Fig 1: Cell cycle regulation of CKS2 and CKS2-SSBP1 complex in HeLa cells by flow cytometry. (A) CKS2 signal in G1-, S-, and G2-phase of the cell cycle. (B) DNA histogram showing the six regions used for gating of data presented in panels C and D. (C) CKS2-SSBP1 PLA signal and DNA content in cells gated as indicated in panel B. (D) Relative CKS2-SSBP1 PLA signal in cells gated as indicated in panel B. PLA signal divided by DNA content is shown. (C, D) Data are shown relative to the first gate in G1-phase (region 1). (A, C, D) Each point represents mean value of at three (A) and five (C, D) independent experiments. The bars represent standard error of the mean. *, statistically significant difference (P < .05) compared to second region in G1-phase (region 2).
Fig 2: Loss of mitochondrial fusion affects replisome composition.(A) Left, representative western blot showing the steady-state levels of mitochondrial replication proteins from isolated MEF mitochondria from control, dMfn KO, Opa1 KO MEFs. Right, representative western blot showing the steady-state levels of mitochondrial replication proteins in isolated heart mitochondria from control and dMfn KO mice at 5–6 weeks of age. (B) Quantification of the steady-state levels of replisome proteins as determined by western blot analysis of mitochondria from control, dMfn KO, and Opa1 KO MEFs. Replisome protein levels were normalized to ATP5A (n = 7 per genotype). (C) Quantification of SSBP1 foci per cell in control, dMfn KO, and Opa1 KO MEFs. Cells were immunostained with anti-SSBP1 and anti-HSP60 antibodies, n = 4 for each genotype, 5–8 cells analyzed per n. (D) Quantification of mitochondrial replication proteins levels in isolated heart mitochondria from control (n = 3) and dMfn KO (n = 3) mice at 5–6 weeks of age, normalized to VDAC. (E) Representative western blot analysis of glycerol gradient fractions from mitochondria isolated from control MEFs. Upper panel corresponds to western blotting and lower panel shows Southern blot (SB) analysis to detect mtDNA by using the pAM1 probe. (F) Representative western blot analysis of fraction 1 from the glycerol density gradient. In total, 4 independent biological samples were used for all genotypes. (G) Quantification of the western blot analysis of fraction 1 from the glycerol density gradient related to (F), n = 4 for all genotypes. (H) Representative confocal image of control and dMfn KO MEFs immunostained with anti-SSBP1 and anti-dsDNA antibodies, n = 3 for each genotype. Scale bars represent 5 µm. (I) Line scan analysis based on intensity profiles of SSBP1 and mtDNA of control and dMfn KO MEFs, n = 3 for each genotype, 7–10 cells analyzed per n. Line scans generated intensity profiles that were separated into three categories, no overlap of intensities between SSBP1-mtDNA (free SSBP1), partial overlap between SSBP1-mtDNA intensities, and complete overlap of intensities between SSBP1-mtDNA. (J) Mander’s Coefficient, related to H, expressing the degree of mtDNA foci colocalizing with SSBP1 foci from confocal acquired images in control (n = 4 independent experiments, 23 cells analyzed per n) and dMfn KO (n = 3 independent experiments, 16 cells analyzed per n), Opa1 KO MEFs (n = 3 independent experiments, 22 cells analyzed per n). For all, error bars indicate ± SEM. (For D and I) Student T-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. For (B, C, G, and J), one-way ANOVA using Turkey’s multiple comparison test relative to control; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 3: SSBP1 protein expression in human retina and fibroblasts.(A) SSBP1 protein expression in human retina. Immunofluorescence labeling was done on retinal cross-sections from healthy human donor. Hoechst was added to label nuclei (blue). SSBP1 localization was done using a specific antibody (green). An antibody against the ATP synthase subunit 5A was used to target mitochondria (red). RPE, retinal pigment epithelium; POS, photoreceptor outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: 20 µm. (B) Quantification of SSBP1 transcript levels in cultured skin fibroblasts from both controls and affected individuals (patient 1, patient 2, and patient 3). mRNA levels were normalized to the reference gene L27. (C) Western blot in lysates from controls (control 1 and control 2) and patient fibroblasts and densitometric analysis of SSBP1 protein abundance in lysates from both controls and patient fibroblasts. GAPDH was used as a loading control. Data are shown as mean ± SEM. ***P < 0.001. (D) Representative ultrastructure of the mitochondria from both controls (C1, C2) and patient fibroblasts by TEM. Scale bar: 1 µm. Single arrows show abnormal mitochondria. Double arrows show lipid droplets. Quantification analyses of abnormal mitochondria (large vacuoles, disturbed cristae) in fibroblasts from both controls and patients. Data are represented as mean percentage of abnormal mitochondria ± SEM in total examined mitochondria. **P < 0.01. All data are representative of 3 independent experiments. One-way ANOVA with Dunnett’s correction was used.
Fig 4: Clinical features of SSBP1 patients.Combined optic atrophy and foveopathy of individual VI-25 from family A. (A) Ocular fundus photographs of the right eye and (B) left eye. Note the symmetrical temporal optic disk pallor (black arrows). (C) Optic SD-OCT and retinal nerve fiber layer (RNFL) deviation map confirm a significant decrease of the temporal RNFL thickness. Temporal part thickness is outside normal limits in red. The green area corresponds to the 5th–95th percentiles, the yellow area corresponds to the 1st–5th percentiles, and the red area corresponds to below the 1st percentile. OD, right eye; OS, left eye. (D) SD-OCT macular analysis of the right eye and (E) of the left eye. SD-OCT reveals a combined foveopathy unsuspected on color photographs. A tiny disruption of both ellipsoid and interdigitation lines is observed in both eyes (blue arrows) beneath the fovea.
Fig 5: Structural effects of SSBP1 mutations.(A) Crystal structure of SSBP1 showing that human SSBP1 consists of a dimer (molecules molA and molB) that contacts a second, symmetrically related dimer (molA' and molB'), giving rise to the tetrameric biological unit. Both Arg38 and Arg107 are displayed in yellow (frame). (B) Zoom-in of mutated residues; Arg38 is replaced by Gln38 (top). The bottom panel is the same, except Arg107 is replaced by Gln 107. In each case, the mutated residue is shown in light gray. (C) SDS/PAGE analysis of SSBP1 monomers and dimers in control (control 1 and control 2) and patient fibroblasts (patient 1, patient 2, patient 3). Actin was used as a loading control. For each cell line, 3 independent biological replicates were loaded. (D) Densitometric quantification of SSBP1 monomer (left) and dimer (right) in pooled controls and patient fibroblasts. Data are shown as scatter plots with mean ± SEM indicated. **P < 0.01; ***P < 0.001, 1-way ANOVA with Dunnett’s correction. Note that some of the same samples (for control 1, patient 1, and patient 2) were run on both membranes and these thus represent technical replicates. The ratios with actin from both membranes were averaged prior to statistical analysis.
Supplier Page from MilliporeSigma for Anti-SSBP1 antibody produced in rabbit