Figure 6. MUL1’s function in mitochondrial morphology and Mfn levels is conserved in human cells.
(A–D‴) HeLa cells transfected with GFP-MUL1 (A–B″) or GFP-MUL1 LD (C–D″) are marked with asterisks, while cells not transfected serve as internal controls. Mitochondria are labeled with mitotracker in red (B and D). (B′ and B″, D′ and D″) Higher magnification images of mitochondria within white boxes in B and D. Cells expressing GFP-MUL1 have clustered mitochondria in the perinuclear region (B). Mitochondria are also small and fragmented (B″), as compared to cells not expressing GFP-MUL1 (B′). Importantly, GFP-MUL1 LD does not result in localization of mitochondria to the perinuclear region (D) or in mitochondrial fragmentation (D′). (E) Western blot analysis of Mfn1 and Mfn2 levels after CHX treatment. HeLa cells expressing scrambled shRNA or MUL1 shMUL1 are treated with CHX. Mfn1 and 2 levels at each time point are normalized with Actin. The relative portion of remaining Mfn1 and 2 as compared to time point 0 was calculated and plotted (E). In cells expressing MUL1 shRNA, Mfn1 and 2 levels after CHX treatment are more stable than those in cells expressing scrambled shRNA. (F) Expression of transfected GFP-MUL1 and GFP-MUL1 LD in HeLa cells, as detected using anti-GFP antibody. (G) Western blot analysis of endogenous MUL1 levels in HeLa cells stably expressing scrambled shRNA and MUL1 shRNA. MUL1 shRNA expressing cells have reduced levels of endogenous MUL1. (H) Human MUL1 sequence and deletion in MUL1 knockout (MUL1−/−) HeLa cells, generated using the CRISPR/Cas 9 system. Sequences targeting MUL1 are highlighted in blue. Red letters indicate start codon. Red dashes represent deleted bases. Deleted eight base pairs include the start codon of MUL1. (I) Western blot analysis of Mfn1 and Mfn2 levels in wild-type and MUL1−/− HeLa cells treated with CHX for the indicated time. Remaining Mfn1 and Mfn2 levels at each time point were plotted below. (J) Western blot showing no MUL1 expression in MUL1−/− HeLa. Arrowhead points to MUL1 protein. Asterisk indicates a non-specific band.