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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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Amphiphysin recruitment to membranes depends on the PRD of dynamin. GUVs (red) were labeled with rhodamine-PE; dynamin (green) was labeled with Alexa-488; amphiphysin (blue) was labeled with Alexa-647. A, GUV in the presence of 1 μm endophilin N-BAR domain. B, GUV in the presence of 1 μm amphiphysin. C, GUV in the presence of 500 nm amphiphysin and 300 nm dynamin. D, GUV in the presence of 1 μm amphiphysin plus 1 μm dynamin-ΔPRD (top panel), 1 μm amphiphysin plus 1 μm dynamin-ΔPRD plus 500 nm full-length dynamin (middle panel), and 1 μm amphiphysin plus 2 μm PRD (bottom panel). E, kymograph showing one particular area of a GUV loaded with dynamin and amphiphysin over 60 min. 25 μm P4 peptide, which disrupts the amphiphysin and dynamin interaction, was added at the beginning of the measurement.
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Figure 4: Amphiphysin recruitment to membranes depends on the PRD of dynamin. GUVs (red) were labeled with rhodamine-PE; dynamin (green) was labeled with Alexa-488; amphiphysin (blue) was labeled with Alexa-647. A, GUV in the presence of 1 μm endophilin N-BAR domain. B, GUV in the presence of 1 μm amphiphysin. C, GUV in the presence of 500 nm amphiphysin and 300 nm dynamin. D, GUV in the presence of 1 μm amphiphysin plus 1 μm dynamin-ΔPRD (top panel), 1 μm amphiphysin plus 1 μm dynamin-ΔPRD plus 500 nm full-length dynamin (middle panel), and 1 μm amphiphysin plus 2 μm PRD (bottom panel). E, kymograph showing one particular area of a GUV loaded with dynamin and amphiphysin over 60 min. 25 μm P4 peptide, which disrupts the amphiphysin and dynamin interaction, was added at the beginning of the measurement.

Mentions: Membrane localization and the assay of CME in cells show a strong relationship between BAR proteins and dynamin. However, to gain a more precise molecular understanding, we developed a system with reduced complexity. We have therefore reconstituted the activities of these proteins on membranes in vitro. Membrane binding of endocytic proteins has classically been shown with spin or flotation assays using small unilamellar vesicles. We considered small unilamellar vesicles an inadequate membrane model system to study dynamin recruitment, because dynamin recruitment to small unilamellar vesicles is robust even in the absence of interaction partners (36). This may be due to enhanced polymerization of dynamin on membranes of high curvature (5). Thus, to visualize dynamin recruitment to membranes, we employed GUVs, which present a virtually flat surface at the protein scale. Shortly after the addition of Alexa-488-labeled dynamin, we observed almost no dynamin recruitment to GUVs (Fig. 2A, upper panel). However, within 30 min, a strong dynamin signal was seen on the membrane (Fig. 2, A, bottom panel, B and C). This suggested the possibility that GUVs represent a suitable model membrane system to study potential factors involved in dynamin recruitment. Addition of labeled endophilin to the assay accelerated the recruitment of dynamin to GUV membranes (Fig. 2D), as seen also in time courses (Fig. 2, E and F). Co-recruitment experiments with dynamin and amphiphysin showed comparable results (Fig. 2, G and H), consistent with earlier results showing an increased rate of GTP hydrolysis by dynamin on membranes in the presence of amphiphysin (26). The addition of endophilin not only led to a more efficient membrane binding of dynamin but also to clustering of both proteins on the membrane (Fig. 2D, arrows, and supplemental Movie 1). Such protein clusters were not observed in experiments with dynamin alone (Fig. 2A) or with dynamin in the presence of amphiphysin (Fig. 4). This observation fits with earlier experiments showing that amphiphysin disassembles dynamin oligomers (13), whereas endophilin more strongly promotes dynamin assembly (20). From these initial experiments, we conclude that BAR+SH3 proteins promote a more efficient recruitment of dynamin to membranes, and given the variety of these proteins, with various membrane specificities, this should facilitate specific dynamin recruitment to many different membrane scission events.


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)

Amphiphysin recruitment to membranes depends on the PRD of dynamin. GUVs (red) were labeled with rhodamine-PE; dynamin (green) was labeled with Alexa-488; amphiphysin (blue) was labeled with Alexa-647. A, GUV in the presence of 1 μm endophilin N-BAR domain. B, GUV in the presence of 1 μm amphiphysin. C, GUV in the presence of 500 nm amphiphysin and 300 nm dynamin. D, GUV in the presence of 1 μm amphiphysin plus 1 μm dynamin-ΔPRD (top panel), 1 μm amphiphysin plus 1 μm dynamin-ΔPRD plus 500 nm full-length dynamin (middle panel), and 1 μm amphiphysin plus 2 μm PRD (bottom panel). E, kymograph showing one particular area of a GUV loaded with dynamin and amphiphysin over 60 min. 25 μm P4 peptide, which disrupts the amphiphysin and dynamin interaction, was added at the beginning of the measurement.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585104&req=5

Figure 4: Amphiphysin recruitment to membranes depends on the PRD of dynamin. GUVs (red) were labeled with rhodamine-PE; dynamin (green) was labeled with Alexa-488; amphiphysin (blue) was labeled with Alexa-647. A, GUV in the presence of 1 μm endophilin N-BAR domain. B, GUV in the presence of 1 μm amphiphysin. C, GUV in the presence of 500 nm amphiphysin and 300 nm dynamin. D, GUV in the presence of 1 μm amphiphysin plus 1 μm dynamin-ΔPRD (top panel), 1 μm amphiphysin plus 1 μm dynamin-ΔPRD plus 500 nm full-length dynamin (middle panel), and 1 μm amphiphysin plus 2 μm PRD (bottom panel). E, kymograph showing one particular area of a GUV loaded with dynamin and amphiphysin over 60 min. 25 μm P4 peptide, which disrupts the amphiphysin and dynamin interaction, was added at the beginning of the measurement.
Mentions: Membrane localization and the assay of CME in cells show a strong relationship between BAR proteins and dynamin. However, to gain a more precise molecular understanding, we developed a system with reduced complexity. We have therefore reconstituted the activities of these proteins on membranes in vitro. Membrane binding of endocytic proteins has classically been shown with spin or flotation assays using small unilamellar vesicles. We considered small unilamellar vesicles an inadequate membrane model system to study dynamin recruitment, because dynamin recruitment to small unilamellar vesicles is robust even in the absence of interaction partners (36). This may be due to enhanced polymerization of dynamin on membranes of high curvature (5). Thus, to visualize dynamin recruitment to membranes, we employed GUVs, which present a virtually flat surface at the protein scale. Shortly after the addition of Alexa-488-labeled dynamin, we observed almost no dynamin recruitment to GUVs (Fig. 2A, upper panel). However, within 30 min, a strong dynamin signal was seen on the membrane (Fig. 2, A, bottom panel, B and C). This suggested the possibility that GUVs represent a suitable model membrane system to study potential factors involved in dynamin recruitment. Addition of labeled endophilin to the assay accelerated the recruitment of dynamin to GUV membranes (Fig. 2D), as seen also in time courses (Fig. 2, E and F). Co-recruitment experiments with dynamin and amphiphysin showed comparable results (Fig. 2, G and H), consistent with earlier results showing an increased rate of GTP hydrolysis by dynamin on membranes in the presence of amphiphysin (26). The addition of endophilin not only led to a more efficient membrane binding of dynamin but also to clustering of both proteins on the membrane (Fig. 2D, arrows, and supplemental Movie 1). Such protein clusters were not observed in experiments with dynamin alone (Fig. 2A) or with dynamin in the presence of amphiphysin (Fig. 4). This observation fits with earlier experiments showing that amphiphysin disassembles dynamin oligomers (13), whereas endophilin more strongly promotes dynamin assembly (20). From these initial experiments, we conclude that BAR+SH3 proteins promote a more efficient recruitment of dynamin to membranes, and given the variety of these proteins, with various membrane specificities, this should facilitate specific dynamin recruitment to many different membrane scission events.

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