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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 membrane tension on the number of dynamin molecules recruited to coated pits at the time of membrane fission. The total number of dynamin molecules recruited to clathrin-coated pits was determined as in Figure 5 using Sum-Dyn2 briefly exposed for 10 min to media of different osmolarities. Five cells were used per condition. (A) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in hyper-osmotic medium at the time of membrane fission (78 pits). (B) Cumulative distribution of the data in A; ∼16% of the pits recruit between 26 and 40 dynamins, and 13% recruited between 40 and 52 dynamins. (C) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in hypo-osmotic medium at the time of membrane fission (210 pits). (D) Cumulative distribution of the data in C; ∼38% of the pits recruit between 26 and 40 dynamins, and ∼27% recruited between 40 and 52 dynamins. (E) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in iso-osmotic medium at the time of membrane fission (312 pits). (F) Cumulative distribution of the data in E; ∼30% of the pits recruited between 26 and 40 dynamins, and ∼30% recruited between 40 and 52 dynamins.
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Figure 7: Effect of membrane tension on the number of dynamin molecules recruited to coated pits at the time of membrane fission. The total number of dynamin molecules recruited to clathrin-coated pits was determined as in Figure 5 using Sum-Dyn2 briefly exposed for 10 min to media of different osmolarities. Five cells were used per condition. (A) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in hyper-osmotic medium at the time of membrane fission (78 pits). (B) Cumulative distribution of the data in A; ∼16% of the pits recruit between 26 and 40 dynamins, and 13% recruited between 40 and 52 dynamins. (C) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in hypo-osmotic medium at the time of membrane fission (210 pits). (D) Cumulative distribution of the data in C; ∼38% of the pits recruit between 26 and 40 dynamins, and ∼27% recruited between 40 and 52 dynamins. (E) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in iso-osmotic medium at the time of membrane fission (312 pits). (F) Cumulative distribution of the data in E; ∼30% of the pits recruited between 26 and 40 dynamins, and ∼30% recruited between 40 and 52 dynamins.

Mentions: Scission of dynamin-coated membrane tubules in vitro requires that the tubule be under longitudinal tension (Roux et al., 2006; Morlot and Roux, 2013; Morlot et al., 2012; Bashkirov et al., 2008; Shnyrova et al., 2013). We examined the effect of membrane tension on the number of dynamin molecules that have accumulated at the time of coated vesicle release in SUM-Dyn2 cells expressing mCherry-LCa. Lowering plasma membrane tension by acute exposure to hyperosmotic medium inhibits coated vesicle formation (and the associated endocytosis of cargo; Heuser and Anderson, 1989; Morlot et al., 2012). Introduction of hyperosmolar medium (lowering membrane tension) caused the total number of dynamins to increase (Figure 7, A and B), suggesting that a flaccid membrane needs more dynamin to rupture than does a tense one. Nonetheless, cells incubated in hypo-osmotic medium (Boulant et al., 2011) and thereby subjected to a higher plasma membrane tension accumulated approximately the same number as did control cells (Figure 7, B–F). Thus there appears to be a threshold requirement for membrane scission, even when tension is unphysiologically high.


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 membrane tension on the number of dynamin molecules recruited to coated pits at the time of membrane fission. The total number of dynamin molecules recruited to clathrin-coated pits was determined as in Figure 5 using Sum-Dyn2 briefly exposed for 10 min to media of different osmolarities. Five cells were used per condition. (A) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in hyper-osmotic medium at the time of membrane fission (78 pits). (B) Cumulative distribution of the data in A; ∼16% of the pits recruit between 26 and 40 dynamins, and 13% recruited between 40 and 52 dynamins. (C) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in hypo-osmotic medium at the time of membrane fission (210 pits). (D) Cumulative distribution of the data in C; ∼38% of the pits recruit between 26 and 40 dynamins, and ∼27% recruited between 40 and 52 dynamins. (E) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in iso-osmotic medium at the time of membrane fission (312 pits). (F) Cumulative distribution of the data in E; ∼30% of the pits recruited between 26 and 40 dynamins, and ∼30% recruited between 40 and 52 dynamins.
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Related In: Results  -  Collection

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Figure 7: Effect of membrane tension on the number of dynamin molecules recruited to coated pits at the time of membrane fission. The total number of dynamin molecules recruited to clathrin-coated pits was determined as in Figure 5 using Sum-Dyn2 briefly exposed for 10 min to media of different osmolarities. Five cells were used per condition. (A) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in hyper-osmotic medium at the time of membrane fission (78 pits). (B) Cumulative distribution of the data in A; ∼16% of the pits recruit between 26 and 40 dynamins, and 13% recruited between 40 and 52 dynamins. (C) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in hypo-osmotic medium at the time of membrane fission (210 pits). (D) Cumulative distribution of the data in C; ∼38% of the pits recruit between 26 and 40 dynamins, and ∼27% recruited between 40 and 52 dynamins. (E) Histogram of the total number of dynamin molecules recruited to coated pits of cells incubated in iso-osmotic medium at the time of membrane fission (312 pits). (F) Cumulative distribution of the data in E; ∼30% of the pits recruited between 26 and 40 dynamins, and ∼30% recruited between 40 and 52 dynamins.
Mentions: Scission of dynamin-coated membrane tubules in vitro requires that the tubule be under longitudinal tension (Roux et al., 2006; Morlot and Roux, 2013; Morlot et al., 2012; Bashkirov et al., 2008; Shnyrova et al., 2013). We examined the effect of membrane tension on the number of dynamin molecules that have accumulated at the time of coated vesicle release in SUM-Dyn2 cells expressing mCherry-LCa. Lowering plasma membrane tension by acute exposure to hyperosmotic medium inhibits coated vesicle formation (and the associated endocytosis of cargo; Heuser and Anderson, 1989; Morlot et al., 2012). Introduction of hyperosmolar medium (lowering membrane tension) caused the total number of dynamins to increase (Figure 7, A and B), suggesting that a flaccid membrane needs more dynamin to rupture than does a tense one. Nonetheless, cells incubated in hypo-osmotic medium (Boulant et al., 2011) and thereby subjected to a higher plasma membrane tension accumulated approximately the same number as did control cells (Figure 7, B–F). Thus there appears to be a threshold requirement for membrane scission, even when tension is unphysiologically high.

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