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

Abstract

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.

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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

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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Competing interests

The authors declare no competing financial interests.

Supplementary information

41596_2017_BFnprot2016173_MOESM30_ESM.avi

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)

41596_2017_BFnprot2016173_MOESM31_ESM.avi

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)

41596_2017_BFnprot2016173_MOESM32_ESM.avi

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)

41596_2017_BFnprot2016173_MOESM33_ESM.avi

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). https://doi.org/10.1038/nprot.2016.173

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