Fig 2: TMEM135 is involved in the balance of mitochondrial fission and fusion.(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.DOI: http://dx.doi.org/10.7554/eLife.19264.009

Fig 3: Tmem135FUN025/FUN025mice are more sensitive to hyperoxic condition.(A–B) Tmem135FUN025/FUN025 (FUN025) mice raised in 75% O2 for two weeks show significant decrease of ONLT and increase of TUNEL positive cells, indicating accelerated photoreceptor cell death by apoptosis. Data from n = 4 WT mice in control air, n = 3 WT mice in 75% O2, n = 3 FUN025 mice in control air, and n = 3 FUN025 mice in 75% O2. Scale bar = 20 µm. (C) Upregulation of GFAP (green) indicating retinal stress is observed in FUN025 mice raised in the normal air, as well as WT mice and FUN025 mice raised in 75% O2 for two weeks. FUN025 mice raised in 75% O2 have the highest increase of GFAP signals in the retina. Scale bar = 20 µm. (D) Western blotting showing that hyperoxia results in upregulation of 4-HNE in both WT and FUN025 retina, and an increase of TMEM135 in FUN025 retina but not in WT retina. Data from n = 3 WT mice in control air, n = 3 WT mice in 75% O2, n = 3 FUN025 mice in control air, and n = 3 FUN025 mice in 75% O2. *p<0.05, **p<0.01, two-way ANOVA. All data are mean ± s.e.m.DOI: http://dx.doi.org/10.7554/eLife.19264.018

Fig 4: Tmem135 plays a role in mitochondrial metabolism.(A) Mitochondrial membrane potential (??M) in Tg-Tmem135 MFs was higher than that in WT MFs, whereas ??M in Tmem135FUN025/FUN025 (FUN025) MFs was comparable to that in WT MFs. Data from n = 3 mice per genotype. n = 1 for WT+CCCP. (B–G) Oxygen consumption rates (OCR) of MFs were determined with a Seahorse XFe24 Extracellular Flux Analyzer in basal and stimulated conditions (n = 3 mice per genotypes). The areas under the curve from different sections of the experiment (B) are shown as individual histograms for basal respiration, ATP production, maximal respiration, spare respiratory capacity, and proton leak (C–G). Data from n = 3 mice per genotype. (H–I) Total ROS and superoxide in FUN025 and Tg-Tmem135 MFs were higher compared to WT cells. Data from n = 3 mice per genotype. (J) Western blot analysis for SOD1, SOD2, SOD3, GPx1and CAT in WT and FUN025 MFs with and without the hydrogen peroxide treatment. Protein levels were normalized using GAPDH expression levels. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA. All data are mean ± s.e.m..DOI: http://dx.doi.org/10.7554/eLife.19264.015

Fig 5: Identification of a Tmem135 mutation in FUN025 mice.(A) Minimal genetic region of FUN025 on chromosome 7 determined by genetic mapping. (B) A point mutation (T > C) in the splice-donor site adjacent to exon 12 of Tmem135 in FUN025 mice. (C) The consensus sequence of mouse splice donor sites, depicting the necessity of the GT sequence at positions 1 and 2 downstream of the exon boundary for the functionality of the site. The C57BL/6J and FUN025 sequences are shown below, demonstrating the disrupted site in FUN025 mice. (D) RT-PCR spanning exon 12 and sequencing the product revealed the absence of this exon in the FUN025 retina. (E) Amino acid sequences of the C-terminus of TMEM135 in C57BL/6J and FUN025 mice. The WT protein is 458 amino acids long, whereas the truncated mutant protein is 406. The change in amino acid sequence is highlighted in red. (F) Consequences of the mutation in the genomic sequence of Tmem135. The mutation adjacent to exon 12 of Tmem135 (red star) results in a non-functional splice donor site, causing skipping of exon 12. This results in a frameshift and an early stop codon (chr7: 96,290,429, NCBI build 37). Locations of the stop codons are highlighted in red. (G) A predicted structure of TMEM135 having five transmembrane domains. The FUN025 mutation is predicted to result in a protein with only three transmembrane domains, whose orientation in the membrane is reversed. The rest of the c-terminal region is absent due to the early stop codon (asterisk). (H) Western blot for TMEM135 in WT and FUN025 brains. GAPDH was used as a loading control.DOI: http://dx.doi.org/10.7554/eLife.19264.005