Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit.
Bottom Line: The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit.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.
Affiliation: Department of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115 Department of Pediatrics, Harvard Medical School, Boston, MA 02115.Show MeSH
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Mentions: Using a TIRF configuration calibrated from single-molecule counting as previously described (Cocucci et al., 2012), we followed the accumulation of dynamin2-EGFP into coated pits at the bottom surface of SUM-Dyn2 cells. We acquired a rapid time series lasting ∼20 s, with 60-ms exposure/frame at an interval of 110 ms. This protocol minimized photobleaching to not more than 20% toward the end of the time series. We analyzed 23 traces from five cells selected to have relatively low amplitude and therefore show phase 1 of dynamin recruitment. We found consecutive stepwise intensity increments of variable magnitude and a dwell time of 1.06 ± 0.57 s (Figure 9A). The number of dynamin2-EGFP molecules recruited during each of the steps was obtained by fitting the intensity distributions with different models that took into account the dynamin2 substitution by dynamin2-EGFP (98%) and alternative contributions of monomer, dimer, tetramer, and so on of dynamin2-EGFP during the steps of recruitment (see Materials and Methods).
Affiliation: Department of Cell Biology, Harvard Medical School, and Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115 Department of Pediatrics, Harvard Medical School, Boston, MA 02115.