Journal: Nature Communications

Article Title: ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division

doi: 10.1038/s41467-021-25621-4

Figure Lengend Snippet: a Soluble GAI-Sac1 can be recruited to mitochondrial fission site using GID1-MFF upon GA 3 -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 GA 3 -AM treatment (10 µM). Scale bar: 10 µm. c Normalized rate of mitochondrial division before and after GA 3 -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 GA 3 -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 GA 3 -AM (10 µM) treatment. Scale bar: 10 µm. j Normalized rate of mitochondrial division in ORP1L KO HeLa cells expressing the cytosolic mTq 2 -GAI-ΔANKORP1L D478A, mApple-TOM20, GFP-ORP1LΔORD, and iRFP-GID1-Rab7 before and after GA 3 -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 mTq 2 -GAI-ΔANKORP1L D478A, mApple-TOM20 and iRFP-GID1-Rab7 before and after GA 3 -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.

Article Snippet: The following constructs were made for this study: 3HA-Rab7, 3HA-Rab7, 3HA-Rab7 Q67L and 3HA-Rab7 T22N were generated by subcloning Rab7, Rab7 Q67L and Rab7 T22N into pcDNA3.1-3xHA-C1; mApple-Drp1 was made by replacing GFP by mApple (from mApple-TOM20) in the GFP-Drp1 plasmid; CFP-GAI-Sac1 was generated by subcloning Sac1 from GFP-ORPSAC1 into CFP-GAI(1-92) (gift from Takanari Inoue, Addgene # 37307) and CFP-GAI-Sac1 C392S was generated from CFP-GAI-Sac1 via site directed mutagenesis; GFP-ORPOSBP was made by replacing amino acid 334 to 950 of ORP1L from GFP-ORP1L by amino acid 184 to 809 of OSBP from mCherry-OSBP (gift from John Brumell, Hospital for Sick Children, Toronto); GFP-ORPOSBP HH/AA was made from GFP-ORPOSBP using site directed mutagenesis; iRFP-GID1-MFF was made by replacing Rab7 from iRFP-GID1-Rab7 by MFF from GFP-MFF (gift from Glia Voeltz, Addgene #49153); GFP-Sac1-MFF and GFP-Sac1 C392S-MFF were made by introducing wild-type and C392S mutant Sac1, from CFP-GAI-Sac1 and CFP-GAI-Sac1 C392S respectively, between GFP and MFF in the GFP-MFF plasmid.

Techniques: De-Phosphorylation Assay, Expressing, Mutagenesis, Construct