Figure 2

Drp1 helical self-assembly is not essential for cooperative GTPase activity. (A) SEC-MALS profiles of full-length Drp1 WT and Drp1 G350D each loaded at 10 μM onto a Superose 6 10/300 GL column. (B) The basal GTPase activity of Drp1 G350D relative to Drp1 WT was measured in the absence of target membranes at 37 °C in solution. (C) Representative EM images of Drp1 WT and Drp1 G350D polymers formed in the presence of GMP-PCP in solution. Scale bar, 200 nm. Inset, magnified images of helical Drp1 WT polymers. Inset scale bar, 50 nm. (D) Lipid-stimulated GTPase activity of Drp1 G350D relative to Drp1 WT was measured at 37 °C upon incubation with 25 mol% CL-containing liposomes. (E) Representative confocal images of RhPE-labeled, 25 mol% CL-containing GUVs (red) upon incubation with BODIPY-FL-labeled (BOD) Drp1 WT or Drp1 G350D (green). Both merged and individual color panels are shown for Drp1 G350D. Only the merged color panel is shown for Drp1 WT (left). Extensive membrane tubulation (arrow) is observed for Drp1 WT in contrast to Drp1 G350D. Scale bar, 5 μm. (F) Trp-dansyl FRET-sensitized increase in dansyl emission intensity upon Drp1 Trp excitation is plotted as F/F₀. F₀ is the background dansyl emission intensity prior to Drp1 addition, and F is dansyl emission intensity at time ‘t’ after Drp1 addition. Representative traces with error bars are shown. (G) Representative EM images of Drp1 WT-decorated membrane tubes drawn from 25 mol% CL-containing liposomes by Drp1 helical self-assembly, prior to (left panel) and after addition of GTP for 2 min (middle two panels), shown in comparison to Drp1 G350D-bound, non-deformed liposomes in the presence of GTP for 2 min (right panel). Arrows point to local membrane tube constriction effected by Drp1 WT upon GTP hydrolysis. Scale bar, 200 nm.