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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 SUM-Dyn2 cells. The number of dynamin molecules recruited to clathrin-coated structures was obtained from the net fluorescence intensities of recruited dynamin2-EGFP (fluorescence intensity for a given spot minus the fluorescence intensity of the background calibrated by the fluorescence of a single molecule), corrected for the calculated substitution (98%) of endogenous dynamin2 by dynamin2-EGFP. Data from five cells, obtained using spinning-disk confocal microscopy; the time series acquired every 1 s with an exposure of 30 ms/frame. (A) Histogram of the total number of dynamin molecules recruited to coated pits at the time of membrane fission (426 pits). Dotted line marks 26 dynamins. (B) Cumulative distribution associated with the values shown in A demonstrating that ∼25% of the coated pits recruited between 26 and 40 dynamin molecules and 28% recruit between 40 and 52. Dotted lines mark 26 and 40 dynamins. The light blue area overlapping the tracings indicates 95% confidence interval for the estimated number of recruited dynamins. (C) Histogram of the number of dynamins recruited during the final burst (150 pits) shows that it peaks at ∼26 dynamins. The number of recruited molecules was calculated as the difference between the maximal peak value and the value averaged from frames imaged 9, 10, and 11 s before the peak. (D) Cumulative distribution of the data in C; ∼30% of the pits recruited between 26 and 40 dynamins and 23% between 40 and 52. Dotted lines mark 26 and 40 dynamins. (E) Histogram of the maximum number of dynamin molecules recruited to abortive pits (n = 8514). Dotted line marks 26 dynamins. (F) Cumulative distribution of the data in E; ∼90% of the pits recruited <26 dynamin molecules. Dotted line marks 26 dynamins.
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Figure 5: Number of dynamin molecules recruited to coated pits at the time of membrane fission in SUM-Dyn2 cells. The number of dynamin molecules recruited to clathrin-coated structures was obtained from the net fluorescence intensities of recruited dynamin2-EGFP (fluorescence intensity for a given spot minus the fluorescence intensity of the background calibrated by the fluorescence of a single molecule), corrected for the calculated substitution (98%) of endogenous dynamin2 by dynamin2-EGFP. Data from five cells, obtained using spinning-disk confocal microscopy; the time series acquired every 1 s with an exposure of 30 ms/frame. (A) Histogram of the total number of dynamin molecules recruited to coated pits at the time of membrane fission (426 pits). Dotted line marks 26 dynamins. (B) Cumulative distribution associated with the values shown in A demonstrating that ∼25% of the coated pits recruited between 26 and 40 dynamin molecules and 28% recruit between 40 and 52. Dotted lines mark 26 and 40 dynamins. The light blue area overlapping the tracings indicates 95% confidence interval for the estimated number of recruited dynamins. (C) Histogram of the number of dynamins recruited during the final burst (150 pits) shows that it peaks at ∼26 dynamins. The number of recruited molecules was calculated as the difference between the maximal peak value and the value averaged from frames imaged 9, 10, and 11 s before the peak. (D) Cumulative distribution of the data in C; ∼30% of the pits recruited between 26 and 40 dynamins and 23% between 40 and 52. Dotted lines mark 26 and 40 dynamins. (E) Histogram of the maximum number of dynamin molecules recruited to abortive pits (n = 8514). Dotted line marks 26 dynamins. (F) Cumulative distribution of the data in E; ∼90% of the pits recruited <26 dynamin molecules. Dotted line marks 26 dynamins.

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).


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 SUM-Dyn2 cells. The number of dynamin molecules recruited to clathrin-coated structures was obtained from the net fluorescence intensities of recruited dynamin2-EGFP (fluorescence intensity for a given spot minus the fluorescence intensity of the background calibrated by the fluorescence of a single molecule), corrected for the calculated substitution (98%) of endogenous dynamin2 by dynamin2-EGFP. Data from five cells, obtained using spinning-disk confocal microscopy; the time series acquired every 1 s with an exposure of 30 ms/frame. (A) Histogram of the total number of dynamin molecules recruited to coated pits at the time of membrane fission (426 pits). Dotted line marks 26 dynamins. (B) Cumulative distribution associated with the values shown in A demonstrating that ∼25% of the coated pits recruited between 26 and 40 dynamin molecules and 28% recruit between 40 and 52. Dotted lines mark 26 and 40 dynamins. The light blue area overlapping the tracings indicates 95% confidence interval for the estimated number of recruited dynamins. (C) Histogram of the number of dynamins recruited during the final burst (150 pits) shows that it peaks at ∼26 dynamins. The number of recruited molecules was calculated as the difference between the maximal peak value and the value averaged from frames imaged 9, 10, and 11 s before the peak. (D) Cumulative distribution of the data in C; ∼30% of the pits recruited between 26 and 40 dynamins and 23% between 40 and 52. Dotted lines mark 26 and 40 dynamins. (E) Histogram of the maximum number of dynamin molecules recruited to abortive pits (n = 8514). Dotted line marks 26 dynamins. (F) Cumulative distribution of the data in E; ∼90% of the pits recruited <26 dynamin molecules. Dotted line marks 26 dynamins.
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Figure 5: Number of dynamin molecules recruited to coated pits at the time of membrane fission in SUM-Dyn2 cells. The number of dynamin molecules recruited to clathrin-coated structures was obtained from the net fluorescence intensities of recruited dynamin2-EGFP (fluorescence intensity for a given spot minus the fluorescence intensity of the background calibrated by the fluorescence of a single molecule), corrected for the calculated substitution (98%) of endogenous dynamin2 by dynamin2-EGFP. Data from five cells, obtained using spinning-disk confocal microscopy; the time series acquired every 1 s with an exposure of 30 ms/frame. (A) Histogram of the total number of dynamin molecules recruited to coated pits at the time of membrane fission (426 pits). Dotted line marks 26 dynamins. (B) Cumulative distribution associated with the values shown in A demonstrating that ∼25% of the coated pits recruited between 26 and 40 dynamin molecules and 28% recruit between 40 and 52. Dotted lines mark 26 and 40 dynamins. The light blue area overlapping the tracings indicates 95% confidence interval for the estimated number of recruited dynamins. (C) Histogram of the number of dynamins recruited during the final burst (150 pits) shows that it peaks at ∼26 dynamins. The number of recruited molecules was calculated as the difference between the maximal peak value and the value averaged from frames imaged 9, 10, and 11 s before the peak. (D) Cumulative distribution of the data in C; ∼30% of the pits recruited between 26 and 40 dynamins and 23% between 40 and 52. Dotted lines mark 26 and 40 dynamins. (E) Histogram of the maximum number of dynamin molecules recruited to abortive pits (n = 8514). Dotted line marks 26 dynamins. (F) Cumulative distribution of the data in E; ∼90% of the pits recruited <26 dynamin molecules. Dotted line marks 26 dynamins.
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).

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