Research ArticleCalcium signaling

Loss of MCU prevents mitochondrial fusion in G1-S phase and blocks cell cycle progression and proliferation

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Science Signaling  30 Apr 2019:
Vol. 12, Issue 579, eaav1439
DOI: 10.1126/scisignal.aav1439
  • Fig. 1 Deletion of MCU reduces cell proliferation in vivo and in vitro.

    (A) Interscapular wounds after skin punch in WT and MCU−/− mice. (B) Quantification of wound area. n = 8 mice per genotype. (C) H&E-stained cross sections of the descending aorta. Scale bars, 100 μm. (D) Quantification of aortic medial cross-sectional areas. n = 12 mice per genotype. (E) Quantification of cell nuclei in the aortic media. (F) Ratio of nuclei per area, derived from (E) and (D). (G) Cell counts for cultured VSMCs transfected with MCU or scrambled siRNA in growth medium with PDGF (20 ng/ml). n = 9 independent experiments. (H) Cell counts for VSMCs treated with the MCU inhibitor RU360 (100 nM) and/or PDGF. n = 8 independent experiments. (I) Cell counts for skin fibroblasts from MCU−/− or WT mice at 72 hours in growth medium with PDGF (20 ng/ml). n = 7 independent experiments. (J) TUNEL staining in VSMCs transfected with MCU or scrambled siRNA and treated with PDGF (20 ng/ml) for 48 hours. n = 6 independent experiments. *P < 0.05, **P < 0.001 by two-way repeated measures analysis of variance (ANOVA) (B), Mann-Whitney U test (D, F, and I), two-tailed t test (E), one-way ANOVA at 48 hours (G and H), and Kruskal-Wallis test (J).

  • Fig. 2 MCU activity is required for cytosolic Ca2+clearance.

    (A) PDGF-evoked Ca2+ transients in WT and MCU−/− VSMCs and WT VSMCs pretreated with 100 nM RU360 for 16 hours (arrow: addition of 10 nM PDGF). (B) AUC for (A). n = 5 independent experiments. (C) Peak amplitude for (A). n = 5 independent experiments with 8, 7, and 6 biological replicates for WT, MCU−/−, and RU360 treatments, respectively). (D) Mitochondrial Ca2+ uptake by Calcuim Green-5N assay in WT and MCU−/− skin fibroblasts. Treatment with digitonin (Dig; 0.005%), Ca2+ (1 μM), and FCCP (25 μM). n = 2 independent experiments. AU, arbitrary units. (E) Thapsigargin (Thap)–induced Ca2+ transients in WT and MCU−/− VSMCs and WT VSMCs pretreated with 100 nM RU360 (arrow: addition of 1 μM thapsigargin). (F) AUC in (E). n = 5 independent experiment with 6, 7, and 5 replicates for WT, MCU−/−, and RU360 treatments, respectively. (G) Quantification of baseline Fura signal in untreated WT VSMCs, WT VSMCs transfected with MCU siRNA, and MCU−/− VSMCs. n = 5 independent experiments with 12, 9, and 8 replicates for WT, MCU−/−, and WT VSMCs transfected with MCU siRNA, respectively. *P < 0.05, **P < 0.01 compared to WT untreated by Kruskal-Willis test (B) and one-way ANOVA (C, F, and G). (H) Cytosolic Ca2+ levels by Fura recording in WT and MCU−/− VSMCs after PDGF treatment (arrows) recorded over 3000 s. n = 4 independent experiments.

  • Fig. 3 MCU−/−skin fibroblasts have blunted cell cycle progression from G1-S phase.

    (A) Representative fluorescence-activated cell sorting analysis for DNA content in synchronized/growth-arrested WT and MCU−/− skin fibroblasts at 0, 16, and 24 hours after release from arrest with 10% fetal bovine serum (FBS) + PDGF (10 ng/ml). (B) Cell cycle phase distribution of fibroblasts from (A). n = 6 independent experiments with 11 and 12 replicates for WT and MCU−/−, respectively. #P < 0.0001 between genotypes, @P < 0.005 between genotypes by two-way ANOVA. (C) Immunoblots for cyclin D and cyclin E in synchronized/growth-arrested skin fibroblasts (0 hours) and at 16 hours after 10% FBS + PDGF treatment (10 ng/ml). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Quantification of cyclin D levels as in (C). n = 7 independent immunoblots. (E) Quantification of cyclin E levels as in (C). (F) Progression to S phase after release from G1 arrest with aphidicolin (Aph). n = 4 independent experiments with a total of 7 and 8 replicates for WT and MCU−/−, respectively. (G) Quantification of (F). n = 4 independent experiments with a total 7 and 8 replicates for WT and MCU−/−, respectively. (H) Confocal microscopy images of skin fibroblasts at baseline and 16 hours after PDGF-induced cell cycle progression [mitochondrial-targeted green fluorescent protein (mtGFP); green]. Scale bars, 20 μm (larger image) and 5 μm (inset). (I) Mitochondrial form factor in skin fibroblasts in (H). n = 5 independent experiments, with the number of cells analyzed indicated in bars. *P < 0.05, **P < 0.01, ****P < 0.001 by Kruskal-Wallis test.

  • Fig. 4 Dynamic regulation of oxygen consumption, mitochondrial and cytosolic Ca2+uptake, and MCU complex subunits during the cell cycle.

    (A) Fura recordings in skin fibroblasts at baseline and 16 hours after serum/PDGF-induced cell cycle progression. n = 8, 7, and 6 biological replicates for WT (16 hours), MCU−/− (16 hours), and all other conditions. (B) Calcium Green-5N assay in skin fibroblasts at baseline and 16 hours after serum/PDGF-induced cell cycle progression. Treatment with digitonin (0.005%; arrow) and Ca2+ (1 μM; asterisks). n = 2 independent experiments. (C) OCR by Clark electrode in skin fibroblasts at baseline and 16 hours after PDGF-induced cell cycle progression. n = 7, 6, and 5 biological replicates for WT (16 hours), MCU−/− (16 hours), and all other conditions. (D) Immunoblots for EMRE, MICU-1, MCUB, and NCLX at baseline and 16 hours after serum/PDGF-induced cell cycle progression. (E) Quantification of EMRE levels as in (D). n = 6 independent experiments. (F) Quantification of MICU-1 levels in (D). n = 5 independent experiments. (G) Quantification of MCUB levels as in (D). n = 6 independent experiments. (H) Quantification of NCLX levels as in (D). n = 4 independent experiments. *P < 0.05, **P < 0.01 by Kruskal-Wallis test.

  • Fig. 5 MCU deficiency increases Drp1 phosphorylation and mitochondrial fission.

    (A) Immunoblots for phosphorylated Drp1 at Ser616 (Drp1 pSer616) and Drp1 in WT and MCU−/− VSMCs treated with PDGF for 20 min. (B) Quantification of Drp1 phosphorylation normalized to Drp1 protein in PDGF-treated VSMCs as in (A). n = 7 independent experiments. (C) Immunoblots for Drp1 (Drp1 pSer616) and Drp1 in tissue samples from the skin and aorta of WT and MCU−/− mice. (D) Quantification of Drp1 phosphorylation at Ser616 normalized to Drp1 protein as in (C). n = 4 independent experiments. (E) Confocal microscopy images of mitochondria (mtGFP; green) and Drp1 (red) in scrambled- or MCU siRNA–treated VSMCs before and at 40 min after PDGF treatment. Scale bars, 5 μm (inset) and 20 μm (larger image). (F) Quantification of the colocalization of Drp1 and mtGFP by Pearson coefficient. n = 4 independent experiments, with the number of cells analyzed indicated in bars. (G) Cell counts for Drp1−/− embryonic fibroblasts transfected with MCU or scrambled siRNA and treated with growth medium with PDGF (10 ng/ml). n = 9 independent experiments. (H) Immunoblots for MCU in Drp1−/− embryonic fibroblasts transfected with MCU or scrambled siRNA as in (G). n = 7 independent experiments. (I) Cell counts in Drp1−/− embryonic fibroblasts with adenoviral overexpression of MCU for 48 hours before culture in growth medium for 72 hours. n = 6 independent experiments. *P < 0.05, **P < 0.01 by Mann-Whitney U test (D), Kruskal-Wallis test (B, F, and I), and Kruskal-Wallis test at 48 hours (G).

  • Fig. 6 Cytosolic CaMKII is activated and controls mitochondrial fission when MCU is deleted.

    (A) Immunoblots for active CaMKII (CaMKII pThr287) and CaMKII in WT and MCU−/− VSMCs treated with PDGF for 20 min. (B) Quantification of CaMKII phosphorylation at Thr287 normalized to CaMKII protein in PDGF-treated WT and MCU−/− VSMCs as in (A). n = 8 independent experiments. (C) Immunoblots for phosphorylated CaMKII (CaMKII pThr287) and CaMKII in tissue samples from the skin and aorta of WT and MCU−/− mice. (D) Quantification of CaMKII phosphorylation at Thr287 normalized to CaMKII protein in the skin and aorta as in (C). n = 4 independent experiments. (E) Immunoblots for phosphorylated Drp1 at Ser616 (Drp1 pSer616) and Drp1 protein in WT VSMCs treated with 30 μM KN-93 for 30 min before the addition of PDGF. (F) Quantification of Drp1 phosphorylation at Ser616 normalized to Drp1 protein in (E). n = 3 independent experiments. (G) Confocal microscopy images of mitochondria (mtGFP; green) in WT and MCU−/− VSMCs after adenoviral overexpression of inactive CaMKII (CaMKII T287A) for 48 hours, followed by treatment with PDGF or control for 20 min. Scale bars, 20 μm (larger image) and 5 μm (inset). (H) Quantification of form factor in WT and MCU−/− VSMCs with overexpression of CaMKII T287A or control in (G). n = 5 independent experiments, with the number of cells analyzed indicated in bars. (I) Immunoblots for phosphorylated, active Drp1 at Ser616 (Drp pSer616) and Drp1 protein in WT and MCU−/− VSMCs with overexpression of CaMKIIN or control for 48 hours, followed by treatment with PDGF or control for 20 min. n = 2 independent experiments. (J) Confocal microscopy images of mitochondria (mtGFP; green) in WT and MCU−/− VSMCs after adenoviral overexpression of CaMKIIN for 48 hours and treatment with PDGF or control for 20 min. Scale bars, 20 μm (larger image) and 5 μm (inset). (K) Quantification of form factor in WT and MCU−/− VSMCs as in (J). (L) Quantification of form factor in WT and MCU−/− VSMCs with overexpression of CaMKIIN as in (J). n = 5 independent experiments, with the number of cells analyzed indicated in bars. *P < 0.05, ***P < 0.005, ****P < 0.001 by Kruskal-Wallis test (B, H, K, and L), Mann-Whitney U test (D), and two-way repeated measures ANOVA (F).

  • Fig. 7 Cytosolic CaMKII inhibition in MCU−/−VSMCs rescues mitochondrial dynamics and cell proliferation.

    (A) OCR in WT and MCU−/− VSMCs after treatment with PDGF (20 ng/ml for 1 hour) with the sequential addition of oligomycin (Oligo; 1 μM), FCCP (1.5 μM), and rotenone/antimycin (2 μM). (B) ECAR in WT and MCU−/− VSMCs after treatment with PDGF (20 ng/ml for 1 hour). n = 5 independent experiments for (A) to (C). (C) Quantification of OCR for mitochondrial respiration calculated as baseline OCR − OCR after oligomycin. (D) OCR in WT and MCU−/− VSMCs with adenoviral overexpression of CaMKIIN after treatment with PDGF (20 ng/ml for 1 hour). (E) ECAR in WT and MCU−/− VSMCs with adenoviral overexpression of CaMKIIN after treatment with PDGF (20 ng/ml for 1 hour). (F) Quantification of OCR for mitochondrial respiration calculated as baseline OCR − OCR after oligomycin. n = 3 independent experiments for control and n = 4 for independent experiments for PDGF treatment in (D) to (F). (G) Lactate concentration in WT and MCU−/− VSMCs after adenoviral overexpression of CaMKIIN, serum starvation for 24 hours, followed by treatment with growth medium containing PDGF for 24 hours. n = 7 independent experiments. (H) Number of WT and MCU−/− VSMCs with overexpression of CaMKIIN and control at 72 hours after PDGF treatment or control. n = 6 independent experiments. Number of biological replicates indicated in bars. *P < 0.05 by Kruskal-Wallis test (C, F, G, and H).

  • Fig. 8 Inhibition of mitochondrial fission in MCU−/−VSMCs augments mitochondrial Ca2+uptake, dynamics, and respiration.

    (A) OCR in WT VSMCs after treatment with P110 (2 μM for 2 hours) and PDGF (20 ng/ml for 1 hours). n = 3 independent experiments. (B) OCR in MCU−/− VSMCs after treatment with P110 and PDGF. n = 3 independent experiments. (C) Confocal microscopy images of mitochondria (mtGFP; green) in WT and MCU−/− VSMCs after treatment with P110 for 2 hours and PDGF or control for 20 min. Scale bars, 20 μm (larger image) and 5 μm (inset). (D) Summary histogram of form factor in control WT and MCU−/− VSMCs. Number of cells analyzed in three independent experiments indicated in bars. (E) Average tracing of mitochondrial Ca2+ by mtPericam in WT or MCU−/− VSMCs after treatment with P110 (2 μM) for 2 hours and acute addition of PDGF (20 ng/ml). n = 3 independent experiments. (F) Summary histogram of form factor in control WT VSMCs with adenoviral overexpression of constitutively active CaMKII [CaMKII T287D; multiplicity of infection (MOI) of 100] or control. Samples pretreated with P110 as indicated. n = 3 experiments, with the number of cells analyzed indicated in bars. (G) Confocal microscopy images of mitochondria (mtGFP; green) in WT and MCU−/− VSMCs after overexpression of OPA1 and MFN1/2 at the baseline and after PDGF treatment. Scale bars, 20 μm (larger image) and 5 μm (inset). (H) Summary histogram of form factor in baseline WT and MCU−/− before and after overexpression of OPA1 and MFN1/2 before and after PDGF treatment. n = 3 independent experiments, with the number of cells analyzed indicated in bars. (I) Immunoblots for OPA-1, Myc (myc-tagged MFN1), MFN2, and GAPDH in WT VSMCs and VSMCs with overexpression of OPA1 and MFN1/2. n = 2 independent experiments. (J) Graphical summary, indicating that MCU deletion increases CaMKII activation, DRP1 phosphorylation, and mitochondrial fission. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 by Kruskal-Wallis test (D, F, and H).

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/579/eaav1439/DC1

    Fig. S1. MCU−/− mice demonstrate postnatal growth retardation.

    Fig. S2. Inhibition of mitochondrial Ca2+ uptake by MCU siRNA and RU360.

    Fig. S3. Cell cycle progression is delayed in MCU−/− VSMCs.

    Fig. S4. Mitochondria in VSMCs with MCU knockdown are fragmented at baseline and do not fragment with PDGF treatment.

    Fig. S5. PKC activity is not altered in tissues from MCU−/− mice.

    Fig. S6. CaMKII associates with mitochondria upon PDGF treatment.

    Fig. S7. PKC inhibition does not alter mitochondrial respiration.

    Fig. S8. MCU knockdown attenuates the metabolic response to PDGF application.

    Fig. S9. MCU expression in MCU−/− VSMCs recovers mitochondrial dynamics and respiration.

  • This PDF file includes:

    • Fig. S1. MCU−/− mice demonstrate postnatal growth retardation.
    • Fig. S2. Inhibition of mitochondrial Ca2+ uptake by MCU siRNA and RU360.
    • Fig. S3. Cell cycle progression is delayed in MCU−/− VSMCs.
    • Fig. S4. Mitochondria in VSMCs with MCU knockdown are fragmented at baseline and do not fragment with PDGF treatment.
    • Fig. S5. PKC activity is not altered in tissues from MCU−/− mice.
    • Fig. S6. CaMKII associates with mitochondria upon PDGF treatment.
    • Fig. S7. PKC inhibition does not alter mitochondrial respiration.
    • Fig. S8. MCU knockdown attenuates the metabolic response to PDGF application.
    • Fig. S9. MCU expression in MCU−/− VSMCs recovers mitochondrial dynamics and respiration.

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