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: In agreement with earlier results (Figure 2D; Merrifield et al., 2002; Ehrlich et al., 2004; Macia et al., 2006; Doyon et al., 2011; Taylor et al., 2011, 2012), most (>90%) of the dynamin2-EGFP spots at the membrane colocalized with mCherry-LCa. Dynamin was generally recruited in two phases, also as previously described, although the distinction was not always as marked as in hCLTAEN/DNM2EN cells. In the first phase, which occurs during early stages of clathrin coat assembly, dynamin accumulated gradually to a plateau of variable magnitude. The second phase was the characteristic burst at the end of assembly, immediately before the loss of the clathrin signal associated with uncoating. Similar results were reported recently using the gene-edited hCLTAEN/DNM2EN and DNM2EN cells (Grassart et al., 2014). In view of the complete substitution in our cells and the very small proportion of dynamin 1, we used the maximum intensity of the total dynamin accumulation to determine the number of molecules recruited at the time of membrane scission. The distribution peaked at ∼40 dynamins and averaged at 50 ± 19 molecules (Figure 5A). Nearly 25% of the events contained between 26 and 40 dynamins, and ∼27% of the events, between 40 and 52 dynamins (Figure 5B).
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.