Fig. 7

Drp1 mediates second phasic mitochondrial fragmentation. a–c Representative images showing the targeting of Drp1 to fragmented mitochondria in Mul1-deficient neurons. Cortical neurons at DIV7-8 were co-transfected with GFP and Mul1-shRNA or scr-shRNA, followed by co-immunostaining of Drp1 (red) and cyto c (green) at DIV14-15 (a) or DIV13 (b) (pre-fission status). GFP (pseudo blue) was used to trace transfected neurons. Arrows point to Drp1 puncta on fragmented mitochondria (a). Average number of Drp1 puncta per 5 µm mitochondrial length was measured from the total number of mitochondria (scr-shRNA, n = 518; Mul1-shRNA, n = 423) in the total number of neurons indicated in the bars. d A representative immunoblot showing an increased Drp1 translocation to mitochondria in Mul1 depleted neurons. Equal amounts (15 µg) of mitochondrial fractions were sequentially immunoblotted on the same membrane with each antibody after stripping. Note that a shorter Drp1 isoform (~58 kDa) was recruited into mitochondrial fraction in Mul1-deficient neurons. e–g Drp1-dependent mitochondrial fragmentation in Mul1-deficient neurons. Cortical neurons at DIV7-8 were co-transfected with Mul1-shRNA, DsRed-Mito and Myc-tagged plasmid (Mock) or Drp1K38A, or co-transfected with Flag-tagged Mul1ΔRing and Mock or Drp1K38A, followed by fixation and imaging at DIV14-15. Note that expressing Drp1K38A locks mitochondrial in hyperfusion status in the late stage of Mul1-deficient neurons. All data were analyzed from the total number of neurons indicated in the bars in three experiments and are expressed as mean ± s.e.m. Unpaired Student’s t-test. Scale Bars: 10 μm