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Electrophysiological Methods for Detection of Membrane Leakage and Hemifission by Dynamin 1

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Dynamin Superfamily GTPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2159))

Abstract

Membrane fusion and fission are indispensable parts of intracellular membrane recycling and transport. Electrophysiological techniques have been instrumental in discovering and studying fusion and fission pores, the key intermediates shared by both processes. In cells, electrical admittance measurements are used to assess in real time the dynamics of the pore conductance, reflecting the nanoscale transformations of the pore, simultaneously with membrane leakage. Here, we described how this technique is adapted to in vitro mechanistic analyses of membrane fission by dynamin 1 (Dyn1), the protein orchestrating membrane fission in endocytosis. We reconstitute the fission reaction using purified Dyn1 and biomimetic lipid membrane nanotubes of defined geometry. We provide a comprehensive protocol describing simultaneous measurements of the ionic conductance through the nanotube lumen and across the nanotube wall, enabling spatiotemporal correlation between the nanotube constriction by Dyn1, leading to fission and membrane leakage. We present examples of “leaky” and “tight” fission reactions, specify the resolution limits of our method, and discuss how our results support the hemi-fission conjecture.

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Acknowledgments

The work was partially supported by the Russian Foundation for Basic Research (project # 17-04-02042) and the Spanish Ministry of Science, Innovation and Universities grants BFU2015-70552-P and PGC2018-099971-B-I00 (MCIU/AEI/FEDER, UE).

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Authors and Affiliations

  1. Federal Research and Clinical Center of Physical-Chemical Medicine, Moscow, Russia

    Pavel V. Bashkirov & Ksenia V. Chekashkina

  2. A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia

    Pavel V. Bashkirov & Ksenia V. Chekashkina

  3. Department of Biochemistry and Molecular Biology, Biophysics Institute (CSIC, UPV/EHU), University of the Basque Country, Leioa, Spain

    Anna V. Shnyrova & Vadim A. Frolov

  4. IKERBASQUE, Basque Foundation for Science, Bilbao, Spain

    Vadim A. Frolov

Authors
  1. Pavel V. Bashkirov
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  2. Ksenia V. Chekashkina
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  3. Anna V. Shnyrova
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  4. Vadim A. Frolov
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Corresponding author

Correspondence to Vadim A. Frolov .

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Editors and Affiliations

  1. School of Medicine, Case Western Reserve University, Cleveland, OH, USA

    Rajesh Ramachandran

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Bashkirov, P.V., Chekashkina, K.V., Shnyrova, A.V., Frolov, V.A. (2020). Electrophysiological Methods for Detection of Membrane Leakage and Hemifission by Dynamin 1. In: Ramachandran, R. (eds) Dynamin Superfamily GTPases. Methods in Molecular Biology, vol 2159. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0676-6_11

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  • DOI: https://doi.org/10.1007/978-1-0716-0676-6_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0675-9

  • Online ISBN: 978-1-0716-0676-6

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