Article Figures & Data
Figures
- Figure 1. Mitochondria relationship with the cilia and effect of mitochondria inhibitors on IMCD3 ciliation.
(A) Representative image of live recording of stable IMCD3 ARL13b-RFP-Mito-PAGFP at confluence. The transparent white circles indicate the radius of 2 μM in the xy dimensions from the base of the cilia. The below images represent slices of the z dimension to show the mitochondria location within the cell. The dashed lines show the distance between the start site of ARL13b and the location of that particular mitochondria indicated with the +. The measurements where performed with Imaris which allows to draw lines in between z-stack and measurements of the distance between two different points in 3D which are reported in the figures. Scale bar = 2 μM. (B) Representative image of live recording of stable IMCD3 ARL13b-Venus loaded with 25 nM TMRE. The transparent white circle/ellipse indicates the mitochondria within the radius of 2 μM from the base of the cilia. (A, B, C, D, E) The image plane was rotated using Imaris to show the mitochondrial subset close to the cilium. The dashed lines show the distance between the start site of ARL13b and the location of that particular mitochondria indicated with the +. Scale bar = 3 μM. (C) Mitochondrial membrane potential of mitochondria within <2 μm from the cilium compared with the overall cell mitochondrial network measured with TMRE in IMCD3 ARL13b-Venus stable cell line. Data are represented by mean + CI; n = 3. (D) Representative image of IMCD3 cell line treated for 48 h with DMSO vehicle or 100 nM of rotenone. Cilia stained with anti-acetylated α-tubulin are indicated with white arrows, whereas not counted cytokinetic bridges in dividing cells are indicated with yellow arrows. (E) Quantification of dose-dependent effect of rotenone on number of ciliated IMCD3 cells. Data are represented by mean + SD; n ≥ 2. (F) ATP/ADP ratio in IMCD3 cells treated 48 h with DMSO vehicle, 25 nM rotenone or 50 nM oligomycin. Data are represented by mean + SD; n ≥ 3. (G) Quantification of IMCD3 ciliated cells treated as above. Data are represented by mean + SD; n = 4. (H) Representative WB image of IFT88 protein in IMCD3 cells treated as above. Quantification is represented by mean + SD; n = 4. Legend: Black empty = DMSO control; Black = rotenone 25 nM; Red = oligomycin 50 nM. Statistical significance between control versus treatments: P < 0.05 (*), <0.01 (**) or <0.001 (***). Rotenone versus oligomycin: P < 0.05 (#).
- Figure S1. Mitochondria relationship with the basal body and effects of rotenone and oligomycin on IMCD3 cilia.
(A) Representative image of live recording of IMCD3 with Mitotracker DeepRed. The transparent white circle/ellipse indicates the mitochondria within the radius of 2 μM from the base of the cilia. (B) Example of image rendering with Imaris software used for TMRE intensity quantification. Rendering colors: mitochondria (green), cilium (red), subset of selected mitochondria (yellow) within 2 μM distance from the primary cilium. (C) Mitochondrial membrane potential of mitochondria within <2 μm from the cilium compared with the overall cell mitochondrial network measured with TMRE in IMCD3 ARL13b-Venus stable cell line. Data are represented by mean + CI; all technical replicates n = 15. (D) Quantification of dose-dependent effect of rotenone on number of ciliated IMCD3 cells. Data are represented by mean + CI; all technical replicates n = 16. (E) ATP/ADP ratio in IMCD3 cells treated 48 h with DMSO vehicle, 25 nM rotenone, or 50 nM oligomycin. Data are represented by mean + CI; all technical replicates n > 5. (F) Quantification of IMCD3 ciliated cells treated as above. Data are represented by mean + CI; all technical replicates n > 16. (G) Representative WB image of IFT88 protein in IMCD3 cells treated as above. Quantification is represented by mean + CI; all technical replicates n > 7. Legend: Black empty = DMSO control; black = rotenone 25 nM; red = oligomycin 50 nM. Statistical significance between control versus treatments: P < 0.05 (*), <0.01 (**), or <0.001 (***). Rotenone versus oligomycin: P < 0.05 (#).
- Figure 2. Reactive oxygen species, but not cells cycle and apoptosis, are in part responsible for decreased ciliation in IMCD3.
(A) Quantification of Mitosox intensity in IMCD3 cells treated for 48 h with DMSO vehicle, 25 nM rotenone, or 50 nM oligomycin. Data are represented by mean + SD; n = 6. (B) Quantification of DCFDA intensity in IMCD3 cells treated as above. Data are represented by mean + SD; n = 5. (C) Quantification of cilia number in IMCD3 cells treated for 48 h with 25 nM of rotenone in the presence of antioxidants (Tiron + Vit.C 1 mM, retinoic acid 1 μM). Data are represented by mean + CI; n = 4. (D) Cell cycle analysis in IMCD3 cells treated as above. In the inset is displayed a representative image of cell cycle plot in control cells. Data are represented by mean + CI; n = 4. (E) Analysis of apoptotic and dead IMCD3 cells treated as above normalized per 100 events of DMSO treated cells. In the inset is displayed a representative plot of control cells. Staurosporin 6 μM has been added 2 h prior the trypsinization as positive control. Data are represented by mean + SEM; n ≥ 2. Legend: Black empty = DMSO control; black = rotenone 25 nM; red = oligomycin 50 nM; blue = staurosporine 6 μM; orange = Tiron + Vit.C 1 mM; pink = Retinoic acid 1 μM. Statistical significance between control versus treatments: P < 0.05 (*), <0.01 (**), or <0.001 (***). Rotenone versus oligomycin: P < 0.05 (#), <0.01 (##), or <0.001 (###).
- Figure S2. Reactive oxygen species (ROS) are partly responsible for decreased number of ciliated IMCD3 cells.
(A) Quantification of Mitosox intensity in IMCD3 cells treated for 48 h with DMSO vehicle, 25 nM rotenone, or 50 nM oligomycin. Data are represented by mean + CI; n > 11. (B) Representation of flow cytometric histogram of IMCD3 cells treated 48 h with DMSO vehicle, 25 nM rotenone, or 50 nM oligomycin stained with Mitosox. Green: unstained, red: control, blue: rotenone, orange: oligomycin. (C) Representation of flow cytometric histogram of IMCD3 cells treated as above and stained with DCFDA. Green: unstained, red: control, blue: rotenone, orange: oligomycin. (D) Quantification of DCFDA intensity in IMCD3 cells treated as above. Data are represented by mean + CI; n = 10. (E) Quantification of dose-dependent effect of 48-h treatment with TBOOH on number of ciliated IMCD3 cells. Data are represented by mean + CI; n = 15. (F) Quantification of DCFDA intensity in IMCD3 cells after 48-h treatment with different doses of TBOOH. Data are represented by mean + CI; n = 6. (G) Quantification of the effect of 48 h treatment with 50 μM paraquat on number of ciliated IMCD3 cells. Data are represented by mean + CI; n = 14. (H) Quantification of cilia number in IMCD3 cells treated for 48 h with 25 nM of rotenone in the presence of antioxidants (Tiron + Vit.C 1 mM, retinoic acid 1 μM). Data are represented by mean + CI; n > 15. (I) Cell cycle analysis in IMCD3 cells treated as above. In the inset is displayed a representative image of cell cycle plot in control cells. Data are represented by mean + CI; n > 8. (J) Analysis of apoptotic and dead IMCD3 cells treated as above normalized per 100 events of DMSO treated cells. In the inset is displayed a representative plot of control cells. Staurosporin 6 μM has been added 2 h prior the trypsinization as positive control. Data are represented by mean + CI; n = 9. Legend: Black empty = DMSO control; black = rotenone 25 nM; red = oligomycin 50 nM; blue = staurosporine 6 μM; orange = Tiron + Vit.C 1 mM; pink = Retinoic acid 1 μM. Statistical significance between control versus treatments: P < 0.05 (*), <0.01 (**), or <0.001 (***). Rotenone versus oligomycin: P < 0.05 (#), <0.01 (##), or <0.001 (###).
- Figure 3. Ciliation is impaired in kidney of diabetic nephropathy mouse model db/db.
(A) Non-fasting plasma creatinine in wild-type and db/db mice at 8 (n = 4) and 24 (n = 3) wk of age. Data are represented by mean + SEM. (B, C) Representative morphological kidney sections (20 μm) counterstained with hematoxylin–eosin of 8- and 24-wk wild-type and db/db mouse. Scale bar = 1 mm in whole kidney pictures and 100 μm in blow up pictures. (D) Representative single stack images of kidney sections stained with ciliary acetylated tubulin (green) and collecting duct cells aquaporin-2 (red). Nuclei (blue) were counterstained with DAPI. (E) Percentage of ciliated collecting duct cells in wild-type and db/db mice at 8 and 24 wk of age. Data represent three medullary images (upper, middle, and lower kidney) in duplicates for each mouse (n = 4). Minimum 10 collecting duct cells have been counted for each image. Data are represented by median+IQr and range (n = 4). Statistical test ANOVA+Bonferroni. (F) Cilia length of collecting ducts cells in wild-type and in db/db mice at 8 and 24 wk of age. Data are represented by median+IQr and range (n = 4). Statistical test ANOVA+Bonferroni. (G) Gene expression measured by qPCR in wild-type and db/db mice at 8 and 24 wk of age. Data are represented by mean + SD (n ≥ 3). (H) Flux analysis recording of the oxygen consumption rate from complex I+III+IV, II+III+IV, or complex IV only of disrupted mitochondrial from frozen samples extracted from the kidney of wild-type and db/db mice at 8 and 24 wk of age. Data are represented by mean + SEM (n ≥ 3). (I) Oxygen consumption rate from complex I+III+IV, II+III+IV, or complex IV of disrupted mitochondrial from frozen samples extracted from kidney of wild-type and db/db mice at 8 and 24 wk of age. Data are represented by mean + SD (n ≥ 3). (J) Representative single stack images of kidney sections stained with ciliary acetylated tubulin (green) and distal tubules cells Na+-K+ ATPase (red). Nuclei (blue) were counterstained with DAPI. Legend: Gray = wild-type mice (wt/wt); black empty = db/db. (K) Percentage of ciliated distal tubules cells in wild-type and db/db mice at 8 and 24 wk of age. Data are represented by median+IQr and range (n = 4). Statistical test ANOVA+Bonferroni; statistical significance between wild-type versus db/db: P < 0.05 (*), <0.01 (**), or <0.001 (***).
- Figure S3. Metabolic features, circulating hormones and cilia length in kidneys of db/db mouse model.
(A) Body weight of female wild-type and db/db mice at 8 and 24 wk of age (n = 4). (B) Non-fasting plasma leptin in wild-type and db/db mice at 8 (n = 4) and 24 (n = 3) wk of age. (C) Non-fasting blood glucose in wild-type and db/db mice at 8 and 24 wk of age (n = 4 each group). (D) Plasma C-Peptide in wild-type and db/db mice at 8 (n = 4) and 24 wk (n = 3) of age. (E) Plasma adiponectin in wild-type and db/db mice at 8 (n = 4) and 24 wk (n = 3) of age. (F) Blood triglycerides in wild-type and db/db mice at 8 (n = 4) and 24 wk (n = 3) of age. (G) Liver triglycerides in wild-type and db/db mice at 8 (n = 4) and 24 wk (n = 3) of age. (H) Blood cholesterol in wild-type and db/db mice at 8 (n = 4) and 24 wk (n = 3) of age. Data are represented by mean + SEM. Statistical significance between wild-type versus db/db: P < 0.05 (*), <0.01 (**), or <0.001 (***). (I) Cilia length of distal tubules cells in wild-type and db/db mice at 8 and 24 wk of age. Data are represented as median+IQr and range (n = 4).
- Figure 4. Rotenone-induced reactive oxygen species production, but not cellular energy status and cell cycle, are responsible for elongating cilia in RPE1 cells.
(A) Representative images of RPE1 cell line treated 48 h with DMSO vehicle or 400 nM rotenone. Cilia stained with anti-acetylated tubulin are indicated with white arrows. Scale bar = 5 μM. (B) Quantification of dose-dependent effect of rotenone on maximum and minimum cilia length in RPE1 cells. Data are represented by mean + CI; n = 3. (C) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) normalized to the baseline after addition of different doses of rotenone and oligomycin in RPE1. Data are represented by mean + CI; n = 3. (D) Number of ciliated RPE1 after 48 h of 800 nM rotenone or 400 nM oligomycin versus DMSO vehicle control. Data are represented by mean + SD; n = 3. (E) Quantification of the median ciliary length in RPE1 treated as above. Data are represented by mean + SD; n = 3. (F) Quantification of the maximum ciliary length in RPE1 treated as above. Data are represented by mean + SD; n = 3. (G) ATP/ADP ratio normalized to the DMSO control in RPE1 cells treated 48 h as above. Data are represented by mean + SD; n = 3. (H) Cell cycle analysis in RPE1 cells treated as above. In the inset is displayed a representative image of cell cycle plot in control cells. Data are represented by mean + CI; n = 4. (I) Quantification of Mitosox intensity in RPE1 cells treated as above. Data are represented by mean + SD; n ≥ 5. (J) Quantification of median and maximum ciliary length in RPE1 cells treated with 10 nM rotenone in glucose-free galactose media. Data are represented by mean + SD; n = 3. Legend: Black empty = DMSO control; black = rotenone 800 nM; red = oligomycin 400 nM; gray = rotenone 10 nM. Statistical significance between control versus treatments: P < 0.05 (*), <0.01 (**). Rotenone versus oligomycin: P < 0.05 (#).
- Figure S4. Mitochondrial CI blockade elongates cilia in human retinal epithelial cells independently of cellular energy status.
(A) Quantification of dose-dependent effect of rotenone on maximum and minimum cilia length in RPE1 cells. Data are represented by mean + CI; n = 12. (B) Quantification of dose-dependent effect of rotenone on median cilia length in RPE1 cells treated for 48 h. Data are represented by mean + CI; n = 12. Statistical significance was calculated using t test between DMSO control and any other rotenone dosage. (C) OCR normalized to the baseline in a representative experiment with acute additions of different doses of rotenone and oligomycin in RPE1 cells. Data are represented by mean + CI; n > 6. (D) Extracellular acidification rate (ECAR) normalized to the baseline in a representative experiment with acute additions of different doses of rotenone and oligomycin in RPE1. Data are represented by mean + CI; n > 6. (E) OCR and extracellular acidification rate (ECAR) normalized to the baseline after addition of different doses of rotenone and oligomycin in RPE1. Data are represented by mean + CI; n = 18. (F) Number of ciliated RPE1 after 48 h of 800 nM rotenone or 400 nM oligomycin versus DMSO vehicle control. Data are represented by mean + CI; n = 15. (G) Quantification of the median ciliary length in RPE1 treated as above. Data are represented by mean + CI; n = 15. (H) Quantification of the maximum ciliary length in RPE1 treated as above. Data are represented by mean + CI; n = 15. (I) ATP/ADP ratio normalized to the DMSO control in RPE1 cells treated 48 h as above. Data are represented by mean + CI; n > 5. (J) Cell cycle analysis in RPE1 cells treated as above. In the inset is displayed a representative image of cell cycle plot in control cells. Data are represented by mean + CI; n > 8. (K) Quantification of Mitosox intensity in RPE1 cells treated as above. Data are represented by mean + CI; n = 10. (L) Representation of flow cytometric histogram of RPE1 cells treated for 48 h with DMSO vehicle, 800 nM rotenone, or 400 nM oligomycin stained with Mitosox. Green: unstained, red: control, blue: rotenone, orange: oligomycin. (M) Representation of flow cytometric histogram of RPE1 cells treated as above and stained with DCFDA. Green: unstained, red: control, blue: rotenone, orange: oligomycin. (N) Quantification of DCFDA intensity in RPE1 cells treated as above normalized to control DMSO. Data are represented by mean + CI; n = 6. (O) Quantification of cilia number in IMCD3 cells treated for 48 h with 25 nM of rotenone in the presence of antioxidants (Tiron + Vit.C 1 mM, retinoic acid 1 μM). Data are represented by mean + CI; n = 14. (P) Quantification of median and maximum ciliary length in RPE1 cells treated with 10 nM rotenone in glucose-free galactose media. Data are represented by mean + CI; n = 10. Legend: Black empty = DMSO control; black = rotenone 800 nM; red = oligomycin 400 nM; gray = rotenone 10 nM. Statistical significance between control versus treatments: P < 0.05 (*), <0.01 (**), or <0.001 (***). Rotenone versus oligomycin: P < 0.05 (#), <0.01 (##), or <0.001 (###).
- Figure S5. Double ciliary staining in RPE1 cells.
(A) Representative images of RPE1 cell line treated for 48 h with 800 nM rotenone or 400 nM oligomycin. The top left box represents the ciliary staining in single channels; blue: DAPI, green: acetylated tubulin, red: ARL13b.
Tables
- Table 1.
List of the 10 most represented mitochondrial proteins found in the ciliary database ordered by ciliary evidence (CiliaDB [Arnaiz et al, 2009]).
Name Ciliary evidence Organisms with ciliary evidence Non flagellary evidence Proteomic studies Evidence in H. sapiens Ubiquinol-cytochrome c reductase core protein II 6 4 3 3 0 NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kD (NADH-coenzyme Q reductase) 5 5 2 1 1 ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide 5 5 1 1 1 Creatine kinase, mitochondrial 1B 5 3 2 2 1 Isocitrate dehydrogenase 2 (NADP+), mitochondrial 5 5 3 3 1 Inner membrane protein, mitochondrial 5 4 3 3 1 Acyl-CoA dehydrogenase, short/branched chain 5 2 3 0 1 Voltage-dependent anion channel 2 5 4 4 3 0 Solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 3 4 4 4 2 1 Solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 4 3 3 3 0
