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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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GTP-dependent vesicle release leads to shrinkage of parent GUVs. GUVs and protein labeled as in Fig. 3. A, time course of a GUV with bound dynamin and endophilin in the absence (−GTP) and presence of GTP. Z-stacks (−GTP and 60 min) show volume shrinkage of the GUV. B, time course of GUV shrinkage under the indicated conditions. C, quantitation of time courses of at least five independent vesicle preparations (mean with S.E.). D, quantitation of the vesiculation in the presence of GTPγS. E, quantitation of vesiculation in the presence of a peptide (P4) that disrupts the amphiphysin-dynamin interaction. Vesicle shrinkage was calculated according to (D0 − D60) × 100/D0. D60 is the vesicle diameter after 60 min, and D0 is the vesicle diameter at the beginning of the experiment.
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Figure 5: GTP-dependent vesicle release leads to shrinkage of parent GUVs. GUVs and protein labeled as in Fig. 3. A, time course of a GUV with bound dynamin and endophilin in the absence (−GTP) and presence of GTP. Z-stacks (−GTP and 60 min) show volume shrinkage of the GUV. B, time course of GUV shrinkage under the indicated conditions. C, quantitation of time courses of at least five independent vesicle preparations (mean with S.E.). D, quantitation of the vesiculation in the presence of GTPγS. E, quantitation of vesiculation in the presence of a peptide (P4) that disrupts the amphiphysin-dynamin interaction. Vesicle shrinkage was calculated according to (D0 − D60) × 100/D0. D60 is the vesicle diameter after 60 min, and D0 is the vesicle diameter at the beginning of the experiment.

Mentions: Different models for membrane scission by dynamin exist. The two published dynamin structures propose a mechanism of how GTP hydrolysis could be coupled to membrane scission (40, 41), and a very recently published model is taking physical parameters of the membrane into account (42). Although our assay does not allow us to test for possible scission mechanisms, we can ask the following question. What is the likely consequence of cooperative membrane binding on dynamin-dependent membrane scission? It has been shown in the past that dynamin is sufficient to release small vesicles from membrane templates in a GTP-dependent manner (6, 43), and dynamin is capable of causing scission of membrane tethers (44, 45). Using GUVs, we observed that addition of dynamin resulted in sporadic tubular structures on the vesicle surfaces and that those structures were released in response to GTP addition (results not shown). However, these events were rather infrequent, and thus we asked if we would see a difference in the presence of BAR domain proteins. In the presence of dynamin and endophilin, GUVs were stable for hours (Fig. 5A, −GTP). However, subsequent addition of GTP led to a visible release of small vesicles from the GUVs (Fig. 5A, arrows). Following the same GUV over a longer period of time revealed a significant volume reduction (Fig. 5A, 60 min), most likely due to the continuous release of small vesicles and the consumption of the GUV.


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)

GTP-dependent vesicle release leads to shrinkage of parent GUVs. GUVs and protein labeled as in Fig. 3. A, time course of a GUV with bound dynamin and endophilin in the absence (−GTP) and presence of GTP. Z-stacks (−GTP and 60 min) show volume shrinkage of the GUV. B, time course of GUV shrinkage under the indicated conditions. C, quantitation of time courses of at least five independent vesicle preparations (mean with S.E.). D, quantitation of the vesiculation in the presence of GTPγS. E, quantitation of vesiculation in the presence of a peptide (P4) that disrupts the amphiphysin-dynamin interaction. Vesicle shrinkage was calculated according to (D0 − D60) × 100/D0. D60 is the vesicle diameter after 60 min, and D0 is the vesicle diameter at the beginning of the experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: GTP-dependent vesicle release leads to shrinkage of parent GUVs. GUVs and protein labeled as in Fig. 3. A, time course of a GUV with bound dynamin and endophilin in the absence (−GTP) and presence of GTP. Z-stacks (−GTP and 60 min) show volume shrinkage of the GUV. B, time course of GUV shrinkage under the indicated conditions. C, quantitation of time courses of at least five independent vesicle preparations (mean with S.E.). D, quantitation of the vesiculation in the presence of GTPγS. E, quantitation of vesiculation in the presence of a peptide (P4) that disrupts the amphiphysin-dynamin interaction. Vesicle shrinkage was calculated according to (D0 − D60) × 100/D0. D60 is the vesicle diameter after 60 min, and D0 is the vesicle diameter at the beginning of the experiment.
Mentions: Different models for membrane scission by dynamin exist. The two published dynamin structures propose a mechanism of how GTP hydrolysis could be coupled to membrane scission (40, 41), and a very recently published model is taking physical parameters of the membrane into account (42). Although our assay does not allow us to test for possible scission mechanisms, we can ask the following question. What is the likely consequence of cooperative membrane binding on dynamin-dependent membrane scission? It has been shown in the past that dynamin is sufficient to release small vesicles from membrane templates in a GTP-dependent manner (6, 43), and dynamin is capable of causing scission of membrane tethers (44, 45). Using GUVs, we observed that addition of dynamin resulted in sporadic tubular structures on the vesicle surfaces and that those structures were released in response to GTP addition (results not shown). However, these events were rather infrequent, and thus we asked if we would see a difference in the presence of BAR domain proteins. In the presence of dynamin and endophilin, GUVs were stable for hours (Fig. 5A, −GTP). However, subsequent addition of GTP led to a visible release of small vesicles from the GUVs (Fig. 5A, arrows). Following the same GUV over a longer period of time revealed a significant volume reduction (Fig. 5A, 60 min), most likely due to the continuous release of small vesicles and the consumption of the GUV.

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