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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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Representative traces of clathrin-coated pits forming in SUM-Dyn2 cells. Plot of the fluorescence intensity traces of dynamin2-EGFP (blue) and mCherry-LCa (red) from SUM-Dyn2 cells recruited during formation of clathrin-coated pits in Sum-Dyn2 cells. The data are from 5-min time series obtained using spinning-disk confocal microscopy every 1 s with an exposure of 30 ms/frame. The traces highlight examples of the variability in the recruitment patterns during the first and second phases of association with clathrin-coated pits. (A) Relatively small amount of dynamin recruited during the first phase, followed by a brief but pronounced sharp recruitment during the second phase. (B) A significant amount of dynamin recruited during the first phase, followed by a distinctive recruitment burst during the second phase. (C) A significant amount of dynamin recruited during the first phase that is poorly resolved from the recruitment preceding membrane scission. (D) Dynamin is recruited as a relatively broad burst toward the end of the budding process.
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Figure 4: Representative traces of clathrin-coated pits forming in SUM-Dyn2 cells. Plot of the fluorescence intensity traces of dynamin2-EGFP (blue) and mCherry-LCa (red) from SUM-Dyn2 cells recruited during formation of clathrin-coated pits in Sum-Dyn2 cells. The data are from 5-min time series obtained using spinning-disk confocal microscopy every 1 s with an exposure of 30 ms/frame. The traces highlight examples of the variability in the recruitment patterns during the first and second phases of association with clathrin-coated pits. (A) Relatively small amount of dynamin recruited during the first phase, followed by a brief but pronounced sharp recruitment during the second phase. (B) A significant amount of dynamin recruited during the first phase, followed by a distinctive recruitment burst during the second phase. (C) A significant amount of dynamin recruited during the first phase that is poorly resolved from the recruitment preceding membrane scission. (D) Dynamin is recruited as a relatively broad burst toward the end of the budding process.

Mentions: We carried out a primary selection of objects containing fluorescent dynamin and clathrin using the cmeAnalysis software with clathrin as the “master” and dynamin as the “slave” (Aguet et al., 2013); we then picked manually from the automated primary selections those objects whose clathrin fluorescent signal followed the expected continuous growth associated with canonical coated pits (see Materials and Methods and representative traces in Figure 4). This selection was important because the clathrin signal was not strong enough for reliable automated selection, and the software often incorrectly classified tracks of coated pits as abortive rather than canonical or incorrectly combined into a single event two or more pits that formed sequentially at a single location (a “hot spot”).


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

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

Representative traces of clathrin-coated pits forming in SUM-Dyn2 cells. Plot of the fluorescence intensity traces of dynamin2-EGFP (blue) and mCherry-LCa (red) from SUM-Dyn2 cells recruited during formation of clathrin-coated pits in Sum-Dyn2 cells. The data are from 5-min time series obtained using spinning-disk confocal microscopy every 1 s with an exposure of 30 ms/frame. The traces highlight examples of the variability in the recruitment patterns during the first and second phases of association with clathrin-coated pits. (A) Relatively small amount of dynamin recruited during the first phase, followed by a brief but pronounced sharp recruitment during the second phase. (B) A significant amount of dynamin recruited during the first phase, followed by a distinctive recruitment burst during the second phase. (C) A significant amount of dynamin recruited during the first phase that is poorly resolved from the recruitment preceding membrane scission. (D) Dynamin is recruited as a relatively broad burst toward the end of the budding process.
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Related In: Results  -  Collection

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Figure 4: Representative traces of clathrin-coated pits forming in SUM-Dyn2 cells. Plot of the fluorescence intensity traces of dynamin2-EGFP (blue) and mCherry-LCa (red) from SUM-Dyn2 cells recruited during formation of clathrin-coated pits in Sum-Dyn2 cells. The data are from 5-min time series obtained using spinning-disk confocal microscopy every 1 s with an exposure of 30 ms/frame. The traces highlight examples of the variability in the recruitment patterns during the first and second phases of association with clathrin-coated pits. (A) Relatively small amount of dynamin recruited during the first phase, followed by a brief but pronounced sharp recruitment during the second phase. (B) A significant amount of dynamin recruited during the first phase, followed by a distinctive recruitment burst during the second phase. (C) A significant amount of dynamin recruited during the first phase that is poorly resolved from the recruitment preceding membrane scission. (D) Dynamin is recruited as a relatively broad burst toward the end of the budding process.
Mentions: We carried out a primary selection of objects containing fluorescent dynamin and clathrin using the cmeAnalysis software with clathrin as the “master” and dynamin as the “slave” (Aguet et al., 2013); we then picked manually from the automated primary selections those objects whose clathrin fluorescent signal followed the expected continuous growth associated with canonical coated pits (see Materials and Methods and representative traces in Figure 4). This selection was important because the clathrin signal was not strong enough for reliable automated selection, and the software often incorrectly classified tracks of coated pits as abortive rather than canonical or incorrectly combined into a single event two or more pits that formed sequentially at a single location (a “hot spot”).

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