS-EPMC33529482256441610.1083/jcb.201109130