An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy

• DOI: 10.1083/jcb.201109130
• Publication Date: 2012-05-08T06:51:53Z
• Authors: Andrew I. Shevchuk; Pavel Novak; Marcus Taylor; Ivan A. Diakonov; Azza Ziyadeh-Isleem; Marc Bitoun; Pascale Guicheney; Max J. Lab; Julia Gorelik; Christien J. Merr...; David Klenerman; Yuri E. Korchev
• Keywords: 0301 basic medicine; Dynamin II; Microscopy; 0303 health sciences; 03 medical and health sciences; COS Cells; Chlorocebus aethiops; Green Fluorescent Proteins; Animals; Coated Pits, Cell-Membrane; Research Articles; Clathrin; Endocytosis

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.