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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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Endophilin recruitment to membranes depends on the PRD of dynamin. GUVs and protein labeled as in Fig. 2. Endophilin N-BAR domain (green) was labeled with Alexa-488. A, GUV in the presence of 1 μm full-length endophilin. B, GUV in the presence of 1 μm endophilin N-BAR domain. C, GUV in the presence of 500 nm full-length endophilin and 300 nm dynamin. D, liposome sedimentation assay in the presence of endophilin-N-BAR (left panel), endophilin full-length (fl) (middle panel), and endophilin full-length plus the PRD of dynamin. S is supernatant, and P is pellet. E, GUV in the presence of 1 μm endophilin plus 1 μm dynamin-ΔPRD (top panel), 1 μm endophilin plus 1 μm dynamin-ΔPRD plus 500 nm full-length dynamin (middle panel), and 1 μm endophilin plus 2 μm PRD (bottom panel).
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Figure 3: Endophilin recruitment to membranes depends on the PRD of dynamin. GUVs and protein labeled as in Fig. 2. Endophilin N-BAR domain (green) was labeled with Alexa-488. A, GUV in the presence of 1 μm full-length endophilin. B, GUV in the presence of 1 μm endophilin N-BAR domain. C, GUV in the presence of 500 nm full-length endophilin and 300 nm dynamin. D, liposome sedimentation assay in the presence of endophilin-N-BAR (left panel), endophilin full-length (fl) (middle panel), and endophilin full-length plus the PRD of dynamin. S is supernatant, and P is pellet. E, GUV in the presence of 1 μm endophilin plus 1 μm dynamin-ΔPRD (top panel), 1 μm endophilin plus 1 μm dynamin-ΔPRD plus 500 nm full-length dynamin (middle panel), and 1 μm endophilin plus 2 μm PRD (bottom panel).

Mentions: Surprisingly, when changing the order of protein addition, we did not detect the recruitment of full-length endophilin to membranes in the absence of dynamin (Fig. 3A). This is similar to our observation in cells where RNAi of dynamin led to a reduced recruitment of endophilin (Fig. 1, D and F). The in vitro system allowed this to be further probed. The N-BAR module alone of endophilin was efficiently recruited (Fig. 3B), ruling out any potential interference of protein and lipid labeling with membrane binding and implying that membrane binding of full-length endophilin is auto-inhibited by its cognate SH3 domain. Addition of endophilin N-BAR in many cases also led to visible tubulation of GUVs (Fig. 4A). When dynamin was added to full-length endophilin, both proteins were recruited to GUV membranes within seconds (Fig. 3C). To further test for auto-inhibition, we performed a series of co-sedimentation assays with SUVs (Fig. 3D). Endophilin N-BAR domain efficiently bound to SUVs, as expected, whereas membrane binding was decreased for full-length endophilin. Almost complete membrane binding was restored in the presence of dynamin PRD (Fig. 3D) that is known to bind to the SH3 domain of endophilin (10). It is tempting to speculate that the stronger membrane binding of full-length endophilin to SUVs might be due to the higher curvature of the small vesicles serving as a binding template or to an increased membrane tension of the GUVs due to the high sucrose concentration within the vesicles.


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)

Endophilin recruitment to membranes depends on the PRD of dynamin. GUVs and protein labeled as in Fig. 2. Endophilin N-BAR domain (green) was labeled with Alexa-488. A, GUV in the presence of 1 μm full-length endophilin. B, GUV in the presence of 1 μm endophilin N-BAR domain. C, GUV in the presence of 500 nm full-length endophilin and 300 nm dynamin. D, liposome sedimentation assay in the presence of endophilin-N-BAR (left panel), endophilin full-length (fl) (middle panel), and endophilin full-length plus the PRD of dynamin. S is supernatant, and P is pellet. E, GUV in the presence of 1 μm endophilin plus 1 μm dynamin-ΔPRD (top panel), 1 μm endophilin plus 1 μm dynamin-ΔPRD plus 500 nm full-length dynamin (middle panel), and 1 μm endophilin plus 2 μm PRD (bottom panel).
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Figure 3: Endophilin recruitment to membranes depends on the PRD of dynamin. GUVs and protein labeled as in Fig. 2. Endophilin N-BAR domain (green) was labeled with Alexa-488. A, GUV in the presence of 1 μm full-length endophilin. B, GUV in the presence of 1 μm endophilin N-BAR domain. C, GUV in the presence of 500 nm full-length endophilin and 300 nm dynamin. D, liposome sedimentation assay in the presence of endophilin-N-BAR (left panel), endophilin full-length (fl) (middle panel), and endophilin full-length plus the PRD of dynamin. S is supernatant, and P is pellet. E, GUV in the presence of 1 μm endophilin plus 1 μm dynamin-ΔPRD (top panel), 1 μm endophilin plus 1 μm dynamin-ΔPRD plus 500 nm full-length dynamin (middle panel), and 1 μm endophilin plus 2 μm PRD (bottom panel).
Mentions: Surprisingly, when changing the order of protein addition, we did not detect the recruitment of full-length endophilin to membranes in the absence of dynamin (Fig. 3A). This is similar to our observation in cells where RNAi of dynamin led to a reduced recruitment of endophilin (Fig. 1, D and F). The in vitro system allowed this to be further probed. The N-BAR module alone of endophilin was efficiently recruited (Fig. 3B), ruling out any potential interference of protein and lipid labeling with membrane binding and implying that membrane binding of full-length endophilin is auto-inhibited by its cognate SH3 domain. Addition of endophilin N-BAR in many cases also led to visible tubulation of GUVs (Fig. 4A). When dynamin was added to full-length endophilin, both proteins were recruited to GUV membranes within seconds (Fig. 3C). To further test for auto-inhibition, we performed a series of co-sedimentation assays with SUVs (Fig. 3D). Endophilin N-BAR domain efficiently bound to SUVs, as expected, whereas membrane binding was decreased for full-length endophilin. Almost complete membrane binding was restored in the presence of dynamin PRD (Fig. 3D) that is known to bind to the SH3 domain of endophilin (10). It is tempting to speculate that the stronger membrane binding of full-length endophilin to SUVs might be due to the higher curvature of the small vesicles serving as a binding template or to an increased membrane tension of the GUVs due to the high sucrose concentration within the vesicles.

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