{Reference Type}: Journal Article
{Title}: Electrophysiological Methods for Detection of Membrane Leakage and Hemifission by Dynamin 1.
{Author}: Bashkirov PV;Chekashkina KV;Shnyrova AV;Frolov VA;
{Journal}: Methods Mol Biol
{Volume}: 2159
{Issue}: 0
{Year}: 2020
暂无{DOI}: 10.1007/978-1-0716-0676-6_11
{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.