Cocucci, EmanueleGaudin, RaphaëlKirchhausen, Tom2015-02-022014Cocucci, Emanuele, Raphaël Gaudin, and Tom Kirchhausen. 2014. “Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit.” Molecular Biology of the Cell 25 (22): 3595-3609. doi:10.1091/mbc.E14-07-1240. http://dx.doi.org/10.1091/mbc.E14-07-1240.1059-1524http://nrs.harvard.edu/urn-3:HUL.InstRepos:13890761Dynamin, 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.en-USMembrane TraffickingJournal Article2015-02-0210.1091/mbc.E14-07-1240