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Cooperative recruitment of dynamin and BIN/amphiphysin/Rvs (BAR) domain-containing proteins leads to GTP-dependent membrane scission.

Meinecke M, Boucrot E, Camdere G, Hon WC, Mittal R, McMahon HT - J. Biol. Chem. (2013)

Bottom Line: Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin.In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis.Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 0QH, United Kingdom.

ABSTRACT
Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.

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Recruitment of dynamin and of BAR domain proteins in live cells.A, effect of amphiphysin (Amph1+2 RNAi) and endophilin + amphiphysin (EndoA1+2+3 + Amph1+2 RNAi) depletion on recruitment of endogenous dynamin2 (dynaminen, green, see white arrows) and clathrin light chain A (clathrinen, red). Bar, 10 μm. B, recruitment of endogenous dynamin at the plasma membrane in cells depleted with three independent pools of siRNA against SNX9 (gray bars), amphiphysin (green bars), endophilin (blue bars), and endophilin + amphiphysin (red bars). C, scatter plots of individual lifetimes of endogenous dynamin2 from three different cells, measured on dataset similar to A and S1A. The median with its interquartile range is shown (black lines), and the mean ± S.D. is written at the bottom; n is the number of events analyzed. D, representative FACS profiles of Tf uptake in cells treated with the indicated siRNA. E, effect of the indicated siRNA on transferrin uptake measured by flow cytometry. AP2 depletion was used as positive control (black bar). The background (cells without Tf) is shown (white bar). The number of cells analyzed is displayed on each bar. F, effect of endophilin overexpression on endogenous dynamin recruitment and of dynamin depletion (DNM1+2 RNAi) on endophilin recruitment. Bar, 10 μm. G, recruitment of endogenous dynamin at the plasma membrane in cells overexpressing SNX9 (gray bar), amphiphysin (green bar), or endophilin (blue bar) measured on datasets similar to F and supplemental Fig. S1B. H, recruitment of SNX9 (gray bar), amphiphysin (green bar) and endophilin (blue bar) in cells depleted of dynamin (DNM1+2 RNAi) measured on datasets similar to F and supplemental Fig. S1B. I, effect of 10 μm pitstop2 on amphiphysin recruitment at the plasma membrane. The control and 10 μmpitstop2 images were taken just before and 5 min after addition of the drug, respectively. Bar, 10 μm. J, recruitment of dynamin (magenta bar), endophilin (blue bar), and amphiphysin (green bar) in cells treated with 10 μm pitstop2 for 5 min, measured on datasets similar to I and supplemental Fig. S1D. In B, E, G, H, and J, the values were normalized to the respective means of the control cells; ns, nonsignificant; *, p < 0.01; **, p < 0.001; ***, p < 0.0001.
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Figure 1: Recruitment of dynamin and of BAR domain proteins in live cells.A, effect of amphiphysin (Amph1+2 RNAi) and endophilin + amphiphysin (EndoA1+2+3 + Amph1+2 RNAi) depletion on recruitment of endogenous dynamin2 (dynaminen, green, see white arrows) and clathrin light chain A (clathrinen, red). Bar, 10 μm. B, recruitment of endogenous dynamin at the plasma membrane in cells depleted with three independent pools of siRNA against SNX9 (gray bars), amphiphysin (green bars), endophilin (blue bars), and endophilin + amphiphysin (red bars). C, scatter plots of individual lifetimes of endogenous dynamin2 from three different cells, measured on dataset similar to A and S1A. The median with its interquartile range is shown (black lines), and the mean ± S.D. is written at the bottom; n is the number of events analyzed. D, representative FACS profiles of Tf uptake in cells treated with the indicated siRNA. E, effect of the indicated siRNA on transferrin uptake measured by flow cytometry. AP2 depletion was used as positive control (black bar). The background (cells without Tf) is shown (white bar). The number of cells analyzed is displayed on each bar. F, effect of endophilin overexpression on endogenous dynamin recruitment and of dynamin depletion (DNM1+2 RNAi) on endophilin recruitment. Bar, 10 μm. G, recruitment of endogenous dynamin at the plasma membrane in cells overexpressing SNX9 (gray bar), amphiphysin (green bar), or endophilin (blue bar) measured on datasets similar to F and supplemental Fig. S1B. H, recruitment of SNX9 (gray bar), amphiphysin (green bar) and endophilin (blue bar) in cells depleted of dynamin (DNM1+2 RNAi) measured on datasets similar to F and supplemental Fig. S1B. I, effect of 10 μm pitstop2 on amphiphysin recruitment at the plasma membrane. The control and 10 μmpitstop2 images were taken just before and 5 min after addition of the drug, respectively. Bar, 10 μm. J, recruitment of dynamin (magenta bar), endophilin (blue bar), and amphiphysin (green bar) in cells treated with 10 μm pitstop2 for 5 min, measured on datasets similar to I and supplemental Fig. S1D. In B, E, G, H, and J, the values were normalized to the respective means of the control cells; ns, nonsignificant; *, p < 0.01; **, p < 0.001; ***, p < 0.0001.

Mentions: Dynaminen levels at the plasma membrane (Fig. 1, B and E) were measured by summing the fluorescence signals on at least 10,000 μm2 of cell membrane from snapshots at various times along live-cell imaging time lapses. Lifetimes of dynaminen were measured on kymographs. Please note that endogenous dynamin is detected for a longer time than transiently expressed versions (28). Relative levels of BAR domain proteins at the plasma membrane (Fig. 1, F and H) correspond to the sum of the fluorescence intensities of at least 100 punctae, normalized to the control levels.


Cooperative recruitment of dynamin and BIN/amphiphysin/Rvs (BAR) domain-containing proteins leads to GTP-dependent membrane scission.

Meinecke M, Boucrot E, Camdere G, Hon WC, Mittal R, McMahon HT - J. Biol. Chem. (2013)

Recruitment of dynamin and of BAR domain proteins in live cells.A, effect of amphiphysin (Amph1+2 RNAi) and endophilin + amphiphysin (EndoA1+2+3 + Amph1+2 RNAi) depletion on recruitment of endogenous dynamin2 (dynaminen, green, see white arrows) and clathrin light chain A (clathrinen, red). Bar, 10 μm. B, recruitment of endogenous dynamin at the plasma membrane in cells depleted with three independent pools of siRNA against SNX9 (gray bars), amphiphysin (green bars), endophilin (blue bars), and endophilin + amphiphysin (red bars). C, scatter plots of individual lifetimes of endogenous dynamin2 from three different cells, measured on dataset similar to A and S1A. The median with its interquartile range is shown (black lines), and the mean ± S.D. is written at the bottom; n is the number of events analyzed. D, representative FACS profiles of Tf uptake in cells treated with the indicated siRNA. E, effect of the indicated siRNA on transferrin uptake measured by flow cytometry. AP2 depletion was used as positive control (black bar). The background (cells without Tf) is shown (white bar). The number of cells analyzed is displayed on each bar. F, effect of endophilin overexpression on endogenous dynamin recruitment and of dynamin depletion (DNM1+2 RNAi) on endophilin recruitment. Bar, 10 μm. G, recruitment of endogenous dynamin at the plasma membrane in cells overexpressing SNX9 (gray bar), amphiphysin (green bar), or endophilin (blue bar) measured on datasets similar to F and supplemental Fig. S1B. H, recruitment of SNX9 (gray bar), amphiphysin (green bar) and endophilin (blue bar) in cells depleted of dynamin (DNM1+2 RNAi) measured on datasets similar to F and supplemental Fig. S1B. I, effect of 10 μm pitstop2 on amphiphysin recruitment at the plasma membrane. The control and 10 μmpitstop2 images were taken just before and 5 min after addition of the drug, respectively. Bar, 10 μm. J, recruitment of dynamin (magenta bar), endophilin (blue bar), and amphiphysin (green bar) in cells treated with 10 μm pitstop2 for 5 min, measured on datasets similar to I and supplemental Fig. S1D. In B, E, G, H, and J, the values were normalized to the respective means of the control cells; ns, nonsignificant; *, p < 0.01; **, p < 0.001; ***, p < 0.0001.
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Figure 1: Recruitment of dynamin and of BAR domain proteins in live cells.A, effect of amphiphysin (Amph1+2 RNAi) and endophilin + amphiphysin (EndoA1+2+3 + Amph1+2 RNAi) depletion on recruitment of endogenous dynamin2 (dynaminen, green, see white arrows) and clathrin light chain A (clathrinen, red). Bar, 10 μm. B, recruitment of endogenous dynamin at the plasma membrane in cells depleted with three independent pools of siRNA against SNX9 (gray bars), amphiphysin (green bars), endophilin (blue bars), and endophilin + amphiphysin (red bars). C, scatter plots of individual lifetimes of endogenous dynamin2 from three different cells, measured on dataset similar to A and S1A. The median with its interquartile range is shown (black lines), and the mean ± S.D. is written at the bottom; n is the number of events analyzed. D, representative FACS profiles of Tf uptake in cells treated with the indicated siRNA. E, effect of the indicated siRNA on transferrin uptake measured by flow cytometry. AP2 depletion was used as positive control (black bar). The background (cells without Tf) is shown (white bar). The number of cells analyzed is displayed on each bar. F, effect of endophilin overexpression on endogenous dynamin recruitment and of dynamin depletion (DNM1+2 RNAi) on endophilin recruitment. Bar, 10 μm. G, recruitment of endogenous dynamin at the plasma membrane in cells overexpressing SNX9 (gray bar), amphiphysin (green bar), or endophilin (blue bar) measured on datasets similar to F and supplemental Fig. S1B. H, recruitment of SNX9 (gray bar), amphiphysin (green bar) and endophilin (blue bar) in cells depleted of dynamin (DNM1+2 RNAi) measured on datasets similar to F and supplemental Fig. S1B. I, effect of 10 μm pitstop2 on amphiphysin recruitment at the plasma membrane. The control and 10 μmpitstop2 images were taken just before and 5 min after addition of the drug, respectively. Bar, 10 μm. J, recruitment of dynamin (magenta bar), endophilin (blue bar), and amphiphysin (green bar) in cells treated with 10 μm pitstop2 for 5 min, measured on datasets similar to I and supplemental Fig. S1D. In B, E, G, H, and J, the values were normalized to the respective means of the control cells; ns, nonsignificant; *, p < 0.01; **, p < 0.001; ***, p < 0.0001.
Mentions: Dynaminen levels at the plasma membrane (Fig. 1, B and E) were measured by summing the fluorescence signals on at least 10,000 μm2 of cell membrane from snapshots at various times along live-cell imaging time lapses. Lifetimes of dynaminen were measured on kymographs. Please note that endogenous dynamin is detected for a longer time than transiently expressed versions (28). Relative levels of BAR domain proteins at the plasma membrane (Fig. 1, F and H) correspond to the sum of the fluorescence intensities of at least 100 punctae, normalized to the control levels.

Bottom Line: Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin.In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis.Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 0QH, United Kingdom.

ABSTRACT
Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.

Show MeSH
Related in: MedlinePlus