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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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Effect of dynamin depletion on the amount of dynamin recruited to coated pits at the time of membrane fission. SUM-Dyn2 expressing mCherry-LCa was depleted of dynamin2 by RNAi treatment for 5 d and then analyzed for the effects on transferrin uptake and on the number of dynamin molecules recruited to the few coated pits that still formed. (A) Representative z-stack projections of planes spaced at 350 nm acquired by live-cell three-dimensional spinning-disk confocal fluorescence microscopy of fluorescently tagged transferrin-A647, mCherry-LCa (clathrin), and dynamin2-EGFP, comparing cells depleted of dynamin2 (−dyn2) with controls (+dyn2); just before imaging, the cells were exposed to 50 μg/ml transferrin-A647 for 7 min at 37°C. The images of the dynamin-depleted cells illustrate absence of the intracellular punctuate pattern characteristic of endocytosed transferrin-A647 and decrease in the overall fluorescence signal of dynamin2-EGFP. (B) Total number of dynamin molecules recruited at the time of membrane fission determined as in Figure 5 from 10 Sum-Dyn2 cells depleted of dynamin2 and unable to internalize transferrin-A647. Histogram is for data from 50 clathrin-coated pits that lacked the first recruitment phase. Dotted line marks 26 dynamins. (C) Histogram is for all the coated pits identified in the 10 cells with and without the first recruitment phase (97 pits). Dotted line marks 26 dynamins.
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Figure 8: Effect of dynamin depletion on the amount of dynamin recruited to coated pits at the time of membrane fission. SUM-Dyn2 expressing mCherry-LCa was depleted of dynamin2 by RNAi treatment for 5 d and then analyzed for the effects on transferrin uptake and on the number of dynamin molecules recruited to the few coated pits that still formed. (A) Representative z-stack projections of planes spaced at 350 nm acquired by live-cell three-dimensional spinning-disk confocal fluorescence microscopy of fluorescently tagged transferrin-A647, mCherry-LCa (clathrin), and dynamin2-EGFP, comparing cells depleted of dynamin2 (−dyn2) with controls (+dyn2); just before imaging, the cells were exposed to 50 μg/ml transferrin-A647 for 7 min at 37°C. The images of the dynamin-depleted cells illustrate absence of the intracellular punctuate pattern characteristic of endocytosed transferrin-A647 and decrease in the overall fluorescence signal of dynamin2-EGFP. (B) Total number of dynamin molecules recruited at the time of membrane fission determined as in Figure 5 from 10 Sum-Dyn2 cells depleted of dynamin2 and unable to internalize transferrin-A647. Histogram is for data from 50 clathrin-coated pits that lacked the first recruitment phase. Dotted line marks 26 dynamins. (C) Histogram is for all the coated pits identified in the 10 cells with and without the first recruitment phase (97 pits). Dotted line marks 26 dynamins.

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.


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

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

Effect of dynamin depletion on the amount of dynamin recruited to coated pits at the time of membrane fission. SUM-Dyn2 expressing mCherry-LCa was depleted of dynamin2 by RNAi treatment for 5 d and then analyzed for the effects on transferrin uptake and on the number of dynamin molecules recruited to the few coated pits that still formed. (A) Representative z-stack projections of planes spaced at 350 nm acquired by live-cell three-dimensional spinning-disk confocal fluorescence microscopy of fluorescently tagged transferrin-A647, mCherry-LCa (clathrin), and dynamin2-EGFP, comparing cells depleted of dynamin2 (−dyn2) with controls (+dyn2); just before imaging, the cells were exposed to 50 μg/ml transferrin-A647 for 7 min at 37°C. The images of the dynamin-depleted cells illustrate absence of the intracellular punctuate pattern characteristic of endocytosed transferrin-A647 and decrease in the overall fluorescence signal of dynamin2-EGFP. (B) Total number of dynamin molecules recruited at the time of membrane fission determined as in Figure 5 from 10 Sum-Dyn2 cells depleted of dynamin2 and unable to internalize transferrin-A647. Histogram is for data from 50 clathrin-coated pits that lacked the first recruitment phase. Dotted line marks 26 dynamins. (C) Histogram is for all the coated pits identified in the 10 cells with and without the first recruitment phase (97 pits). Dotted line marks 26 dynamins.
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Figure 8: Effect of dynamin depletion on the amount of dynamin recruited to coated pits at the time of membrane fission. SUM-Dyn2 expressing mCherry-LCa was depleted of dynamin2 by RNAi treatment for 5 d and then analyzed for the effects on transferrin uptake and on the number of dynamin molecules recruited to the few coated pits that still formed. (A) Representative z-stack projections of planes spaced at 350 nm acquired by live-cell three-dimensional spinning-disk confocal fluorescence microscopy of fluorescently tagged transferrin-A647, mCherry-LCa (clathrin), and dynamin2-EGFP, comparing cells depleted of dynamin2 (−dyn2) with controls (+dyn2); just before imaging, the cells were exposed to 50 μg/ml transferrin-A647 for 7 min at 37°C. The images of the dynamin-depleted cells illustrate absence of the intracellular punctuate pattern characteristic of endocytosed transferrin-A647 and decrease in the overall fluorescence signal of dynamin2-EGFP. (B) Total number of dynamin molecules recruited at the time of membrane fission determined as in Figure 5 from 10 Sum-Dyn2 cells depleted of dynamin2 and unable to internalize transferrin-A647. Histogram is for data from 50 clathrin-coated pits that lacked the first recruitment phase. Dotted line marks 26 dynamins. (C) Histogram is for all the coated pits identified in the 10 cells with and without the first recruitment phase (97 pits). Dotted line marks 26 dynamins.
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.

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