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Use of the supported membrane tube assay system for real-time analysis of membrane fission reactions


The process of membrane fission is fundamental to diverse cellular processes such as nutrient uptake, synaptic transmission and organelle biogenesis, and it involves the localized application of curvature stress to a tubular membrane intermediate, forcing it to undergo scission. Alternative techniques for creating such substrates necessitate the use of micromanipulators or sophisticated optical traps and require a high level of technical expertise. We present a facile method to generate an array of membrane tubes supported on a passivated glass coverslip, which we refer to as supported membrane tubes (SMrTs). SMrT templates are formed upon hydration of a dry lipid mix in physiological buffer and subsequent flow-induced extrusion of the lipid reservoir into long membrane tubes with variable dimensions. Following surface passivation of coverslips, these templates can be formed from a variety of lipids, with as little as 1–2 nmol of lipid in a matter of 2 h, and can be used in membrane-curvature-sensitive fission assays.

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Figure 1: Schematic of the generation of SMrT templates.
Figure 2: Covalent modification of glass surfaces with PEG.
Figure 3: Flow cell assembly and characterization.
Figure 4: Tube radius estimation.
Figure 5: Typical results.


  1. Koenig, J., Saito, K. & Ikeda, K. Reversible control of synaptic transmission in a single gene mutant of Drosophila melanogaster. J. Cell Biol. 96, 1517–1522 (1983).

    CAS  Article  Google Scholar 

  2. van der Bliek, A.M. et al. Mutations in human dynamin block an intermediate stage in coated vesicle formation. J. Cell Biol. 122, 553–563 (1993).

    CAS  Article  Google Scholar 

  3. Praefcke, G.J.K. & McMahon, H.T. The dynamin superfamily: universal membrane tubulation and fission molecules? Nat. Rev. Mol. Cell Biol. 5, 133–147 (2004).

    CAS  Article  Google Scholar 

  4. Schmid, S.L. & Frolov, V.A. Dynamin: functional design of a membrane fission catalyst. Annu. Rev. Cell Dev. Biol. 27, 79–105 (2011).

    CAS  Article  Google Scholar 

  5. Ferguson, S.M. & De Camilli, P. Dynamin, a membrane-remodelling GTPase. Nat. Rev. Mol. Cell Biol. 13, 75–88 (2012).

    CAS  Article  Google Scholar 

  6. Miwako, I., Schröter, T. & Schmid, S.L. Clathrin- and dynamin-dependent coated vesicle formation from isolated plasma membranes. Traffic 4, 376–389 (2003).

    CAS  Article  Google Scholar 

  7. Takei, K. et al. Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes. Cell 94, 131–141 (1998).

    CAS  Article  Google Scholar 

  8. Kinuta, M. et al. Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol. Proc. Natl. Acad. Sci. USA 99, 2842–2847 (2002).

    CAS  Article  Google Scholar 

  9. Boucrot, E. et al. Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains. Cell 149, 124–136 (2012).

    CAS  Article  Google Scholar 

  10. Pucadyil, T.J. & Schmid, S.L. Supported bilayers with excess membrane reservoir: a template for reconstituting membrane budding and fission. Biophys. J. 99, 517–525 (2010).

    CAS  Article  Google Scholar 

  11. Neumann, S., Pucadyil, T.J. & Schmid, S.L. Analyzing membrane remodeling and fission using supported bilayers with excess membrane reservoir. Nat. Protoc. 8, 213–222 (2013).

    CAS  Article  Google Scholar 

  12. Pucadyil, T.J. & Schmid, S.L. Real-time visualization of dynamin-catalyzed membrane fission and vesicle release. Cell 135, 1263–1275 (2008).

    CAS  Article  Google Scholar 

  13. Morlot, S. et al. Membrane shape at the edge of the dynamin helix sets location and duration of the fission reaction. Cell 151, 619–629 (2012).

    CAS  Article  Google Scholar 

  14. Bashkirov, P.V. et al. GTPase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission. Cell 135, 1276–1286 (2008).

    CAS  Article  Google Scholar 

  15. Roux, A. et al. Membrane curvature controls dynamin polymerization. Proc. Natl. Acad. Sci. USA 107, 4141–4146 (2010).

    CAS  Article  Google Scholar 

  16. Shnyrova, A.V. et al. Geometric catalysis of membrane fission driven by flexible dynamin rings. Science 339, 1433–1436 (2013).

    CAS  Article  Google Scholar 

  17. Dar, S., Kamerkar, S.C. & Pucadyil, T.J. A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function. Nat. Cell Biol. 17, 1588–1596 (2015).

    CAS  Article  Google Scholar 

  18. Holkar, S.S., Kamerkar, S.C. & Pucadyil, T.J. Spatial control of epsin-induced clathrin assembly by membrane curvature. J. Biol. Chem. 290, 14267–14276 (2015).

    CAS  Article  Google Scholar 

  19. Pucadyil, T.J. & Holkar, S.S. Comparative analysis of adaptor-mediated clathrin assembly reveals general principles for adaptor clustering. Mol. Biol. Cell 27, 3156–3163 (2016).

    CAS  Article  Google Scholar 

  20. Hsieh, W.-T. et al. Curvature sorting of peripheral proteins on solid-supported wavy membranes. Langmuir 28, 12838–12843 (2012).

    CAS  Article  Google Scholar 

  21. Jung, H., Robison, A.D. & Cremer, P.S. Detecting proteinligand binding on supported bilayers by local pH modulation. J. Am. Chem. Soc. 131, 1006–1014 (2009).

    CAS  Article  Google Scholar 

  22. Kunding, A.H., Mortensen, M.W., Christensen, S.M. & Stamou, D. A fluorescence-based technique to construct size distributions from single-object measurements: application to the extrusion of lipid vesicles. Biophys. J. 95, 1176–1188 (2008).

    CAS  Article  Google Scholar 

  23. Bassereau, P., Sorre, B. & Lévy, A. Bending lipid membranes: experiments after W. Helfrich's model. Adv. Colloid Interface Sci. 208, 47–57 (2014).

    CAS  Article  Google Scholar 

  24. Frolov, V.A., Lizunov, V.A., Dunina-Barkovskaya, A.Y., Samsonov, A.V. & Zimmerberg, J. Shape bistability of a membrane neck: a toggle switch to control vesicle content release. Proc. Natl. Acad. Sci. USA 100, 8698–8703 (2003).

    CAS  Article  Google Scholar 

  25. Koster, G., VanDuijn, M., Hofs, B. & Dogterom, M. Membrane tube formation from giant vesicles by dynamic association of motor proteins. Proc. Natl. Acad. Sci. USA 100, 15583–15588 (2003).

    CAS  Article  Google Scholar 

  26. Baumgart, T., Capraro, B.R., Zhu, C. & Das, S.L. Thermodynamics and mechanics of membrane curvature generation and sensing by proteins and lipids. Annu. Rev. Phys. Chem. 62, 483–506 (2011).

    CAS  Article  Google Scholar 

  27. Sorre, B. et al. Nature of curvature coupling of amphiphysin with membranes depends on its bound density. Proc. Natl. Acad. Sci. USA 109, 173–178 (2012).

    CAS  Article  Google Scholar 

  28. Roux, A. et al. A minimal system allowing tubulation with molecular motors pulling on giant liposomes. Proc. Natl. Acad. Sci. USA 99, 5394–5399 (2002).

    CAS  Article  Google Scholar 

  29. Leduc, C. et al. Cooperative extraction of membrane nanotubes by molecular motors. Proc. Natl. Acad. Sci. USA 101, 17096–17101 (2004).

    CAS  Article  Google Scholar 

  30. Roux, A., Uyhazi, K., Frost, A. & De Camilli, P. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature 441, 528–531 (2006).

    CAS  Article  Google Scholar 

  31. Rossier, O., Cuvelier, D., Borghi, N., Puech, P.H. & Derényi, I. Giant vesicles under flows: extrusion and retraction of tubes. Langmuir 19, 575–584 (2003).

    CAS  Article  Google Scholar 

  32. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  Article  Google Scholar 

  33. Piehler, J., Brecht, A., Valiokas, R., Liedberg, B. & Gauglitz, G. A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces. Biosens. Bioelectron. 15, 473–481 (2000).

    CAS  Article  Google Scholar 

  34. Bernazzani, L. et al. On the interaction of sodium dodecyl sulfate with oligomers of poly(ethylene glycol) in aqueous solution. J. Phys. Chem. B 108, 8960–8969 (2004).

    CAS  Article  Google Scholar 

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S.D. and S.C.K. acknowledge the Council for Scientific and Industrial Research (CSIR) for fellowships. T.J.P. is an Intermediate Fellow of the Wellcome Trust-DBT India Alliance and thanks the Alliance and IISER Pune for funding.

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Authors and Affiliations



S.D., S.C.K. and T.J.P. developed the method. S.D. performed all experiments with dynamin. S.C.K. performed flow cell calibration. S.D., S.C.K. and T.J.P analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Thomas J Pucadyil.

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The authors declare no competing financial interests.

Supplementary information


A time-lapse sequence showing how flow rates can be estimated. The first 2 s of the video shows fluorescent liposomes moving in solution, following which the pump is turned on at a fine dial setting of 8. (AVI 2023 kb)


A time-lapse sequence showing how the lipid at the source is extruded into membrane tubes in response to buffer flow. The first 30 s of the video is acquired at a low flow rate to visualize tubules and buds formed at the source. These structures are subsequently extruded into membrane tubes upon increasing the flow rate. (AVI 3302 kb)


A time-lapse sequence showing the effect of flowing 0.5 μM dynamin in the presence of 1 mM GTP and 1 mM MgCl2 in HKS supplemented with oxygen scavengers into SMrT templates. (AVI 2752 kb)


A time-lapse sequence showing the effect of flowing 1 mM GTP and 1 mM MgCl2 in HKS into preassembled dynamin on SMrT templates. (AVI 1832 kb)

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Dar, S., Kamerkar, S. & Pucadyil, T. Use of the supported membrane tube assay system for real-time analysis of membrane fission reactions. Nat Protoc 12, 390–400 (2017).

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