(A) Morphology of mitochondria (MitoTracker; red) in WT, Tmem135FUN025/FUN025 (FUN025) and Tg-Tmem135 MFs. Scale bar = 10 μm. (B) Scoring of mitochondrial network morphologies in WT, FUN025 and Tg-Tmem135 MFs. Data from n = 290 WT, n = 304 FUN025, and n = 372 Tg-Tmem135 cells; 3 mice per genotype. (C–E) Quantification of size, number and coverage of mitochondria in WT, FUN025 and Tg-TMEM135 MFs. Data from n = 49 WT, n = 104 FUN025, and n = 29 Tg-Tmem135 cells; 3 mice per genotype. (F) Knocking down Tmem135 by siRNA against Tmem135 results in increased mitochondrial size in WT MFs. Data from n = 72 cells with scrambled siRNA, n = 81 cells with Tmem135 siRNA. Two tailed, unpaired, Student’s t-test. (G) EM revealed the morphology of mitochondria in RPE and inner segments of photoreceptor cells from wild-type and FUN025 mice. m = mitochondria; m.g. = melanin granules; n = necleus; o.s. = outer segments; i.s. = inner segments. Scale bar = 1 μm. (H) Quantification of mitochondria size in RPE and inner segments of photoreceptor cells from wild-type and FUN025 mice. RPE data from 300 mitochondria from three wild-type mice and 400 mitochondria from four FUN025 mice. Inner segments data from 200 mitochondria from four wild-type mice and 200 mitochondria from four FUN025 mice. (I) Immunofluorescence of DRP1 (anti DRP1 antibody, green) and mitochondria (MitoTracker, red) in WT and FUN025 MFs. Scale bar = 10 μm. (J–K) Quantification of DRP1 puncta density and fluorescence intensity on mitochondria. (L) Time-lapse fluorescence imaging (modified as described in Materials and methods) of a living HT22 cell expressing TMEM135-GFP and labeled with MitoTracker at the indicated time points. TMEM135 puncta that are located relatively close to the mitochondrial surface are shown in magenta, whereas those relatively far from the mitochondrial surface are shown in turquoise. Mitochondria (gray) was made partial transparent in order to see TMEM135 puncta located on the back of mitochondria. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA. All data are mean ± s.e.m.