An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy
| Autor: | Pavel Novák, David Klenerman, Max J. Lab, Pascale Guicheney, Andrew Shevchuk, Yuri E. Korchev, Marcus J. Taylor, Christien J. Merrifield, Azza Ziyadeh-Isleem, Julia Gorelik, Ivan Diakonov, Marc Bitoun |
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| Rok vydání: | 2012 |
| Předmět: |
Green Fluorescent Proteins
02 engineering and technology Endocytosis Clathrin Green fluorescent protein law.invention Dynamin II 03 medical and health sciences law Confocal microscopy Report Chlorocebus aethiops Microscopy Animals Research Articles 030304 developmental biology 0303 health sciences biology Conductance Coated Pits Cell-Membrane Cell Biology 021001 nanoscience & nanotechnology Fluorescence COS Cells Biophysics biology.protein Electron microscope 0210 nano-technology |
| Zdroj: | The Journal of Cell Biology The Journal of Cell Biology : JCB |
| ISSN: | 1540-8140 0021-9525 |
| DOI: | 10.1083/jcb.201109130 |
| Popis: | Simultaneous ion conductance and confocal microscopy in live cells reveal a new form of asymmetric clathrin-coated pit closure. 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. |
| Databáze: | OpenAIRE |
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