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Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit.

Cocucci E, Gaudin R, Kirchhausen T - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

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.

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Number of dynamin molecules recruited to coated pits at the time of membrane fission in hCLTAEN/DNM2EN cells. The number of dynamin molecules recruited to clathrin-coated structures was obtained as described in Figure 5 from three cells. The substitution of endogenous dynamin2 by dynamin2-EGFP in hCLTAEN/DNM2EN cells was ∼50%. (A) Histogram of the total number of dynamin molecules recruited to coated pits at the time of membrane fission (337 pits). (B) Cumulative distribution of the data in A; ∼19% of the coated pits recruited between 26 and 40 dynamin molecules, and ∼23% recruited between 40 and 52 dynamins. (C) Histogram of the number of dynamins recruited during the final burst (337 pits) shows a peak at ∼26 dynamins. Dotted line marks 26 dynamins. (D) Cumulative distribution of the data in C; ∼27% of the pits recruited between 26 and 40 dynamins, and ∼24% recruited between 26 and 40. Dotted lines mark 26 and 40 dynamins. (E) Histogram of the maximum number of dynamin molecules recruited to abortive pits (n = 1523). Dotted line marks 26 dynamins. (F) Cumulative distribution of the shown in E; ∼70% of the pits recruited <26 dynamin molecules. Dotted line marks 26 dynamins.
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Figure 6: Number of dynamin molecules recruited to coated pits at the time of membrane fission in hCLTAEN/DNM2EN cells. The number of dynamin molecules recruited to clathrin-coated structures was obtained as described in Figure 5 from three cells. The substitution of endogenous dynamin2 by dynamin2-EGFP in hCLTAEN/DNM2EN cells was ∼50%. (A) Histogram of the total number of dynamin molecules recruited to coated pits at the time of membrane fission (337 pits). (B) Cumulative distribution of the data in A; ∼19% of the coated pits recruited between 26 and 40 dynamin molecules, and ∼23% recruited between 40 and 52 dynamins. (C) Histogram of the number of dynamins recruited during the final burst (337 pits) shows a peak at ∼26 dynamins. Dotted line marks 26 dynamins. (D) Cumulative distribution of the data in C; ∼27% of the pits recruited between 26 and 40 dynamins, and ∼24% recruited between 26 and 40. Dotted lines mark 26 and 40 dynamins. (E) Histogram of the maximum number of dynamin molecules recruited to abortive pits (n = 1523). Dotted line marks 26 dynamins. (F) Cumulative distribution of the shown in E; ∼70% of the pits recruited <26 dynamin molecules. Dotted line marks 26 dynamins.

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.


Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit.

Cocucci E, Gaudin R, Kirchhausen T - Mol. Biol. Cell (2014)

Number of dynamin molecules recruited to coated pits at the time of membrane fission in hCLTAEN/DNM2EN cells. The number of dynamin molecules recruited to clathrin-coated structures was obtained as described in Figure 5 from three cells. The substitution of endogenous dynamin2 by dynamin2-EGFP in hCLTAEN/DNM2EN cells was ∼50%. (A) Histogram of the total number of dynamin molecules recruited to coated pits at the time of membrane fission (337 pits). (B) Cumulative distribution of the data in A; ∼19% of the coated pits recruited between 26 and 40 dynamin molecules, and ∼23% recruited between 40 and 52 dynamins. (C) Histogram of the number of dynamins recruited during the final burst (337 pits) shows a peak at ∼26 dynamins. Dotted line marks 26 dynamins. (D) Cumulative distribution of the data in C; ∼27% of the pits recruited between 26 and 40 dynamins, and ∼24% recruited between 26 and 40. Dotted lines mark 26 and 40 dynamins. (E) Histogram of the maximum number of dynamin molecules recruited to abortive pits (n = 1523). Dotted line marks 26 dynamins. (F) Cumulative distribution of the shown in E; ∼70% of the pits recruited <26 dynamin molecules. Dotted line marks 26 dynamins.
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Related In: Results  -  Collection

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Figure 6: Number of dynamin molecules recruited to coated pits at the time of membrane fission in hCLTAEN/DNM2EN cells. The number of dynamin molecules recruited to clathrin-coated structures was obtained as described in Figure 5 from three cells. The substitution of endogenous dynamin2 by dynamin2-EGFP in hCLTAEN/DNM2EN cells was ∼50%. (A) Histogram of the total number of dynamin molecules recruited to coated pits at the time of membrane fission (337 pits). (B) Cumulative distribution of the data in A; ∼19% of the coated pits recruited between 26 and 40 dynamin molecules, and ∼23% recruited between 40 and 52 dynamins. (C) Histogram of the number of dynamins recruited during the final burst (337 pits) shows a peak at ∼26 dynamins. Dotted line marks 26 dynamins. (D) Cumulative distribution of the data in C; ∼27% of the pits recruited between 26 and 40 dynamins, and ∼24% recruited between 26 and 40. Dotted lines mark 26 and 40 dynamins. (E) Histogram of the maximum number of dynamin molecules recruited to abortive pits (n = 1523). Dotted line marks 26 dynamins. (F) Cumulative distribution of the shown in E; ∼70% of the pits recruited <26 dynamin molecules. Dotted line marks 26 dynamins.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus