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: We do not know whether the gradually accumulated dynamin contributes to scission. To estimate the number of dynamins specifically recruited during the burst phase, we subtracted the maximum fluorescence from the plateau 10 s before the peak of the burst. The values plotted, as a histogram and as a cumulative distribution in Figure 5, C and D, showed a peak at 26–28 dynamins with an average of 33 ± 14 dynamins. In the low-intensity, abortive pits (events lasting <25 s), we saw no dynamin burst and detected only small and variable amounts of phase one dynamin recruitment (Figure 5, E and F). We obtained similar results from hCLTAEN/DNM2EN cells (Figure 6). Because substitution of dynamin2 by dynamin2-EGFP was only 50% in those cells, the agreement supports the assumption of random incorporation of tagged and untagged dynamin molecules in a ratio depending only on their relative concentrations in the cytosol.
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.