A Highlights from MBoC Selection: Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit |
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Authors: | Emanuele Cocucci Rapha?l Gaudin Tom Kirchhausen |
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Institution: | National Institutes of Health;aDepartment of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children''s Hospital, Boston, MA 02115;bDepartment of Pediatrics, Harvard Medical School, Boston, MA 02115 |
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Abstract: | Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission. |
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