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Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB

Nature Cell Biology volume 17, pages 288–299 (2015)Cite this article

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Abstract

The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signalling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release through mucolipin 1 (MCOLN1) activates calcineurin, which binds and dephosphorylates TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis through TFEB required MCOLN1-mediated calcineurin activation. These data link lysosomal calcium signalling to both calcineurin regulation and autophagy induction and identify the lysosome as a hub for the signalling pathways that regulate cellular homeostasis.

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Figure 1: Calcineurin regulates TFEB nuclear translocation.
Figure 2: Calcineurin binds and dephosphorylates TFEB.
Figure 3: Ca2+ elevation induces TFEB nuclear translocation through calcineurin.
Figure 4: Starvation induces lysosomal Ca2+ release through MCOLN1.
Figure 5: MCOLN1-mediated calcium release induces TFEB nuclear translocation.
Figure 6: Calcineurin regulates the lysosomal/autophagic pathway.
Figure 7: MCOLN1 regulates the lysosomal/autophagic pathway.
Figure 8: Model of Ca2+-mediated regulation of TFEB.

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Acknowledgements

We thank J. Meldolesi, T. Pozzan, D. Rubinsztein and R. Polishchuk for helpful suggestions and critical review of the manuscript. We thank G. Diez-Roux and A. Burton for their support in manuscript preparation. We are also grateful to R. De Cegli and D. Carrella for their support in the statistical analysis of the results. The TIGEM Bioinformatic and High Content Screening Facilities are gratefully acknowledged for their technological contributions to the project. We also thank J. D. Molkentin for the CanB KO MEFs, B. A. Rothermel for the HA–ΔCnA, and P. Aza-Blanc for suggestions on the reverse transfection protocol. We acknowledge the support of the Italian Telethon Foundation grant numbers TGM11CB6 (A.B.); the Beyond Batten Disease Foundation (A.B.); European Research Council Advanced Investigator grant no. 250154 (CLEAR) (A.B.); US National Institutes of Health (R01-NS078072) (A.B.); Telethon-Italy (TCP04009) (M.S.), European Research Council Consolidator grant no. 282310-(MyoPHAGY) (M.S.).

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Authors and Affiliations

  1. Telethon Institute of Genetics and Medicine (TIGEM), Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy

    Diego L. Medina, Simone Di Paola, Ivana Peluso, Rossella Venditti, Sandro Montefusco, Anna Scotto-Rosato, Carolina Prezioso, Alison Forrester, Carmine Settembre, Marco Sandri, Maria Antonietta De Matteis & Andrea Ballabio

  2. Department of Biomedical Science, University of Padova, Padova 35121, Italy

    Andrea Armani, Diego De Stefani, Marco Sandri & Rosario Rizzuto

  3. CNR Neuroscience Institute, Padova 35121, Italy

    Diego De Stefani & Rosario Rizzuto

  4. Dulbecco Telethon Institute at TIGEM, Naples 80078, Italy

    Carmine Settembre & Marco Sandri

  5. Department of Translational Medicine, Medical Genetics, Federico II University, Via Pansini 5, 80131 Naples, Italy

    Carmine Settembre & Andrea Ballabio

  6. Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA

    Wuyang Wang, Qiong Gao & Haoxing Xu

  7. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA

    Andrea Ballabio

  8. Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, Texas 77030, USA

    Andrea Ballabio

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  1. Diego L. Medina
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Contributions

D.L.M., S.D.P., I.P., S.M., A.S-R., C.P., R.V., A.F. and C.S. performed all experiments in cultured cells. A.A. and M.S. performed in vivo experiments in the murine muscle. W.W., Q.G., D.D.S., H.X. and R.R. performed electrophysiology experiments. D.L.M., M.A.D.M. and A.B. designed the overall study. D.L.M. and A.B. supervised the work. All authors discussed the results and made substantial contributions to the manuscript.

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Correspondence to Diego L. Medina or Andrea Ballabio.

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Integrated supplementary information

Supplementary Figure 1 Starvation and calcineurin overexpression induce NFAT nuclear translocation and de-phosphorylation.

HeLa cells were transiently transfected with a vector carrying NFAT fused to the GPF. (a) The plot shows the Nuc/Cyt ratio of NFAT-GFP. 1, 3, and 6 hours of starvation increased the nuclear translocation of NFAT. Thapsigargin (300 nM, 1 hour) was used as positive control. Bar graphs show mean ± s.d. for n = 3 independent experiments. (b) Starvation (1 and 3 hours, respectively) induces NFAT de-phosphorylation as measured by the mobility of NFAT detected by using anti-GFP antibodies. ΔCaN overexpression in fed conditions also induced NFAT-GFP mobility down-shift (right blot) (n = 3 independent experiments). (c) The overexpression of HA-tagged dominant positive calcineurin (ΔCaN) in HeLa cells transfected with NFAT-GFP induces NFAT nuclear translocation. ΔCaN positive cells were identified using anti-HA antibodies. The graph represents the nucleus/cytosol ratio of NFAT-GFP in both negative and positive calcineurin populations. Bar graphs show mean ± s.d. for n = 3 independent experiments. Scale bar 10 μm. Source data are provided in Supplementary Table 4.

Supplementary Figure 2 PPP3CB binds TFEB.

(a–c) Co-immunoprecipitation experiments performed both in HeLaTFEB–GFP (a,c) and in HEK-293TFEB–GFP cells (b) transfected with PPP3CB-FLAG and treated as indicated (fed or STV during 3 hr). 2 mg of lysates were immunoprecipitated with anti-GFP antibodies or rabbit IgG as a control and immunoblotted with the indicated antibodies. 50 μg of lysates were used to the input. n = 5 independent experiments were performed. (d) Histidine-TFEB pull down assay performed as described in Methods section confirms the interaction between TFEB and calcineurin. The input proportions of each protein are indicated. n = 3 independent experiments.

Supplementary Figure 3 Calcineurin interacts with TFEB and regulates its localization.

(a) Proximity Ligation Assay (PLA) to study TFEB interactions. HeLa cells were pre-treated 10 minutes before fixation with 1 μM ER-tracker (green) as marker for the cytoplasm or with Hoechst 33258 (blue) for nuclei. After fixation, cells were processed for investigating TFEB proximity interactions. Golgin 97 was used as negative control, and mTOR as a positive one. PLA detection red dots indicate positive interactions. White squares contain higher magnification images (n = 3 independent experiments). (b) Validation of the TFEB antibody. HeLa cells were control treated (Mock), or treated with TFEB siRNAs for 72 hours, processed for IF and stained with anti-TFEB (SantaCruz) (green, as a marker of silencing efficiency) or with Hoechst 33258 (blue, as nuclear staining). Mean values ± s.d. of TFEB fluorescence intensity under Mock or TFEB-KD conditions. n = 100 cells pooled from 2 independent experiments, scale bars, 10 μm. (c) Cell lysates (50 μg/sample) were analyzed by SDS-PAGE and immunobloting with anti-TFEB (Cell Signaling). β-actin was used as a loading control. n = 5 independent experiments (d) Cellular localization at the steady state of the endogenous proteins used in the PLA experiment: TFEB (green) (PPP3CB, Golgin97, mTOR, red) in HeLa cells. Hoechst 33258 (blue), was used as nuclear staining. n = 60 cells pooled from 2 independent experiments. Scale bars, 10 μm. (e), Nuclear translocation of endogenous TFEB is reduced in PPP3CB/R1 knock-down cells. Representative images from HC assay of HeLa cells transfected with SCRMBL or a pool of PPP3CB/R1 siRNAs, and then subjected to the indicated conditions. A similar experiment was performed in MCF-7 cells, providing similar results. The plot shows the percentage of nuclear TFEB translocation of PPP3CB/R1 silenced HeLa and MCF-7, compared with their corresponding control-treated cells transfected with scramble siRNA oligonucleotides (n = 3 independent experiments were performed). Scale bar 10 μm. (f) Nuclear fractions from cells treated as indicated for 3 h, WT MEFs (PPP3R1+/+) or PPP3R1−/− were immunobloted, and endogenous TFEB protein was detected using anti-TFEB antibodies. n = 2 independent experiments. (g) Normal mTOR phosphorylation activity in WT MEFs (PPP3R1+/+) or PPP3R1−/−, treated as indicated for 3 h, was assessed by immunoblotting analysis of mTOR substrate P70S6K. n = 2 independent experiments. Source data are provided in Supplementary Table 4.

Supplementary Figure 4 TPCN2 and MICU1 calcium channels do not regulate TFEB localization.

(a) Depletion of the lysosomal two-pore channel TPCN2 does not affect TFEB nuclear translocation during starvation (1 hour) (left graph). Quantification of the efficiency of TPCN2 gene silencing by qPCR analysis (right graph). The plot shows the mean ± s.d. for n = 3 independent experiments. (b) Overexpression of the essential mitochondrial Ca2+ regulator MICU1 does not impact on the subcellular localization of TFEB in fed and starvation conditions (1 hr), as compared to untransfected cells. High content imaging analysis was used to quantify TFEB localization using HeLa-TFEB-GFP cells. The plot shows the mean ± s.d. for n = 3 independent experiments. Source data are provided in Supplementary Table 4.

Supplementary Figure 5 MCOLN1 regulates TFEB phosphorylation and localization in a calcineurin-dependent fashion.

(a) Depletion of the lysosomal calcium channel MCOLN1 reduces starvation mediated TFEB nuclear translocation. WT HeLa and HeLa-shMCOLN1cells were starved at different time-points. Following starvation, 50 μg of protein extracts were prepared and probed for endogenous TFEB using anti-TFEB antibodies. TFEB mobility downshift after starvation is reduced in MCOLN1 depleted cells during starvation (black arrows point-out the phosphorylated forms, while red arrows show the downshift corresponding to de-phosphorylated TFEB forms). n = 3 independent experiments. (b) MCOLN1-mediated induction of TFEB nuclear localization is calcineurin-dependent. HeLaTFEB–GFP cells were transfected with FLAG-tagged MCOLN1 gene in combination with siRNAs against PPP3CB and PPP3R1 (siCaN) or scramble siRNA oligonucleotides (SCRMBL). TFEB subcellular localization was assessed in FLAG-positive population using HC imaging. The graph shows the percentage of nuclear TFEB in siCaN knock-down cells compared with control SCRMBL-transfected cells. The plot shows the mean ± s.d. for n = 5 independent experiments. (c) The MCOLN1 agonist SF-51 induces TFEB nuclear translocation. We quantified the effect of SF-51 treatment on HeLaTFEB–GFP cells using an HC assay at the time points indicated in the plot. The graph shows the percentage of treated cells with nuclear TFEB compared to DMSO control cells. The plot shows the mean ± s.d. for n = 3 independent experiments. Source data are provided in Supplementary Table 4.

Supplementary Figure 6 Calcineurin regulates the transcriptional response to starvation.

(a) The figure shows genes that were significantly regulated by starvation ordered by gene ID. A value of “1” was assigned to expression levels under fed conditions. n = 3 independent experiments. (b) The TFEB-mediated transcriptional response to starvation is inhibited in PPP3R1−/− MEFs. PPP3R1−/− and wild type MEFs were starved for 6 hrs or left in fed conditions as indicated, and the mRNA levels of TFEB target genes analyzed by quantitative PCR analysis (qPCR). mRNA expression levels of PPP3R1 were determined to confirm downregulation of PPP3R1 in PPP3R1−/− MEFs. n = 3 independent experiments. (c) HeLa cells were transfected with control SCRMBL siRNA oligonucleotides or depleted of PPP3CB and PPP3R1. 72 hours after silencing, cells were left untreated of starved. Expression levels of TFEB target genes were investigated by qPCR (n = 3 independent experiments). ∗p < 0.05, source data are provided in Supplementary Table 4.

Supplementary Figure 7 Calcineurin and MCOLN1 regulate starvation-mediated increase of autophagosome number.

(a) HeLa cells were left untreated, starved or starved in presence of FK506 (25 μM) or BAPTA-AM (25 μM). Subsequently, cells were incubated with anti-LC3 antibody to detect endogenous LC3 by immunofluorescence. HC imaging analysis was used to quantify the number of LC3-positive vesicles. As shown in the graph, the administration of the calcineurin inhibitor FK506 and calcium chelator BAPTA-AM caused a decrease of LC3-positive puncta during starvation. (n = 2 independent experiments). Scale bar 10 μm. ∗, p < 0.05. (b) Silencing of MCOLN1 reduces the earlier marker of autophagy WIPI. HeLa cells were silenced against MCOLN1 for 72 h and left untreated or starved for 4 h. IF against WIPI endogenous protein was performed. Confocal images of 30 cells per condition were analyzed. The plot shows the mean ± s.em. of WIPI-positive puncta per cell (n = 30 cells pooled from three independent experiments). Source data are provided in Supplementary Table S4.

Supplementary Figure 8 Uncropped scans of key western blots.

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Agonist-dependent activation of MCOLN1 induces TFEB nuclear translocation.

Time-Lapse of mock control HeLa cells showing the induction of TFEB nuclear translocation during the tretament with the agonist of MCOLN1 SF-51. (MOV 1563 kb)

The silencing of calcineurin reduces MCOLN1-dependent TFEB nuclear localization during SF-51 treatment.

Time-Lapse of HeLa cells knock-down for calcineurin (siCaN) showing a reduction of TFEB nuclear translocation during the tretment with the agonist of MCOLN1 SF-51. (MOV 1516 kb)

The silencing of MCOLN1 reduces TFEB nuclear localization during SF-51 treatment.

Time-Lapse of HeLa cells knock-down for MCOLN1 (siMCOLN1) showing a reduction of TFEB nuclear translocation during the treatment with its agonist SF-51. (MOV 1298 kb)

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Medina, D., Di Paola, S., Peluso, I. et al. Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. Nat Cell Biol 17, 288–299 (2015). https://doi.org/10.1038/ncb3114

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