Figure 5.
CCDC51 spatially demarcates a subset of mitochondrial fission events. (A) Single-plane confocal images are shown at the indicated time intervals (s = seconds) of yeast cells expressing endogenously-tagged GFP-Mdm33 and mito-dsRed. Arrows mark sites of GFP-Mdm33 focal enrichment relative to mitochondrial fission events. The table at bottom characterizes mitochondrial fission events marked by Mdm33 and their expected frequency due to chance (n = 73 fission events characterized). Asterisks (****P < 0.0001) represent a Fisher’s exact test. See also Videos 4 and 5. (B) Single-plane confocal images are shown as in A of U2OS cells expressing low levels of GFP-CCDC51 and mito-mCherry. Arrows mark sites of GFP-CCDC51 focal enrichment relative to mitochondrial fission events. See also Videos 6, 7, and 8. The table at bottom characterizes mitochondrial fission events marked by CCDC51 and their expected frequency due to chance (n = 78 fission events characterized). Asterisks (****P < 0.0001) represent a Fisher’s exact test. (C) Representative single-plane confocal images as in B of U2OS cells expressing GFP-CCDC51, mCherry-Drp1, and mito-TagBFP. Arrows mark examples of GFP-CCDC51 and mCherry-Drp1 co-localization at sites of mitochondrial fission. See also Videos 9 and 10. (D) Single-plane confocal images of the indicated CRISPRi cells stained with MitoTracker Red and PicoGreen (mtDNA). Arrows mark examples of mtDNA nucleoids that are spatially linked to mitochondrial fission events. The table at the right characterizes the frequency of mtDNA coordination with fission and the spatial distribution of fission events with relation to mitochondrial morphology (n = 80 fission events per condition). N.S. indicates not statistically significant results of a Fisher’s exact test of mtDNA proximity to fission events. (E–G) 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 HSP60 and (E) Drp1, (F) MFF, or (G) dsDNA (mtDNA). Yellow arrows (E and F) mark examples of co-localization of GFP-CCDC51 foci with enrichments of Drp1 and MFF, respectively. White arrows in G mark examples of mtDNA nucleoids that localize adjacent to, but are not co-localized with, GFP-CCDC51 foci (yellow arrows). (H) A graph categorizing the spatial relationship between GFP-CCDC51 foci and enrichments of Drp1, MFF, and mtDNA from cells as in E–G. Data represent 100 GFP-CCDC51 foci characterized per condition. Scale bars: (A, B, and D) 3 µm; (C) 2 µm; and (E–G) 5 µm.

CCDC51 spatially demarcates a subset of mitochondrial fission events. (A) Single-plane confocal images are shown at the indicated time intervals (s = seconds) of yeast cells expressing endogenously-tagged GFP-Mdm33 and mito-dsRed. Arrows mark sites of GFP-Mdm33 focal enrichment relative to mitochondrial fission events. The table at bottom characterizes mitochondrial fission events marked by Mdm33 and their expected frequency due to chance (n = 73 fission events characterized). Asterisks (****P < 0.0001) represent a Fisher’s exact test. See also Videos 4 and 5. (B) Single-plane confocal images are shown as in A of U2OS cells expressing low levels of GFP-CCDC51 and mito-mCherry. Arrows mark sites of GFP-CCDC51 focal enrichment relative to mitochondrial fission events. See also Videos 6, 7, and 8. The table at bottom characterizes mitochondrial fission events marked by CCDC51 and their expected frequency due to chance (n = 78 fission events characterized). Asterisks (****P < 0.0001) represent a Fisher’s exact test. (C) Representative single-plane confocal images as in B of U2OS cells expressing GFP-CCDC51, mCherry-Drp1, and mito-TagBFP. Arrows mark examples of GFP-CCDC51 and mCherry-Drp1 co-localization at sites of mitochondrial fission. See also Videos 9 and 10. (D) Single-plane confocal images of the indicated CRISPRi cells stained with MitoTracker Red and PicoGreen (mtDNA). Arrows mark examples of mtDNA nucleoids that are spatially linked to mitochondrial fission events. The table at the right characterizes the frequency of mtDNA coordination with fission and the spatial distribution of fission events with relation to mitochondrial morphology (n = 80 fission events per condition). N.S. indicates not statistically significant results of a Fisher’s exact test of mtDNA proximity to fission events. (E–G) 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 HSP60 and (E) Drp1, (F) MFF, or (G) dsDNA (mtDNA). Yellow arrows (E and F) mark examples of co-localization of GFP-CCDC51 foci with enrichments of Drp1 and MFF, respectively. White arrows in G mark examples of mtDNA nucleoids that localize adjacent to, but are not co-localized with, GFP-CCDC51 foci (yellow arrows). (H) A graph categorizing the spatial relationship between GFP-CCDC51 foci and enrichments of Drp1, MFF, and mtDNA from cells as in E–G. Data represent 100 GFP-CCDC51 foci characterized per condition. Scale bars: (A, B, and D) 3 µm; (C) 2 µm; and (E–G) 5 µm.

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