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. 2021 Sep 9;12:5354. doi: 10.1038/s41467-021-25621-4

Fig. 7. The recruitment of the PI(4)P phosphatase Sac1 to mitochondria impairs their division.

Fig. 7

a Soluble GAI-Sac1 can be recruited to mitochondrial fission site using GID1-MFF upon GA3-AM treatment, leading to dephosphorylation of PI(4)P at the mitochondrial division site. b Representative image of a HeLa cell expressing CFP-GAI-Sac1, iRFP-GID1-MFF (not imaged) and mApple-TOM20 before and after GA3-AM treatment (10 µM). Scale bar: 10 µm. c Normalized rate of mitochondrial division before and after GA3-AM (10 µM) treatment in HeLa cells overexpressing the wild-type CFP-GAI-Sac1 or the inactive C392S mutant, iRFP-GID1-MFF and mApple-TOM20. When treated with GA3-AM (10 µM) cells were imaged between 5 and 25 min of treatment. Cells from three independent experiments. Two-way ANOVA, Sidak’s multiple comparisons test. d In GFP-Sac1-MFF, Sac1 was fused to MFF to anchor it directly to the outer mitochondrial membrane leading to dephosphorylation of PI(4)P at the mitochondrial division site. e Representative maximum projection images of HeLa cells expressing GFP, GFP-MFF, GFP-Sac1-MFF or the catalytic inactive GFP-Sac1 C292S-MFF and mApple-TOM20. Inset shows the morphology of the mitochondrial network. Scale bars: 10 µm and 5 µm (inset). f–h Mitochondrial morphology was quantified for f mean area per mitochondrion, g mitochondrial number per region of interest (ROI), and h number of junctions per mitochondria. Cells from three independent experiments. One-way ANOVA with Tukey’s Multiple Comparison Test. i Representative images of ORP1L KO HeLa cells expressing the indicated constructs before and after GA3-AM (10 µM) treatment. Scale bar: 10 µm. j Normalized rate of mitochondrial division in ORP1L KO HeLa cells expressing the cytosolic mTq2-GAI-ΔANKORP1L D478A, mApple-TOM20, GFP-ORP1LΔORD, and iRFP-GID1-Rab7 before and after GA3-AM (10 µM) treatment. Cells from three independent experiments. Two-sided unpaired t-test. k Normalized rate of mitochondrial division in ORP1L KO HeLa cells expressing the cytosolic mTq2-GAI-ΔANKORP1L D478A, mApple-TOM20 and iRFP-GID1-Rab7 before and after GA3-AM (10 µM) treatment. Cells from three independent experiments. Two-sided unpaired t-test. c, f–h, j, k All graphs show the mean ± SEM. ns non-significant: f p = 0.7632, g p = 0.3592, h p = 0.7935, and k p = 0.5192.