MT association sequesters Drp1 away from mitochondria and inhibits mitochondrial fission and apoptosis. (A and B) HeLa cells transfected with mitoRFP and the indicated GFP-Drp1 splice variants were fixed and assessed for colocalization of Drp1 with mitochondria. Representative epifluorescence images are shown in A (means ± SEM [up]/SD [down] of 125–184 cells in B). (C) HeLa cells transfected as indicated and treated with vehicle, the NO donor 2,2′-(hydroxynitrosohydrazono)bis-ethanimine (DETA-NO; 1 mM, 18 h), or the uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 1 µM, 30 min) in combination with the ATP synthase inhibitor oligomycin (0.1 µM) were immunolabeled for GFP and mitochondria (cytochrome oxidase II) and analyzed for mitochondrial morphology (means ± SEM [up]/SD [down] of ∼100 cells). (D–F) Rat primary astroglia with replacement of endogenous with GFP-Drp1-010 or -001 were treated for up to 6 h with 1 µM staurosporine and scored for shape of mitochondria (mitoRFP) and apoptosis (condensation/fragmentation of nuclei stained with Hoechst 33342). (D) Representative epifluorescence images with arrowheads pointing to Drp1 foci on mitochondria. (E) Mitochondrial morphometry (means ± SEM of 127–140 astrocytes). (F) Counts of apoptotic nuclei (means ± SD of four wells) from representative experiments. (G and H) Drp1 KO MEFs transfected as indicated were challenged with 1 µM staurosporine (9 h) and GFP-positive cells with apoptotic nuclei (arrows [G] were counted [H]; means ± SEM, n = 3). ○, P < 0.05; Ø, P < 0.01; ⊗, P < 0.0005; *, P < 10⁻⁷ compared with all cytosolic Drp1 splice variants.