Figure 3.
Overexpression of CCDC51 promotes Drp1-dependent mitochondrial fission. (A) Maximum projection confocal microscopy images of the indicated MitoTracker Red-stained U2OS CRISPRi cells expressing empty vector or low levels of GFP-CCDC51. Insets display MitoTracker staining corresponding to the dashed boxes. (B) A graph of mitochondrial morphology categorization from cells as in A. Data shown represent 50 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (***P < 0.001) represent an unpaired two-tailed t test of tubular mitochondrial morphology. (C) A representative single-plane confocal microscopy image is shown of a CCDC51 CRISPRi cell, as in A, expressing GFP-CCDC51 and labeled with MitoTracker Red. Arrows mark sites of GFP-CCDC51 concentration at discrete foci along mitochondria. (D) A representative single-plane confocal microscopy image is shown of a U2OS cell fixed and immunolabeled for CCDC51 and HSP60. The arrow marks a site of CCDC51 enrichment. (E) Representative single-plane confocal images are shown of U2OS cells expressing empty vector (top) or high levels of GFP-CCDC51 (bottom) that were fixed and immunolabeled for TOMM20 and HSP60. Arrows mark examples of GFP-CCDC51 enrichment at discrete foci. (F) A graph is shown of mitochondrial morphology characterization from cells as in E. Data shown represents 50 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (****P < 0.0001) represent an unpaired two-tailed t test of tubular mitochondrial morphology. (G) As in F for wild-type and Drp1 KO HeLa cells. Corresponding images are shown in Fig. S3 B. (H) Representative maximum intensity confocal images are shown of HeLa Drp1 KO cells expressing empty vector (top) or high levels of GFP-CCDC51 (bottom) and co-expressing TIMM50-mCherry and BFP-OMP25. Arrows mark sites of focal accumulation of GFP-CCDC51. See also Fig. S3 C for corresponding wild-type cells. Scale bars: (A) 15 µm (5 µm in inset); (C) 5 µm; (D) 5 µm; (E) 5 µm; and (H) 5 µm.

Overexpression of CCDC51 promotes Drp1-dependent mitochondrial fission. (A) Maximum projection confocal microscopy images of the indicated MitoTracker Red-stained U2OS CRISPRi cells expressing empty vector or low levels of GFP-CCDC51. Insets display MitoTracker staining corresponding to the dashed boxes. (B) A graph of mitochondrial morphology categorization from cells as in A. Data shown represent 50 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (***P < 0.001) represent an unpaired two-tailed t test of tubular mitochondrial morphology. (C) A representative single-plane confocal microscopy image is shown of a CCDC51 CRISPRi cell, as in A, expressing GFP-CCDC51 and labeled with MitoTracker Red. Arrows mark sites of GFP-CCDC51 concentration at discrete foci along mitochondria. (D) A representative single-plane confocal microscopy image is shown of a U2OS cell fixed and immunolabeled for CCDC51 and HSP60. The arrow marks a site of CCDC51 enrichment. (E) Representative single-plane confocal images are shown of U2OS cells expressing empty vector (top) or high levels of GFP-CCDC51 (bottom) that were fixed and immunolabeled for TOMM20 and HSP60. Arrows mark examples of GFP-CCDC51 enrichment at discrete foci. (F) A graph is shown of mitochondrial morphology characterization from cells as in E. Data shown represents 50 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (****P < 0.0001) represent an unpaired two-tailed t test of tubular mitochondrial morphology. (G) As in F for wild-type and Drp1 KO HeLa cells. Corresponding images are shown in Fig. S3 B. (H) Representative maximum intensity confocal images are shown of HeLa Drp1 KO cells expressing empty vector (top) or high levels of GFP-CCDC51 (bottom) and co-expressing TIMM50-mCherry and BFP-OMP25. Arrows mark sites of focal accumulation of GFP-CCDC51. See also Fig. S3 C for corresponding wild-type cells. Scale bars: (A) 15 µm (5 µm in inset); (C) 5 µm; (D) 5 µm; (E) 5 µm; and (H) 5 µm.

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