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: Complete elimination of dynamin2 by disruption of all dynamin2 alleles showed that its presence is essential for budding of endocytic clathrin-coated vesicles (Liu et al., 2008). Accordingly, we found that dynamin depletion in SUM-Dyn2 cells by RNA interference (RNAi) treatment for 5 d hindered clathrin-dependent uptake of transferrin (Figure 8A). We inferred that depletion was incomplete because the fluorescence intensity of dynamin2-EGFP remaining in the cytosol was 25 ± 10% (average ± SD, 10 cells) and because a small fraction of dynamic pits remained that contained both clathrin and dynamin2-EGFP. Under these conditions of partial depletion, we asked what would be the number of dynamins required for coated-vesicle budding. We analyzed 97 events in 10 cells and found that ∼50% of the budding coated pits had no detectable recruitment during the first phase and on average recruited 28 ± 10 dynamins (Figure 8, B and C); the distribution of the total number of dynamins at the time of pinching (Figure 8, D and E) averaged at 32 ± 13 molecules. The results suggest that a very small number of dynamins in the first phase of recruitment is enough to allow the pit to mature and that 26–28 dynamins (one rung) are sufficient for 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.