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AMPK-mediated activation of MCU stimulates mitochondrial Ca2+ entry to promote mitotic progression

Abstract

The capacity of cells to alter bioenergetics in response to the demands of various biological processes is essential for normal physiology. The coordination of energy sensing and production with highly energy-demanding cellular processes, such as cell division, is poorly understood. Here, we show that a cell cycle-dependent mitochondrial Ca2+ transient connects energy sensing to mitochondrial activity for mitotic progression. The mitochondrial Ca2+ uniporter (MCU) mediates a rapid mitochondrial Ca2+ transient during mitosis. Inhibition of mitochondrial Ca2+ transients via MCU depletion causes spindle checkpoint-dependent mitotic delay. Cellular ATP levels drop during early mitosis, and the mitochondrial Ca2+ transients boost mitochondrial respiration to restore energy homeostasis. This is achieved through mitosis-specific MCU phosphorylation and activation by the mitochondrial translocation of energy sensor AMP-activated protein kinase (AMPK). Our results establish a critical role for AMPK- and MCU-dependent mitochondrial Ca2+ signalling in mitosis and reveal a mechanism of mitochondrial metabolic adaptation to acute cellular energy stress.

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Data availability

All data supporting the findings of this study are available from the corresponding authors upon reasonable request. Statistical source data for Figs. 17 and Supplementary Figs. 17 are provided in Supplementary Table 4.

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Acknowledgements

The authors thank Y.X. Zheng, H.T. Yu and Z.G. Liu for discussions and critical reading of the manuscript. They also thank D.D. Stefani for providing 4mt-GCaMP6 plasmids, Y.Q. Shen for the mtAequorin plasmid, and K. Wang and X. Xu for assistance with microscopy assays. This work was supported by the National Natural Science Foundation of China (grant numbers 81522034, 31570840, 81521064, 31571419, 31370915 and 81790252), the China National Basic Research Program (2014CB910603), the International S&T Cooperation Program of China (2015DFA31610), Beijing Nova Program (Z151100000315085, Z161100004916166), Beijing Talents Foundation (2016000021223ZK24), National Key Research and Development Program (2017YFC1601100) and a NIH grant (R01HL142589).

Author information

H.Z. designed and carried out most of the experiments. H.Z. and Teng. L. performed calcium measurements, and H.Z., Teng. L. and F.Z. contributed to cell cycle analyses. H.Z., K.W. and G.X. performed phosphorylation experiments, and H.Z. J.C. and F.Z. carried out metabolic analyses. H.Z. and X.P. performed in vivo experiments, and L.L. and Ting. L. contributed to constructing plasmids. L.C., J.Z., Q.X., T.Z., H.-Y.L., A.-L.L., T.F., X.-M.Z. and X.P. contributed to interpreting the results. T.F. contributed to editing the manuscript. X.P. and X.-M.Z. conceived the study, supervised the research and wrote the paper.

Competing interests

The authors declare no competing interests.

Correspondence to Xue-Min Zhang or Xin Pan.

Integrated supplementary information

  1. Supplementary Figure 1

    Mitochondrial Ca2+ transients in cell cycle. a, Representative images of HeLa cells expressing mitochondrial Ca2+ probe 4mt-GCaMP6 (Green, left panel) co-stained with MitoTracker Red (middle panel). The merged image is shown in the right panel. b, Representative image of HeLa/RFP-H2B cells expressing 4mt-GCaMP6. White arrows indicate mitotic cells. Some of the mitotic cells exhibit higher mitochondrial Ca2+ levels. c, Selected frames of a mitochondrial Ca2+ transient during mitosis in HeLa/RFP-H2B cell expressing 4mt-GCaMP6. The cells were synchronized by a thymidine block and released to fresh medium without CGP37157. The mitotic progression was then imaged by fluorescence time-lapse microscopy. The time on the images is in minute. See Supplementary Video 1. d, Mitochondrial Ca2+ levels in HeLa cells pretreated with DMSO or 5 μM CGP37157 were measured by fluorescence of 4mt-GCaMP6 following 100 μM histamine stimulation. n = 25 cells for each condition. e, HeLa cells pretreated with DMSO or 5 μM CGP37157 were analyzed for the lengths of time from NEB to anaphase onset based on time-lapse imaging. n = 57 DMSO-pretreated cells and 47 CGP37157-pretreated cells. f, Mitochondrial Ca2+ transient frequency (times per hour) in the cells at interphase or in mitosis. n = 171 interphase cells and 84 mitotic cells from 3 independent experiments. g, Quantification of the distribution of mitochondrial Ca2+ transients in prometaphase or metaphase. n = 3 independent experiments. (h-k) Representative mitotic mitochondrial Ca2+ transient in rat kangaroo kidney epithelial Ptk1 cells (h), NIH3T3 mouse embryonic fibroblast cells (i), hepatoma carcinoma cell HepG2 (j) and LM3 (k). All scale bars indicate 10 μm. Data in a-e,h-k represent three independent experiments with similar results. Data are shown as mean ± s.e.m. Unpaired two-sided student’s t-test was performed in e,g; unpaired two sided Mann Whitney test was performed in f. Source data are provided in Supplementary Table 4.

  2. Supplementary Figure 2 MCU is required for mitotic progression in many mammalian cell lines.

    a, Representative images of mitochondrial Ca2+ dynamics (4mt-GCaMP6) in control or MCU knockdown HeLa/RFP-H2B cells during mitosis, as described in Fig. 1c-e. b, Resting mitochondrial Ca2+ levels (4mt-GCaMP6) during mitosis were measured in control and MCU knockdown HeLa/RFP-H2B cells. n = 51 control cells and 40 MCU silenced cells. The experiment was repeated twice independently. c, The lengths of time from NEB to anaphase onset in control cells, MCU siRNA #1 cells and #2 cells were analyzed, as described in Fig. 2c. n = 147, 105, 80 cells (from left to right). Data are from two independent experiments. MCU protein expression was verified by Western Blot. d, The lengths of time from NEB to anaphase onset in RPE, MB-231, U251 and U2OS cells transfected with indicated siRNAs. n = 36, 15, 56, 49, 57, 56, 50, 50 cells (from left to right). The experiment was repeated twice. MCU protein expression was verified by Western Blot. e, EdU staining (green) of HeLa cells transfected with indicated siRNAs. The experiment was repeated three times independently. f, Percentage of EdU-positive HeLa cells based on images in e. n = 3 independent experiments. g, Relative mean traces of mitochondrial Ca2+ level (indicated by luminescence of mtAequorin) in HeLa cells expressing vector, siRNA-resistant MCUWT or MCUMut (MCUD261E, D264E) transfected with indicated siRNAs. 100 μM histamine was added as the arrow indicated. The peak value of the control cells was set to 1. h, Quantification of the maximal amplitudes of the traces in g. n = 3 replicate samples. Data are representative of three independent experiments. i, MCU protein expression was verified by Western Blot. The experiment was repeated three times independently with similar results. All scale bars indicate 10 μm. All data are shown as mean ± s.e.m. One-way ANOVA was performed in c,h; Unpaired two-sided student’s t-test was performed in f; Unpaired two sided Mann Whitney test was performed in b,d. Scanned images of unprocessed blots are shown in Supplementary Fig. 8. Source data are provided in Supplementary Table 4.

  3. Supplementary Figure 3

    a, Embryos were obtained by heterozygous crosses. Wild type (+/+) and MCU knockout embryos (-/-) harvested at E13.5. At this time point, some MCU-/- embryos have already died (#4) while other show a mild size reduction (#5) or appear normal (#6). b, Embryos were obtained by heterozygous crosses and isolated at the indicated time of gestation. Genotypes of embryos were determined by PCR. Number of each genotype was recorded and the analyzed percentage was indicated in bracket. c, Selected frames from time-lapse movies of mitotic progression and subsequent cell death of representative primary wild-type and MCU knockout fetal liver cells stained with SiR-Hoechst. The time on the images is in minute. Scale bar, 10 μm. Cell death percentage in mitosis were analyzed (right panel). Data are shown as mean ± s.e.m. n = 3 pairs of mice. Unpaired two-sided student’s t-test was performed. d, TUNEL staining of E12.5 whole embryos with high power view of corresponding fetal liver shown directly below (left panel). The quantification of percent TUNEL positive cells was shown (right panel). Scale bar, 100 μm. Data are shown as mean ± s.e.m. n = 3 pairs of mice. Unpaired two-sided student’s t-test was performed. e, Available CD1 mice were obtained by heterozygous crosses. Genotypes were determined by PCR. Number of each genotype was recorded and analyzed percentage was shown. Source data are provided in Supplementary Table 4.

  4. Supplementary Figure 4 Mitochondrial Ca2+ transients boost mitochondrial ATP production and don’t alter cytosolic Ca2+ signaling.

    a, Snapshot of cytosolic Ca2+ (upper, green) and mitochondrial Ca2+ (lower, red) transient images from time-lapse movies of representative HeLa cells co-expressing GCaMP6 and 4mt-RCaMPh during mitosis. The time on the images is in minute. Scale bar, 10 μm. (b and c) Amplitude (b) and frequency (c) quantification of cytosolic Ca2+ transients in control and MCU knockdown mitotic cells. n = 55 control cells and 62 MCU silenced cells from three independent experiments. d, Resting cytosolic Ca2+ levels (GCaMP6) during mitosis were measured in control and MCU knockdown HeLa cells. n = 62 control cells and 74 MCU silenced cells. The experiment was repeated three times independently. e, Representative traces of mitochondrial Ca2+ (4mt-RCaMPh) and mitochondrial ATP (4mt-ATeam1.03) dynamics in the presence of 10 μM oligomycin or not. 100 μM histamine was added as the arrow indicated. The experiment was repeated three times independently. All data are shown as mean ± s.e.m. Unpaired two-sided student’s t-test was performed in b,d; Unpaired two-sided Mann Whitney test was performed in c. Source data are provided in Supplementary Table 4.

  5. Supplementary Figure 5 MCU-mediated ATP production is required for proper microtubule dynamics during mitosis.

    (a and b) Images from time-lapse movies of a representative HeLa cell expressing ATeam1.03 (pseudocolored) after PBS or 5 mM ATP addition. Cytosolic ATP level change was measured by the emission ratio (YFP/CFP) of ATeam1.03 and shown in b. n = 12 cells for PBS and 14 cells for ATP treatment. The time on the images is in minute. Scale bar, 10 μm. The experiment was repeated three times independently. c, HeLa cells were treated with the indicated siRNAs before taxol arrest. For the last 2 h before shake-off, MG132 was added. Then taxol-arrested cells were shaken off and treated with ZM447439 for the indicated times. Cdc20 was immunoprecipitated, and co-precipitated Mad2 was analyzed by immunoblotting. The experiment was repeated twice independently. d, Quantification of spindle tubulin levels in Fig. 3f. n = 45, 44, 43, 53, 45, 45 cells (from left to right). The experiment was repeated twice independently. e, HeLa cells were pretreated with 5 mM ATP or AMP for 4 hrs followed by washout. Cytosolic Ca2+ levels were measured by fluorescence of GCaMP6 following 10 μM histamine stimulation. f, Quantification of the maximal amplitudes of the traces in e. n = 30 cells for each group. Data are representative of three independent experiments with similar results. All data are shown as mean ± s.e.m. All P values were calculated using one-way ANOVA. Source data are provided in Supplementary Table 4.

  6. Supplementary Figure 6 AMPK phosphorylates MCU at Ser57.

    a, 293T cells were transfected with vectors expressing Flag-tagged MCU or Flag-tagged MCUS57A. Cell lysates were analyzed by Western Blot. The experiment was repeated three times independently. b, HeLa cells were transfected with siRNAs as indicated. After 72h, cell lysates were analyzed by Western Blot. The experiment was repeated three times independently. c, WT (parental) or AMPK DKO HeLa cells were treated with DMSO, 300 μM A-769662, 2 mM metformin for 2–4 hrs or 5μM Ionomycin for 1 h followed by Western blotting analysis with indicated antibodies. The experiment was repeated three times independently. d, In vitro kinase assay using active, recombinant AMPK (α1, β1, γ1) holoenzyme with GST-MCUWT or GST-MCU NTD (1–165aa) as substrates in the presence of ATP for 45 min followed by Western blotting analysis with indicated antibodies. The experiment was repeated three times independently. e, HeLa cell mitochondria were isolated and proteins were extracted with 0.1 M Na2CO3 at pH 11.5. Both the soluble fractions (S) and the insoluble pellet (P) were analyzed by Western Blot. The experiment was repeated twice independently. f, Isolated mitochondria from mitotic HeLa cells were treated with Proteinase K or not and stained with indicated antibodies. Scale Bar, 1 μm. The experiment was repeated twice independently. g, Asynchronous (Asy) and nocodazole-arrested (M) extracts from WT (parental) or AMPK DKO U2OS and HEK293 cells were immunoblotted with indicated antibodies. The experiment was repeated twice independently. h, Representative immunofluorescent images of control and MCU siRNA transfected cells stained for pMCU-Ser57 (green), mitochondria (HSP60, red) and DNA (Hoechst, blue), respectively. Scale Bar, 10 μm. The experiment was repeated three times independently. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.

  7. Supplementary Figure 7 MCU phosphorylation regulates Ca2+ uptake activity.

    a, Representative immunofluorescent images of MCUWT-GFP, MCUS57A-GFP and MCUS57D-GFP transfected HeLa cells stained for mitochondria inner membrane (COXIV, red) and DNA (Hoechst, blue), respectively. Scale Bar, 10 μm. b, HEK293T cells were transfected with MCUWT-Flag, MCUS57A-Flag and MCUS57D-Flag. Cell lysates were fractionated by gel filtration chromatography and immunoblotted. The experiment was repeated twice independently. c, Representative traces of mitochondrial Ca2+ dynamics following 10 μM histamine treatment indicated by fluorescence of 4mt-GCaMP6 in WT (parental), AMPK DKO HeLa cells or HeLa cells pretreated with 300μM A769662 for 4 hrs followed by washout. Data are shown as mean ± s.e.m. n = 30 cells for each group. d, Quantification of the maximal amplitudes of the mitochondrial Ca2+ traces in c. n = 30 cells for each group. One-way ANOVA was performed. e, Phosphorylation of MCU and AMPK in c was analyzed by Western Blot. f, Representative traces of mitochondrial Ca2+ dynamics following 10 μM histamine treatment indicated by fluorescence of 4mt-GCaMP6 in MCU depleted cells stably transfected with MCUWT, MCUS57A or MCUS57D pretreated with or not 300μM A769662 for 4 hrs followed by washout. g. Quantification of the maximal amplitudes of the mitochondrial Ca2+ traces in f. n = 30 cells for each group. Data are shown as mean ± s.e.m. One-way ANOVA was performed. Data in a,c-g represent three independent experiments with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8. Source data are provided in Supplementary Table 4.

  8. Supplementary Figure 8 Unprocessed scans of western blots.

    Pictures of individual blots shown throughout this study.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–8 and legends for Supplementary Tables 1–4 and Supplementary Videos 1–7.

  2. Reporting Summary

  3. Supplementary Table 1

    List of oligonucleotides used in this study.

  4. Supplementary Table 2

    List of antibodies used in this study.

  5. Supplementary Table 3

    List of reagents used in this study.

  6. Supplementary Table 4

    Statistics source data.

  7. Supplementary Video 1

    A representative mitotic mitochondrial Ca2+ transient in HeLa cell #1.

  8. Supplementary Video 2

    A representative mitotic mitochondrial Ca2+ transient in HeLa cell #2.

  9. Supplementary Video 3

    A representative M phase progression in HeLa GFP–H2B cell transfected with control siRNA.

  10. Supplementary Video 4

    A representative M phase progression in HeLa GFP–H2B cell transfected with MCU #1 siRNA.

  11. Supplementary Video 5

    A representative M phase progression in HeLa GFP–H2B cell transfected with MCU #2 siRNA.

  12. Supplementary Video 6

    RFP–Mad2 disappearance in a representative HeLa GFP–H2B cell transfected with control siRNA.

  13. Supplementary Video 7

    RFP–Mad2 disappearance in a representative HeLa GFP–H2B cell transfected with MCU siRNA.

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Fig. 1: A MCU-mediated mitochondrial Ca2+ transient occurs in mitosis.
Fig. 2: A mitochondrial Ca2+ transient is required for mitotic progression in vitro and in vivo.
Fig. 3: Mitochondrial Ca2+ transients provide acute ATP for mitotic progression.
Fig. 4: The mitochondrial Ca2+ transient is a response to the acute low energy status during mitosis.
Fig. 5: Energy status regulates mitotic Ca2+ transients through AMPK.
Fig. 6: AMPK translocates to the mitochondria and phosphorylates MCU at Ser57 in mitosis.
Fig. 7: AMPK-mediated MCU phosphorylation regulates Ca2+ uptake activity and mitotic progression.
Supplementary Figure 1
Supplementary Figure 2: MCU is required for mitotic progression in many mammalian cell lines.
Supplementary Figure 3
Supplementary Figure 4: Mitochondrial Ca2+ transients boost mitochondrial ATP production and don’t alter cytosolic Ca2+ signaling.
Supplementary Figure 5: MCU-mediated ATP production is required for proper microtubule dynamics during mitosis.
Supplementary Figure 6: AMPK phosphorylates MCU at Ser57.
Supplementary Figure 7: MCU phosphorylation regulates Ca2+ uptake activity.
Supplementary Figure 8: Unprocessed scans of western blots.