Dynamin, a crucial factor in endocytosis1,2,3, is a member of a family of GTPases that participates in membrane fission4,5,6. It was initially proposed to act as a machine that constricts and cuts the neck of nascent vesicles in a GTP-hydrolysis-dependent reaction4,5, but subsequent studies suggested alternative models2,7,8. Here we monitored the effect of nucleotides on dynamin-coated lipid tubules in real time. Addition of GTP, but not of GDP or GTP-γS, resulted in twisting of the tubules and supercoiling, suggesting a rotatory movement of the helix turns relative to each other during GTP hydrolysis. Rotation was confirmed by the movement of beads attached to the tubules. Twisting activity produced a longitudinal tension that was released by tubule breakage when both ends of the tubule were anchored. Fission also occurred when dynamin and GTP were added to lipid tubules that had been generated from liposomes by the motor activity of kinesin on microtubules. No fission events were observed in the absence of longitudinal tension. These findings demonstrate a mechanoenzyme activity of dynamin in endocytosis, but also imply that constriction is not sufficient for fission. At the short necks of endocytic vesicles, other factors6,9,10 leading to tension may cooperate with the constricting activity of dynamin to induce fission11,12,13.
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We thank V. Unger, G. Di Paolo, M. Solimena and K. Erdmann for discussion and critical reading of this manuscript. We thank F. Wilson for technical support. This work was supported by National Institutes of Health grants and a G. Harold and Leila Y. Mathers Charitable Foundation grant to P.D.C. A.R. was supported by the European Molecular Biology Organization (EMBO) Long-Term Postdoctoral Fellowship programme and the Cross-Disciplinary Fellowship programme of the Human Frontier Science Program (HFSP).Author Contributions A.R. and P.D.C. conceived the project, designed the experiments and evaluated the results. A.R. performed the experiments alone, with the exception of the giant-liposome assay (K.U. and A.R.) and electron microscopy (A.F., K.U. and A.R.). A.R. and P.D.C. wrote the paper.
Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
This file contains Supplementary Methods, Supplementary Figures 1–5 and Supplementary Movie Legends. (PDF 1200 kb)
Growth of dynamin coated tubules on sheets of lipid membranes observed by DIC microscopy. Speed, 30X. (AVI 8910 kb)
Effect of 1 mM GTP on a network of dynamin-coated lipid tubules. Real-time (15 fps) DIC microscopy. (AVI 3846 kb)
Effect of 1 mM GTP on a dynamin-coated lipid tubule anchored at static point at both ends. Real-time (15 fps) DIC microscopy. (AVI 2852 kb)
Effect of 1 mM GTP on a dynamin-coated lipid tubule free to retract. Real-time (15 fps) DIC microscopy. (AVI 1644 kb)
Effect of 1 mM GTP, in the absence of ATP, on membrane tubules generated from giant liposomes by kinesin on microtubules and subsequently coated with dynamin. Speed, 60X (AVI 1860 kb)
Effect of 1mg/ml dynamin co-added with 0.5 mM GTP in the presence of ATP on membrane tubules generated by kinesin on microtubules. Speed, 15X (AVI 9278 kb)
1 mM GTP induces supercoiling of loops of dynamin-coated tubules. Same field as Fig. 4a. Real-time (15 fps) DIC microscopy. (AVI 1386 kb)
Rotation of a streptavidin bead (diameter 260 nm) attached to a biotin-dynamin coated tubule after addition of 200 μM GTP. Real time (30fps). (AVI 8750 kb)
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Roux, A., Uyhazi, K., Frost, A. et al. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature 441, 528–531 (2006). https://doi.org/10.1038/nature04718
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