Drp1-Mediated Lipid Membrane Dynamics

Drp1 and the fluorescent constructs were dialyzed overnight at 4 °C against 20 mM HEPES pH 7.4 buffer with 150 mM NaCl and spun down at 100,000 × g to remove aggregates. Cardiolipin (CL) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (both from Avanti Polar Lipids) were aliquoted in a 25:75 molar ratio into a clean glass test tube, dried under vacuum for 30 min and hydrated in 20 mM HEPES pH 7.4 buffer with 150 mM KCl for 1 h at 50 °C. Samples were vortexed vigorously to prepare multilamellar vesicles. Unilamellar vesicles were prepared by extruding the multilamellar vesicles through a 100-nm pore size filter (Avanti Polar Lipids). Drp1 (1 μM) was mixed with GTP (Jena Biosciences, Germany) (1 mM) in the absence or presence of liposomes (100 μM) in 20 mM HEPES pH 7.4 buffer with 150 mM KCl and 1 mM MgCl₂ and incubated at 37 °C. Aliquots were taken at regular intervals and quenched with 5 mM EDTA. Inorganic phosphate released was assayed with the malachite green reagent according to ref. ^{15 (link)}.