Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB.

A RT I C L E S

Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB Diego L. Medina1,9, Simone Di Paola1, Ivana Peluso1, Andrea Armani2, Diego De Stefani2,3, Rossella Venditti1, Sandro Montefusco1, Anna Scotto-Rosato1, Carolina Prezioso1, Alison Forrester1, Carmine Settembre1,4,5, Wuyang Wang6, Qiong Gao6, Haoxing Xu6, Marco Sandri1,2,4, Rosario Rizzuto2,3, Maria Antonietta De Matteis1 and Andrea Ballabio1,5,7,8,9 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. Lysosomes are membrane-bound organelles present in all cell types. Their role in degradation and recycling processes has been extensively characterized1–3. The acidic pH of the lysosomal lumen and the presence of a broad variety of hydrolases able to degrade an ample spectrum of substrates make these organelles extraordinary machineries for the recycling of cellular waste. Extracellular substrates reach the lysosome mainly through the endocytic and phagocytic pathways, whereas intracellular substrates are delivered to the lysosome by the autophagic pathway by the fusion of autophagosomes with lysosomes4,5. Thus, lysosomes are the ‘terminal end’ of most cellular catabolic pathways. The role of the lysosomes in degradation and recycling processes has always been considered as a cellular ‘housekeeping’ function and little attention has been paid to the regulation of these processes and to the possible influence of environmental cues, such as starvation and physical exercise. The discovery that the transcription factor EB (TFEB) is a master regulator of lysosomal and autophagic function and of energy metabolism6–8 suggested that environmental cues may control

lysosomal function through the induction of a broad transcriptional program. TFEB activity is regulated by phosphorylation9–13, which keeps TFEB inactive in the cytoplasm; in contrast, dephosphorylated TFEB travels to the nucleus to activate transcriptional target genes. TFEB phosphorylation is mediated mainly by mTORC1, a major kinase complex that controls cell growth and negatively regulates autophagy. Interestingly, mTORC1 exerts its activity on the lysosomal surface and is positively regulated by lysosomal nutrients14,15. The regulation of TFEB by lysosomal mTORC1 and the shuttling of TFEB to the nucleus revealed a lysosome-tonucleus signalling mechanism9. Thus, these studies indicate that the lysosome controls cellular homeostasis using both post-translational and transcriptional mechanisms14–17. Another aspect of lysosomal function underestimated in the past is the ability to store Ca2+ and participate in calcium signalling processes. Several calcium channels reside on the lysosomal membrane. Recent studies have investigated the role of these lysosomal calcium channels in fundamental cellular processes and

1

Telethon Institute of Genetics and Medicine (TIGEM), Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy. 2Department of Biomedical Science, University of Padova, Padova 35121, Italy. 3CNR Neuroscience Institute, Padova 35121, Italy. 4Dulbecco Telethon Institute at TIGEM, Naples 80078, Italy. 5Medical Genetics, Department of Translational Medicine, Federico II University, Via Pansini 5, 80131 Naples, Italy. 6Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA. 7Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA. 8Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, Texas 77030, USA. 9 Correspondence should be addressed to D.L.M. or A.B. (e-mail: [email protected] or [email protected]) Received 29 September 2014; accepted 16 January 2015; published online 27 February 2015; DOI: 10.1038/ncb3114

288

NATURE CELL BIOLOGY VOLUME 17 | NUMBER 3 | MARCH 2015

© 2015 Macmillan Publishers Limited. All rights reserved

STV (1 h)

STV + FK506

STV + CsA

STV + FK506 + CsA

P = 0.002 P = 0.0004 P = 0.0005

100 50 0

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Percentage of TFEB nuclear translocation versus DMSO in STV

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Percentage of nuclei positive fibres

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Percentage of TFEB translocation versus SCRMBL in starvation

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Figure 1 Calcineurin regulates TFEB nuclear translocation. (a,b) siRNAmediated inhibition of PPP3CB (PPP3CB siRNA) suppresses starvationinduced nuclear translocation in HeLaTFEB–GFP cells. Scale bar, 10 µm. (b) The left plot represents the percentage of nuclear TFEB in starved (STV) cells (3 h) silenced with each single PPP3CB oligonucleotide or the pool of the 3 oligonucleotides compared with starved control cells transfected with scramble (SCRMBL) siRNA oligonucleotides. qPCR analysis of PPP3CB mRNA levels confirms the efficacy of siRNA-mediated silencing (right plot). Bar graphs show mean ± s.d. for n = 3 independent experiments. (c) Pharmacological inhibition of calcineurin reduces starvation-mediated TFEB nuclear localization. Images from a high-content assay using normally fed or 1 h starved HeLaTFEB–GFP cells treated with FK506 (5 µM) and/or CsA (10 µM). Plot represents the percentage of nuclear TFEB translocation compared with DMSO-treated fed and starved cells. Bar graphs show mean ± s.d. for n = 3 independent experiments. Scale bar, 10 µm. (d) HeLaTFEB–GFP cells were transfected with a constitutively active form of calcineurin (HA-tagged-1CaN). The plot represents the percentage of TFEB nuclear translocation. Data show the mean ± s.d. of n = 3 independent experiments. Scale bar, 10 µm. (e) HeLa cells were transfected with an empty vector or HA-tagged-1CaN. Subcellular localization of endogenous

TFEB was analysed for each condition using specific anti-TFEB antibodies. The plot represents the percentage of TFEB nuclear translocation. Bar graphs show mean ± s.d. of n = 3 independent experiments. Scale bar, 10 µm. (f) Calcineurin overexpression upregulates TFEB target genes. Top two panels: qPCR showing the efficiency of TFEB silencing (upper) and 1CaN overexpression (lower). Bottom panel: overexpression of 1CaN in HeLaTFEB–GFP cells (1CaN) and in HeLaTFEB–GFP cells transfected with an siRNA against TFEB (TFEB siRNA+1CaN) showing that calcineurin enhances the expression of TFEB target genes in a TFEB-dependent manner. n = 3 independent experiments, mean ± s.d., ∗ P < 0.05. (g) Exercise induces TFEB nuclear translocation through calcineurin. Muscles were transfected by electroporation with TFEB–GFP (green), 1CaN, its regulatory subunit (CnB) and the calcineurin inhibitor Myc–CAIN. An antibody against dystrophin (Dys, red) was used to visualize the muscle fibres. The yellow arrowheads indicate TFEB nuclear translocation in fibres stimulated by 1CaN+CnB and exercise, respectively. The plot shows the percentages of TFEB-positive nuclei in muscles under the following conditions: sedentary (control), sedentary with calcineurin (1CaN+CnB), exercised (run), exercised with CAIN (run + CAIN). The graph represents mean ± s.d. of n = 3 mice. Scale bar, 50 µm. Source data are provided in Supplementary Table 4.

their involvement in disease mechanisms18. In addition, the recent discoveries of calcium microdomains, which mediate local calcium signals from several intracellular compartments (for example, mitochondria)19, suggest that a similar situation may be found at the lysosomal surface.

In the present study, while searching for a phosphatase that dephosphorylates TFEB, we discovered another example of lysosomal signalling. We revealed a calcium signalling mechanism that starts at the lysosome and controls autophagy through calcineurin-mediated induction of TFEB.



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Figure 2 Calcineurin binds and dephosphorylates TFEB. (a) Proximity ligation assay (PLA). HeLa cells were pre-treated 10 min before fixation with 1 µM ER-tracker (green) to visualize the cytoplasm and with Hoechst 33258 (blue) to stain nuclei. After fixation, cells were processed to detect TFEB proximity interactions. Positive interactions between TFEB and PPP3CB (red dots) are clearly visible (white squares contain higher-magnification images). n = 3 independent experiments were performed. Scale bar, 10 µm. (b) PLA performed on HeLa cells transfected with PPP3CB–Myc, TFEB–GFP or both. PPP3CB and TFEB interactions are shown by the red dots. n = 3 independent experiments were performed. Scale bar, 10 µm. (c) Calcineurin knockdown reduces the starvation-induced downshift of endogenous TFEB electrophoretic mobility. HeLa cells expressing siRNA against PPP3CB were treated as indicated. Specific antibodies against TFEB were used to detect the endogenous protein. n = 3 independent experiments were performed. (d) Calcineurin overexpression reduces TFEB electrophoretic mobility. TFEB–FLAG vector alone or in combination with a constitutively active form of calcineurin (HA–1CaN) was co-transfected in fed HeLa cells. Calcineurin overexpression was confirmed by using anti-HA antibodies. n = 3 independent experiments were performed. (e) Calcineurin dephosphorylates

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Ser 142 and Ser 211 of immunoprecipitated TFEB. Lambda phosphatase (λ-PPT) was used as a positive control. Specific antibodies against S142-TFEB and a 14-3-3 motif, which binds phosphorylated TFEB serine residue Ser 211, were used to detect TFEB phosphorylation. Bar graphs show mean ± s.d. of n = 3 independent experiments. NS, not significant. (f) Calcineurin regulates TFEB downstream of mTOR. Nuclear translocation of TFEB is reduced in stable HeLaTFEB–GFP cells after silencing of PPP3CB, its essential regulatory subunit PPP3R1 or both (PPP3CB/R1). Representative images from high-content assay of HeLaTFEB–GFP cells reverse transfected with SCRMBL, PPP3CB, PPP3R1 or PPP3CB/R1 siRNAs, and then subjected to the indicated conditions. The left plot shows the mean ± s.d. of the percentage of nuclear TFEB translocation in knockdown cells compared with their corresponding control-treated cells transfected with scramble siRNA oligonucleotides. n = 3 independent experiments were performed. Scale bar, 10 µm. siRNA-mediated silencing was confirmed by qPCR analysis of PPP3CB and PPP3R1 mRNA levels (right plot). The bar graph shows the mean ± s.d. of n = 3 independent experiments. Uncropped scans of western blots are provided in Supplementary Fig. 8. Source data are provided in Supplementary Table 4.



A RT I C L E S Calcineurin modulates TFEB subcellular localization Previous studies demonstrated that mTOR-mediated phosphorylation of TFEB serine residue Ser 211 promotes the interaction of TFEB with the 14-3-3 protein and results in a cytoplasmic localization. Conversely, conditions that lead to mTOR inhibition, such as starvation and lysosomal stress, promote TFEB nuclear translocation and transcriptional activation of lysosomal and autophagic genes6,7,9,14,15,17. Although the role of the kinases that mediate TFEB phosphorylation has been defined by previous studies9–13, the phosphatase(s) involved in its dephosphorylation have remained elusive. To identify the phosphatase(s) that dephosphorylate(s) TFEB we performed a high-content screening of a phosphatase short interfering RNA (siRNA) library using a cellular assay based on cytoplasm-to-nucleus shuttling of TFEB during starvation9. We tested the effects of the specific inhibition of each of 231 phosphatases or putative phosphatases on TFEB subcellular localization. The most significant hit identified by the primary screening was the calcineurin catalytic subunit isoform beta (PPP3CB; Gene ID:5532; ref. 20), thus we focused subsequent studies exclusively on this phosphatase. Figure 1a shows that inhibition of PPP3CB suppressed starvationinduced nuclear translocation of TFEB. The ability of PPP3CB to inhibit TFEB nuclear translocation was further confirmed by using a set of 3 different PPP3CB siRNAs, as well as the pool of 3, in the HeLaTFEB–GFP cell line (Fig. 1b). In addition, the calcineurin inhibitors cyclosporin A and FK506, both jointly and individually, reduced TFEB nuclear translocation during starvation (Fig. 1c). Importantly, overexpression of 1CaN, a constitutively active form of calcineurin, induced TFEB nuclear translocation in normally fed cells, suggesting that calcineurin activity was not only necessary but sufficient to induce TFEB nuclear translocation (Fig. 1d,e). Consistent with its effect on TFEB nuclear translocation, 1CaN induced the expression of TFEB transcriptional target genes in a TFEB-dependent manner (Fig. 1f). Both starvation and calcineurin overexpression induced nuclear translocation as well as dephosphorylation of the nuclear factor of activated T cells (NFAT), which is known to depend on calcineurin activity21 (Supplementary Fig. 1). In vivo studies showed that transfection by electroporation of both calcineurin subunit 1CaN and its essential regulatory subunit PPP3R1 (CnB; ref. 22) in adult muscles from wild-type mice significantly induced TFEB nuclear translocation (Fig. 1g). In addition, exercise, a physiological stimulus that activates calcineurin23,24, promoted TFEB nuclear translocation and this effect was blunted when an endogenous calcineurin inhibitor, CAIN, was electroporated into muscle cells25 (Fig. 1g). These lossof-function and gain-of-function data demonstrate that calcineurin plays a crucial role in the regulation of TFEB subcellular localization, both in cell culture and in skeletal muscle, during energy-demanding conditions, such as starvation and physical exercise. Calcineurin binds and dephosphorylates TFEB These data prompted us to investigate the relationship between calcineurin and TFEB. PPP3CB–TFEB interaction was detected by coimmunoprecipitation experiments, performed in both HeLaTFEB–GFP and in HEK293TFEB–GFP cells (Supplementary Fig. 2a–c) and by a histidine-pulldown assay using purified His–TFEB and purified catalytic and regulatory calcineurin subunits (CaN; Supplementary Fig. 2d). In the His-pulldown assay the interaction between purified

TFEB and CaN plus calmodulin (CaM), a protein known to interact with calcineurin22, seemed to be stronger than the one between TFEB and CaN alone, whereas no interaction was detected between TFEB and CaM alone (Supplementary Fig. 2d). Notably, the interaction between PPP3CB and TFEB was detected both in fed and starved cells, suggesting that it is independent from the phosphorylation status of TFEB, as previously shown for the binding of calcineurin to NFAT (refs 21,26; Supplementary Fig. 2a,c). Finally, a physical interaction between PPP3CB and TFEB was also detected by proximity ligation assay27 (PLA) performed on endogenous proteins in non-transfected living cells (Fig. 2a). Similar data were obtained by PLA on exogenous overexpressed proteins (Fig. 2b). The interaction between mTOR and TFEB (ref. 9) was used as a positive control of the assay, and the absence of interaction between the Golgi protein Golgin97 and TFEB was used as a negative control (Supplementary Fig. 3a). The specificities of the antibodies used in the PLA were tested by both immunofluorescence and immunoblotting (Supplementary Fig. 3b–d). These data demonstrate that calcineurin physically interacts with TFEB and suggest that TFEB is a calcineurin substrate. Thus, we tested whether calcineurin dephosphorylates TFEB. siRNA-mediated inhibition of calcineurin abolished the downshift in TFEB molecular weight that is associated with TFEB dephosphorylation during starvation7 (Fig. 2c). In addition, expression of a constitutively active mutant form of calcineurin in normally fed cells resulted in TFEB molecular weight downshift (Fig. 2d). An in vitro calcineurin phosphatase assay revealed that calcineurin dephosphorylates two critical serine residues, Ser 211 and Ser 142, known to be involved in TFEB nuclear translocation9–12 (Fig. 2e). These results identify TFEB as a calcineurin substrate. Thus, calcineurin dephosphorylates TFEB and induces its nuclear translocation by a mechanism similar to the one described for NFAT (refs 21,26). Starvation and inhibition of mTOR are known to promote TFEB nuclear translocation9–11. However, we found that siRNAmediated inhibition of calcineurin significantly reduced the nuclear translocation of both endogenous and overexpressed TFEB in HeLa, HeLaTFEB–GFP and MCF-7 cells, either starved or treated with Torin1, a strong mTOR inhibitor28, demonstrating that the regulation of TFEB subcellular localization by calcineurin is independent from mTOR activity and suggesting that calcineurin acts downstream of mTOR in the regulation of TFEB (Fig. 2f and Supplementary Fig. 3e). Calcium modulates TFEB localization As calcineurin is activated by intracellular calcium20,29, we assessed the calcium-dependence of TFEB regulation by artificially modulating cytoplasmic Ca2+ using Ca2+ chelators and ionophores. The Ca2+ chelator BAPTA-AM significantly reduced TFEB nuclear translocation in both starved and Torin1-treated cells (Fig. 3a). Conversely, thapsigargin, an inhibitor of ER Ca2+ -ATPase, and ionomycin, a Ca2+ ionophore, significantly induced TFEB nuclear translocation in normally fed cells and kept TFEB in the nucleus after starvation/re-feeding (Fig. 3b–d). Thapsigargin treatment also reduced the phosphorylation levels of TFEB serine residues Ser 142 and Ser 211 in fed cells (Fig. 3e,f). Importantly, thapsigargin-induced nuclear translocation of TFEB was suppressed when PPP3CB and its essential regulatory



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Percentage of TFEB nuclear translocation versus SCRMBL SC P R P 3C M PP PP B BL P3 3R siR C 1 NA B/ si R1 RN si A RN FK A 50 6

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Figure 3 Ca2+ elevation induces TFEB nuclear translocation through calcineurin. (a) Chelation of intracellular Ca2+ suppresses TFEB nuclear translocation in both starvation (1 h) and Torin1 treatment (1 µM). The graph represents the percentage of TFEB nuclear translocation in starved and Torintreated HeLaTFEB–GFP cells in the presence or absence of BAPTA-AM (10 µM; mean ± s.d. for n = 3 independent experiments). (b) Nuclear and cytosol fractions extracted from HeLa cells overexpressing TFEB–FLAG, and treated as indicated, were subjected to immunoblot to detect TFEB using antiFLAG antibodies. (c) Intracellular Ca2+ elevation inhibits nutrient-dependent relocation of TFEB from the nucleus to the cytosol. HeLaTFEB–GFP cells were starved for 1 h to induce TFEB nuclear translocation. Subsequently, the culture medium was changed to complete normal medium, which normally induces TFEB relocation to the cytosol, or complete medium plus calcium ionophores (1 µM ionomycin or 300 nM thapsigargin) for 20 min. The plot shows the percentage of nuclear TFEB in treated cells compared with corresponding control cells (mean ± s.d. for n = 3 independent experiments). (d) Thapsigargin induces endogenous TFEB translocation. Endogenous TFEB subcellular localization under feeding, starvation, and thapsigargin-treatment conditions was analysed using a high-content assay. The plot shows the

percentage of TFEB nuclear translocation compared with fed control cells (mean ± s.d., n = 4 independent experiments). (e,f) Thapsigargin reduces TFEB phosphorylation at key serine residues Ser 142 and Ser 211. (e) Immunoblot analysis of TFEB phosphorylation using an anti-phospho S142-TFEB antibody. Protein lysates from HeLa cells transfected with TFEB–GFP vector and subjected to the indicated treatments were blotted against anti-phospho-S142-TFEB antibody; anti-GFP antibody was used to detect total TFEB. The plot shows the mean ± s.d. for n = 4 independent experiments. (f) Indirect assessment of phosphorylation of TFEB Ser 211. Immunoprecipitation of GFP from HeLa cells overexpressing TFEB–GFP was followed by immunoblot using antibodies against the specific 14-3-3 motif that recognizes phosphorylated Ser 211. The plot shows the mean ± s.d. for n = 2 independent experiments. (g) Silencing of PPP3CB and its regulatory subunit PPP3R1 suppresses thapsigargin-induced nuclear translocation of TFEB. The graph represents the percentage of TFEB nuclear translocation in silenced cells versus cells transfected with a scramble oligonucleotide in the treatments indicated (mean ± s.d. for n = 3 independent experiments). All scale bars in this figure, 10 µm. Uncropped western blots are provided in Supplementary Fig. 8, and source data in Supplementary Table 4.

subunit PPP3R1 were silenced (Fig. 3g) and was blunted in PPP3R1−/− cells30 (Supplementary Fig. 3f), thus proving that the effects of Ca2+ on TFEB are mediated by calcineurin. Previous studies reported that long-term (24 h) thapsigargin treatment inhibited mTOR activity

through Ca2+ -dependent induction of AMPK (ref. 31). However, we found that up to 3 h treatment with thapsigargin did not affect mTOR activity, measured by mTOR-mediated phosphorylation of P70S6 kinase (Supplementary Fig. 3g), indicating that intracellular Ca2+

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Figure 4 Starvation induces lysosomal Ca2+ release through MCOLN1. (a) Starvation does not induce bulk cytosolic Ca2+ elevation in HeLa cells transfected with the Ca2+ -sensitive probe aequorin. Bulk cytosolic [Ca2+ ] was monitored during perfusion with complete L-15 medium, HBSS and HBSS supplemented with 100 µM histamine as indicated. n = 8 coverslips from two independent transfections. (b) Stable HeLaTFEB–GFP cells were left untreated or pretreated for 30 min with the Ca2+ chelators BAPTAAM or EGTA-AM (5 µM each). After washing, cells were left untreated or starved for 1 h. After treatment, cells were fixed and a high-content imaging analysis was performed. The plot shows the percentage of TFEB nuclear translocation in BAPTA-treated cells compared with untreated and EGTA treated (mean ± s.d., n = 3 independent experiments). Scale bar, 10 µm. (c) Average and s.d. of [Ca2+ ]cyt evoked by maximal histamine stimulation in wild-type HeLa cells. Agonist stimulation was carried out in complete L15 medium or after a three-minute starvation with HBSS (2.45 ± 0.19 µM, HeLa wild-type in L-15 medium; 2.406 ± 0.23 µM, HeLa wild-type in HBSS; mean ± s.d.; n = 8 coverslips from two independent transfections). (d) Representative traces of the cytosolic GCaMP6s and the perilysosomal ML1-GCaMP3 calcium probes. HeLa cells were transfected with the indicated probe and ratiometric imaging (474 and 410 nm excitation) was performed.

Cells were continuously perfused with the indicated solutions. The bottom left plot represents the average perilysosomal calcium peak values induced by perfusion of the indicated buffer, as recorded by the GCaMP3–ML1 probe (ML1–GCaMP3 ratio: 1.149 ± 0.051, L-15; 1.508 ± 0.060, HBSS; 7.500 ± 0.456, histamine; mean ± s.e.m.; n = 18 cells from two independent transfections; ∗ , P < 0.001 when compared with L-15). The bottom right plot represents the average cytosolic calcium peak values induced by perfusion of the indicated buffer, as recorded by the GCaMP6s probe (ML1–GCaMP3 ratio, ex 474/410 nm: 0.404 ± 0.026, L-15; 0.389 ± 0.022, HBSS; 7.473 ± 0.428, histamine; mean ± s.e.m.; n = 12 cells from two independent transfections). (e) Ca2+ release (measured with 1F /F0 fluorescence intensity) was detected right after the L-15 medium (containing 2 mM [Ca2+ ], amino acids and 10% FBS) was switched to Tyrode’s solution (2 mM [Ca2+ ]) in HEK293 cells stably expressing GCaMP3–ML1 (ref. 36). The agonist of MCOLN1 ML-SA1 (10 µM) was applied to induce MCOLN1-mediated Ca2+ release. n = 3 independent experiments. (f) Similarly, the lysosomotropic drug GPN blunted starvation-mediated calcium release detected by GCaMP3– ML1. The bar graph shows the mean ± s.d. of n = 3 independent experiments, ∗ , P < 0.05. Source data are provided in Supplementary Table 4.

elevation promotes TFEB nuclear translocation through the activation of calcineurin and further suggesting that calcineurin-dependent TFEB dephosphorylation acts downstream of mTOR in the TFEB regulatory pathway.

Starvation induces lysosomal Ca2+ release through MCOLN1 Our data indicate that starvation-induced TFEB nuclear translocation requires the activity of calcineurin and that an increase of cytosolic calcium induces TFEB nuclear translocation. Thus, we studied the



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Figure 5 MCOLN1-mediated calcium release induces TFEB nuclear translocation. (a,b) Silencing of MCOLN1 reduces starvation-mediated TFEB nuclear translocation. (a) HeLa cells or a HeLa cell line stably-transfected with a MCOLN1 shRNA was left untreated or transiently transfected with a TFEB–GFP plasmid. The left plot shows the percentage of TFEB–GFP nuclear translocation in starved (3 h) HeLa-MCOLN1 shRNA cells compared with starved HeLa cells (n = 3 independent experiments). Similarly, the middle plot shows the results on endogenous TFEB nuclear translocation (n = 4 independent experiments). The right plot shows the efficiency of MCOLN1 silencing (n = 3 independent experiments). The graphs show the mean ± s.d. (b) Silencing of MCOLN1 reduces starvation-mediated TFEB nuclear translocation. HeLa and HeLa-MCOLN1 shRNA cells were transfected with a TFEB–3×FLAG construct. Following starvation, 50 µg of nuclear and 100 µg of cytosolic extracts were prepared and probed using anti-Flag antibody (n = 3 independent experiments). (c) Endogenous TFEB nuclear translocation analysis of human wild-type and mucolipidosis IV fibroblasts in fed conditions and after a 3 h starvation of serum and amino acids. The graph shows the percentage of TFEB nuclear translocation in human MLIV cells when compared with WT cells in starvation conditions (mean ± s.d. of n = 4 independent experiments). Scale bar, 10 µm. (d) Overexpression of MCOLN1 induces TFEB nuclear translocation. Stable HeLaTFEB–GFP cells

were transfected with plasmids carrying wild-type or constitutively active mutant forms of FLAG-tagged MCOLN1. TFEB subcellular localization was assessed in FLAG-positive (red-stained cells) and -negative cell populations. The graph shows the percentage of TFEB nuclear translocation in the different transfected cell groups, compared with fed and starved (3 h) conditions (mean ± s.d. for n = 5 independent experiments). Scale bar, 10 µm. (e) Frames of time-lapse experiments in HeLaTFEB–GFP cells treated with the MCOLN1 agonist SF-51 (200 µM) after the transfection of PPP3CB+PPP3R1 (CaN) siRNA, MCOLN1 or scramble siRNAs (Supplementary Videos 1–3). Yellow arrowheads indicate TFEB nuclear localization. The plot shows the kinetics of TFEB nuclear localization during the time of recording (n = 3 independent experiments). (f) Exercise induces TFEB nuclear translocation through MCOLN1. Muscles were electroporated with TFEB–GFP (green) and shRNAs against MCOLN1 (red). Antibodies against dystrophin (Dys, red) were used to reveal the muscle fibres. The arrowheads indicate TFEB nuclear localization in muscle fibres. The plot shows the percentages of TFEBpositive nuclei in muscles under the following conditions: sedentary (control), exercised (run), exercised with MCOLN1 shRNA (run + MCOLN1 shRNA) (mean ± s.d. of n = 3 mice). Scale bar, 50 µm. Uncropped western blots and source data are provided in Supplementary Fig. 8 and Supplementary Table 4.

effects of starvation on cytosolic Ca2+ . Starvation did not induce changes in the overall levels of cytosolic Ca2+ , as recorded by the aequorin-based probe localized in the bulk cytoplasm (Fig. 4a). Furthermore, although BAPTA-AM treatment inhibited TFEB nuclear translocation, a slower Ca2+ -chelator (that is, EGTA-AM; ref. 32) did not, suggesting that starvation rapidly triggers local calcium signals (Fig. 4b). The ER Ca2+ content was not significantly affected after

starvation, as detected by measuring cytosolic calcium transients evoked by the IP3 -mobilizing hormone histamine in fed and starved cells (Fig. 4a,c). We postulated that a calcium release from the lysosome may be responsible for local calcineurin activation. Starvation clearly elicited a Ca2+ signal in HeLa cells transiently transfected with a Ca2+ sensor linked to the lysosomal calcium channel mucolipin 1 (MCOLN1),

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Figure 6 Calcineurin regulates the lysosomal/autophagic pathway. (a) The transcriptional response of lysosomal/autophagic genes is reduced in PPP3R1−/− KO MEFS (bottom) compared with wild-type (top) cells (ranked by fold-change). A value of 1 was assigned to expression levels in fed conditions (n = 3 independent experiments). (b,c) Immunoblots against LAMP1 (b) and LC3 (c) from wild-type and PPP3R1−/− MEFs in fed and starved (3 h) conditions. The plot shows the quantification of Lamp1 and LC3-II proteins levels normalized by actin loading control (mean ± s.d. for n = 2 and n = 5 independent experiments, respectively). NS, not significant. (d) high-content analysis of LC3-positive vesicles in wild-type and PPP3R1−/− MEFs in fed and starved conditions. The bar graph shows the mean ± s.d. of LC3positive vesicles in the different treatment conditions for n = 6 independent experiments. Scale bar, 10 µm. (e) Analysis of the overexpression of the autophagy-related PI(3)P reporter GFP–2×FYVE (ref. 55) during starvation in (Mock) HeLa cells, and cells silenced for PPP3CB/PPP3R1. GFP–2×FYVEpositive vesicles were counted using ImageJ software. Approximately 50 cells per condition were analysed by confocal imaging. Data shows the mean ± s.d. for n = 2 independent experiments. Scale bar, 10 µm. (f) Depletion of

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calcineurin reduces the autophagic flux. HeLa cells were transfected with SCRMBL siRNA or siRNA against both PPP3CB and PPP3R1 (CaN siRNA). After 48 h, cells were transfected with a plasmid carrying the autophagy substrate p62 fused to GFP for 24 h, and treated as indicated. The graph shows the levels of p62–GFP normalized by actin at the different times (mean ± s.d.). (g) Analysis of mTOR activity during starvation in normal cells and in cells silenced against calcineurin (CaN siRNA). Two mTOR substrates, phospho-ULK and phospho-p70S6K, as well as the total protein, were detected by immunoblotting using specific antibodies (n = 2 independent experiments). (h,i) Overexpression of 1CaN increased LC3II protein levels in a TFEB-dependent manner. RPE-1 cells (h) and HeLa cells (i) were transfected with TFEB or scramble siRNAs and after 48 h they were transfected with 1CaN for 24 h. Cells were left in fed conditions and treated or not with bafilomycin. Fifty micrograms of protein extracts we then immunoblotted and tested for the amount of LC3-II using specific antibodies (n = 4 independent experiments). Bar graphs show the mean ± s.d. Uncropped western blots and source data are provided in Supplementary Fig. 8 and Supplementary Table 4.



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A RT I C L E S also known as transient receptor potential calcium channel mucolipin subfamily member 1 (TRPML1), which is localized on the lysosomal surface (ML1-GCaMP3; Fig. 4d). Similar results were obtained in HEK293 cells stably overexpressing ML1-GCaMP3 (Fig. 4e). Consistent with a lysosomal contribution to this calcium signalling mechanism, the increase in Ca2+ levels induced by starvation detected by ML1-GCaMP3 was not affected by the removal of extracellular Ca2+ (ruling out the contribution of plasma membrane calcium channels), but was blunted by treatment with the lysosomotropic agent GPN (Fig. 4f). Notably, neither the silencing of the lysosomal twopore channel TPCN2 (ref. 33) nor the overexpression of a critical regulator of mitochondrial Ca2+ uptake, MICU1 (ref. 34), altered TFEB subcellular localization (Supplementary Fig. 4a,b). These data strongly suggest that starvation specifically induces lysosomal Ca2+ release through MCOLN1 and that this generates a lysosomal calcium microdomain that is responsible for calcineurin activation. We then analysed the involvement of MCOLN1 in the regulation of TFEB nuclear translocation35–38. Short hairpin (shRNA)-mediated inhibition of MCOLN1 significantly reduced cytoplasm-to-nucleus shuttling of both endogenous and transiently transfected TFEB– GFP after starvation, further suggesting that this process requires Ca2+ release from the lysosome (Fig. 5a). Similar results were obtained by nucleus/cytoplasm fractionation (Fig. 5b). Consistently, depletion of MCOLN1 reduced the downshift of endogenous TFEB molecular weight during starvation, suggesting a defective dephosphorylation (Supplementary Fig. 5a). Mutations in the MCOLN1 gene cause mucolipidosis type 4 (MLIV), a lysosomal storage disease39,40. We found that starvation-induced nuclear translocation of TFEB is inhibited in fibroblasts from MLIV patients (Fig. 5c). Furthermore, overexpression of wild-type MCOLN1 and of gain-of-function MCOLN1 mutants in HeLaTFEB–GFP cells promoted TFEB nuclear translocation (Fig. 5d) in a calcineurin-dependent fashion (Supplementary Fig. 5b) and similar results were obtained by using a recently described MCOLN1-specific agonist, SF51 (ref. 36; Fig. 5e and Supplementary Videos 1–3 and Supplementary Fig. 5c). Importantly, inhibition of MCOLN1 in skeletal muscles in vivo by transfection of MCOLN1 shRNA suppressed the exercisemediated nuclear translocation of TFEB (Fig. 5f), demonstrating the physiological relevance of lysosomal calcium signalling on TFEB regulation in vivo. Together these data suggest that although calcineurin–TFEB interaction may occur anywhere in the cytoplasm, focal Ca2+ release from the lysosome through MCOLN1 modulates TFEB activity through local calcineurin activation. Thus, MCOLN1 plays a crucial role in the activation of calcineurin near the lysosomal surface, through the generation of a lysosomal Ca2+ microdomain.

and the amplitude of the response were significantly reduced in PPP3R1−/− compared with wild-type cells (Fig. 6a and Supplementary Fig. 6a and Supplementary Tables 2 and 3). Transcriptome analysis results were confirmed by real-time PCR experiments on selected TFEB target genes (Supplementary Fig. 6b). Similar results were obtained in HeLa cells in which both PPP3R1 and PPP3CB were silenced (Supplementary Fig. 6c). Next we tested whether calcineurin-mediated TFEB activity had an effect on lysosomal/autophagic function. Lack of calcineurin in PPP3R1−/− cells suppressed the starvation-induced elevation of the lysosomal marker LAMP1 (Fig. 6b) and autophagosomal protein LC3, and blunted the increase in LC3-II levels and in the number of LC3-positive vesicles (Fig. 6c,d). Similar results were obtained by pharmacological inhibition of calcineurin (Supplementary Fig. 7a). In addition, the silencing of calcineurin significantly reduced the number of phosphatidylinositol 3-phosphate (PI(3)P)-positive vesicles, indicating an impairment in autophagosome formation42, during starvation (Fig. 6e). Furthermore, silencing of both PPP3R1 and PPP3CB in HeLa cells resulted in a reduction of the autophagic flux, measured by the degradation of the autophagic substrate p62 (Fig. 6f). This autophagy impairment was not due to the inhibition of mTOR signalling, as we observed normal phosphorylation kinetics of two different mTOR substrates (ULK, and P70S6K; Fig. 6g). Conversely, overexpression of 1CaN increased LC3II protein levels in a TFEB-dependent manner (Fig. 6h,i). Together these data identify calcineurin as a new regulator of the lysosomal/autophagic pathway. Consistent with a role of MCOLN1 and lysosomal Ca2+ in the activation of calcineurin, the silencing of MCOLN1 significantly reduced the number of PI(3)P-positive vesicles (Fig. 7a). Similar results were observed using immunofluorescence against endogenous WIPI protein, a marker of early steps in autophagosome formation, in cells silenced for MCOLN1 (Supplementary Fig. 7b). In addition, we found that MCOLN1 overexpression resulted in a significant increase in the levels of the autophagosome marker LC3, which were further increased by bafilomycin, indicating an induction of autophagy, rather than an impairment of autophagosome degradation (Fig. 7b,c). Thus, lysosomal calcium signalling through MCOLN1 controls autophagy through calcineurin-mediated activation of TFEB. Interestingly, MCOLN1 is known to be a direct transcriptional target of TFEB (refs 6,38,41). This suggests the presence of a positive feedback loop by which TFEB regulates the expression of MCOLN1, which in turn promotes TFEB activity through Ca2+ mediated activation of calcineurin. Consistently, overexpression of TFEB markedly increased the effects of MCOLN1 agonists, probably owing to an increase in the number of MCOLN1 channels, as observed with lysosomal patch clamp (Fig. 7d,e).

Calcineurin and MCOLN1 regulate autophagy through TFEB

DISCUSSION

As TFEB transcriptionally regulates autophagy during starvation7,41, we tested whether the transcriptional response of lysosomal/ autophagic genes to starvation required calcineurin. Whole-genome gene expression profiling experiments revealed that starvation perturbed the transcriptomes of both wild-type and PPP3R1−/− mouse embryonic fibroblasts (MEFs) in a statistically significant manner. We specifically analysed the expression behaviour of 322 lysosomal/autophagic genes (Supplementary Table 1) in the two cell lines. Remarkably, both the number of genes responsive to starvation

The regulation of autophagy is thought to be a biphasic process, with a fast induction that relies on post-translational and protein– protein interaction events, and a sustained response that is mediated by transcriptional mechanisms17. Our results identify a calcium signalling pathway that originates from the lysosome and regulates autophagy by activating a TFEB-mediated transcriptional program. It has been well established that autophagy is negatively regulated by the mTOR kinase43. The pathway regulating autophagy identified in our study is mediated by the Ca2+ -dependent phosphatase

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Figure 7 MCOLN1 regulates the lysosomal/autophagic pathway. (a) Representative images and analysis of the overexpression of the autophagyrelated PI(3)P reporter GFP–2×FYVE (ref. 55) during starvation in wildtype (Mock) HeLa cells, and in cells silenced for MCOLN1. GFP–2×FYVEpositive vesicles were counted using ImageJ software. Approximately 50 cells were pooled per condition and analysed by confocal microscopy imaging. Data show the mean ± s.d. of n = 2 independent experiments. (b) MCOLN1 overexpression leads to a significant increase in LC3 levels. HeLa cells were transfected with Myc-tagged MCOLN1 or gain-of-function mutants R427P and V432P respectively. Immunofluorescence was performed using antiLC3 antibodies. The number of LC3 spots per cell was quantified using

high-content imaging analysis. Mean values ± s.d. are plotted for n = 4 independent experiments. Scale bar, 10 µm. (c) Fifty micrograms of protein extracts from HeLa cells treated with bafilomycin (BAFA) and overexpressing an empty vector of a Myc–MCOLN1 plasmid for 24 h was immunoblotted against LC3 (n = 3 independent experiments). (d,e) Overexpression of TFEB increases the effects of MCOLN1. Lysosomal patch-clamp was performed in both control (d) and TFEB-overexpressing cells (e). Note that the effect of different concentrations of the MCOLN1 agonist ML-SA1 is strongly enhanced probably owing to an increase in the number of MCOLN1 channels. n = 3 independent experiments. Uncropped scans of western blots are provided in Supplementary Fig. 8. Source data are provided in Supplementary Table 4.

calcineurin and seems to operate independently from mTOR. Figure 8 shows a model representing how starvation and exercise regulate TFEB using both the mTOR and calcineurin pathways. In normal feeding/sedentary conditions mTORC1 phosphorylates TFEB on the lysosomal surface, enabling its interaction with 14-3-3 proteins, thus preventing its nuclear translocation. Conversely, during starvation and physical exercise mTORC1 dissociates from the lysosomal surface and its activity is inhibited. At the same time, calcineurin activity is locally induced near the lysosome by lysosomal calcium release through MCOLN1. This leads to both a decreased rate of TFEB phosphorylation, through mTORC1 inhibition, and an induction of TFEB dephosphorylation, through calcineurin induction. Dephosphorylated TFEB is free to travel to the nucleus and to induce the expression of lysosomal and autophagic genes (Fig. 8). Previous studies reported that exercise induces autophagy in muscle44–46. Our results provide a mechanistic link between calcineurin activation and autophagy induction during exercise. So far, the transcription factors of the NFAT family, master regulators of T-cell activation47, have been considered the main transcriptional mediators of calcineurin function. The discovery that TFEB, a master controller of autophagy, is a calcineurin substrate expands the current view on the mechanism of action of calcineurin.

Interestingly, we found that starvation induces calcineurin-mediated dephosphorylation and nuclear translocation of both TFEB and NFAT, suggesting a coordinated regulation of the two different transcriptional networks. Previous studies implicated calcineurin in a variety of both physiological and pathological processes, such as neuronal synaptic transmission, adaptative immune response and muscle remodelling after physical exercise48,49. Our data suggest that some of these processes may be linked to the lysosomal/autophagic pathway through TFEB. In addition, other calcineurin substrates may also play a role in autophagy. Calcium plays a crucial role in the regulation of the activity of transcription factors through the induction of calcium-dependent kinases and phosphatases50. Until now conflicting results have been reported on the mechanism by which calcium regulates autophagy. Here we dissected a Ca2+ -dependent pathway that originates from the lysosome and regulates autophagy at the transcriptional level. Recent studies have revealed the presence of calcium microdomains, mediated by different calcium channels, which activate calcineurin in the vicinity of different cellular compartments (that is, sarcolemma, mitochondria, sarcoplasmatic reticulum and perinuclear sarcoplasmatic reticulum) in the heart51. Our study demonstrates that local calcineurin activation can also



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in the vicinity of the lysosome. This leads to local calcineurin (Cn) activation and TFEB dephosphorylation. Dephosphorylated TFEB is no longer able to bind 14-3-3 proteins and can freely translocate to the nucleus where it transcriptionally activates the lysosomal/autophagic pathway.

occur near the lysosome through the lysosomal calcium channel MCOLN1. In addition, our data also revealed a positive feedback loop by which TFEB is activated by the calcium channel MCOLN1, which is known to be a TFEB transcriptional target41. In conclusion, the identification of a mechanism by which lysosomal Ca2+ regulates autophagy through the activation of the phosphatase calcineurin and the transcription factor TFEB further supports the role of the lysosome as a signalling hub. It is likely that future studies will lead to the identification of other signalling pathways originating from the lysosomal surface.

AUTHOR 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.

METHODS Methods and any associated references are available in the online version of the paper. Note: Supplementary Information is available in the online version of the paper 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–1CnA, 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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299

METHODS

DOI: 10.1038/ncb3114

METHODS Drugs and cellular treatments. The following drugs were used: Torin 1 (1 µM, otherwise indicated) from Tocris Bioscience; FK506 (5 µM, otherwise indicated), cyclosporine A (10 µM, otherwise indicated), chloroquine (100 µM), thapsigargin (300 nM), ionomycin (1 µM) and EGTA from Sigma; BAPTA-AM (5–25 µM) from Invitrogen; EGTA-AM (5 µM) from Santa Cruz Biotechnology; bafilomycin A1 (300 nM); SF51 (200 µM) from VITAS-M Laboratory. Starvation medium was HBSS, 10 mM HEPES.

Cell culture, plasmids and siRNA transfection. HeLa, MCF-7, HEK293 and RPE cells were purchased from ATCC and cultured in DMEM or RPMI 1640 media supplemented with 10% fetal bovine serum, 200 µM L-glutamine, 100 µM sodium pyruvate, in 5% CO2 at 37◦ . Cells were silenced with TFEB siRNA oligonucleotides (Thermo, Dhamacon D-009798-03), PPP3CB siRNA oligonucleotides (Ambion, 4390824), PPP3R1 siRNA oligonucleotides (Invitrogen, HSS108410-3, HSS1830463, HSS108411-3) or non-targeting siRNAs (Thermo, D-001810-10-05), by direct or reverse transfection, using Lipofectamine RNAiMAX reagent (Invitrogen) according to the protocol from the manufacturer. siRNA-transfected cells were collected after 72 h, if not otherwise stated. Immortalized MEFs from conditional PPP3R1flox/flox (PPP3R1+/+ ) mice, a gift from J. Molkentin (Cincinnati Children’s Hospital Medical Center, USA), were cultured in DMEM, 10% CS, 200 µM L-glutamine, 100 µM sodium pyruvate. PPP3R1flox/flox (PPP3R1+/+ ) MEFs were used as control, and lentiviral-mediated Cre transduction was used to generate PPP3R1−/− MEFs (ref. 30). The stable HeLaMCOLN1shRNA cell line was described in ref. 38. The stable HeLaTFEB–GFP cell line was described in ref. 9. ML-IV human fibroblasts were a gift from G. Borsani (University of Brescia, Italy). Human full-length TFEB–GFP and TFEB–FLAG were previously described7. Human full-length PPP3CB and MCOLN1 tagged with Myc–FLAG were from OriGene. The plasmid carrying a constitutively active form of the human calcineurin catalytic subunit (1Can) was donated by B. Rothermel (The University of Texas Southwestern Medical Center, USA). NFAT–GFP was a gift from T. Baldari (University of Siena, Italy)52; Micu1 plasmid was described in ref. 34. Cells were transfected using Lipofectamine LTX – Plus reagent (Invitrogen) or Trans-IT reagent (Mirus-Bio) according to the protocol from the manufacturers.

Antibodies and western blotting. For western blot the following antibodies were used: anti-FLAG (M2, cat. F1804, 1:2,000), β-actin (AC-15, cat. A5441, 1:4,000), β-tubulin (TUB 2.1, cat.T4026, 1:2,000) and -ULK (cat. A7481, 1:1,000) from SigmaAldrich; anti-LC3 (cat. NB100-2220, 1:1,000) from Novus Biologicals; anti-PARP1 (cat. ALX-10-302, 1:4,000) from ENZO Life Sciences; anti-TFEB (cat. 4240, 1:1,000), -p-Ser 14-3-3 binding motif (cat. 9601, 1:1,000), -p-P70S6K (cat. 108D2, 1:1,000), p-ULK (cat. 6888, 1:1,000) from Cell Signaling Technologies; anti-HA (16B12, cat. MMS-101P, 1:7,500) from Covance; anti-P70S6K (cat. KAP-CC035, 1:1,000) from Stressgen; anti-PPP3CB (cat. TA308438, 1:1,000) from OriGene; anti-pan 14-3-3 (B11, cat. sc-133232, 1:1,000), -GAPDH (6C5, cat. sc-32233, 1:10,000) and -c-Myc (9E10, cat. sc-40, 1:1,000) from Santa Cruz Biotechnology; anti-GFP (1:10,000) was provided by M.A.D.M. (TIGEM); anti-p-Ser142-TFEB (1:1,000) was produced as described previously9. Total cell lysates were prepared by solubilization in RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl and 0.01 M sodium phosphate, at pH 7.2) containing protease (Roche) and phosphatase (Sigma) inhibitors. The soluble fraction was recovered in the supernatant after centrifugation at 12,000g for 15 min, and protein concentration was measured by the Bradford method. Nuclear/cytosolic fractions were isolated as previously described9. After SDS–PAGE and immunoblotting, the proteins recognized by the specific antibodies were visualized by chemiluminescence methods (ECL Western Blotting Substrate, Pierce) using peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Calbiochem). Membranes were developed using a Chemidoc UVP imaging system (Ultra-Violet Products) and densitometric quantification was performed in unsaturated images using ImageJ (NIH).

Proximity ligation assay. The PLA assay was performed using the Duolink in situ reagents (Olink Biosciences) according to the manufacturer’s specifications. Golgin 97 polyclonal antibody used for immunofluorescence was raised in rabbit by immunization with recombinant glutathione S-transferase (GST)-fusion full-length protein. The antibody was affinity-purified over a column conjugated with a His fusion of the protein. Other antibodies used in this study were from the following sources: monoclonal antibody TFEB (17, cat. sc-101532, 1:50) from Santa Cruz Biotechnology and anti-mTOR (cat. 7010, 1:200) from Cell Signaling Technology. Alexa-conjugated secondary antibodies and ER-tracker were from Life Technologies. Immunofluorescence experiments were performed as previously described53. Briefly, HeLa cells were fixed in 4% paraformaldehyde (PFA) for 10 min and

permeabilized in 0.1% (w/v) saponin, 0.5% (w/v) BSA and 50 mM NH4 Cl in PBS (blocking buffer). Cells were incubated with the indicated primary antibodies for 1 h and subsequently incubated with secondary antibodies. For confocal imaging, the samples were examined under a Zeiss LSM 700 confocal microscope. Optical sections were obtained under a ×63 immersion objective at a definition of 1,024 × 1,024 pixels (average of eight or sixteen scans), adjusting the pinhole diameter to 1 Airy unit for each emission channel to have all of the intensity values between 1 and 254 (linear range). For image analyses, mock-treated and TFEB-KD HeLa cells were fixed and stained as described above and cells acquired with the same laser parameters using the same image magnification.

Co-immunoprecipitation assays. HeLa cells expressing the indicated tagged proteins were rinsed twice with ice-cold PBS and lysed in ice-cold lysis buffer (400 mM NaCl, 25 mM Tris-HCl, pH 7.4, 1 mM EDTA and 1% Triton X-100) containing protease and phosphatase inhibitors. The soluble fractions from cell lysates were isolated by centrifugation at 12,000g for 15 min in a microfuge. For coimmunoprecipitations, each lysate was incubated with the corresponding antibody in binding buffer (200 mM NaCl, 25 mM Tris-HCl, pH 7.4, 1 mM EDTA) with constant rotation overnight at 4 ◦ C. Then, 40 µl of a 50% slurry of Protein-G beads (Sigma-Aldrich) was added to lysates and incubated with rotation for an additional 2 h at 4 ◦ C. After incubation, the resins were washed 5 times in binding buffer and the samples were eluted in Laemmli buffer and separated by SDS–PAGE.

Histidine-pulldown assay. Purified His–TFEB (300 nM), CaN (ProAlt) and CaM (Enzo Life Sciences) and GST (100 nM each) were incubated in binding buffer (20 mM Tris, pH 8; 100 mM NaCl; 2 mM CaCl2 ; 6 mM MgCl2 ; 0.2% Triton X-100; 30 mM imidazole; protease and phosphatase inhibitor) overnight at 4 ◦ C. His–TFEB was pulled-down by adding 20 µl of Ni-NTA resin (Qiagen) for 2 h at 4 ◦ C. After incubation, the resins were washed 5 times in binding buffer and the samples were eluted in Laemmli buffer and separated by SDS–PAGE.

In vitro calcineurin phosphatase assay. Phosphorylated FLAG–TFEB was obtained by immunoprecipitation from fed HeLa cells. Protein-G beads containing FLAG–TFEB were incubated for 40 min at 30 ◦ C in 24 µl of the corresponding phosphatase reaction buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2 , 2 mM dithiothreitol, protease inhibitors with 1 mM CaCl2 or 10 mM EGTA) containing purified calcineurin (100U; ENZO Life Sciences) and calmodulin (4 mM; ENZO Life Sciences). Samples were eluted by adding 8 µl of 4× Laemmli buffer, resolved by SDS–PAGE and analysed by immunoblotting. Antibodies anti-p-Ser 14-3-3 binding motif10, anti-P-Ser142-TFEB (ref. 9) and TFEB (Cell Signaling Technologies) were used to detect phosphorylated and total TFEB forms, respectively.

RNA extraction and quantitative PCR. Total RNA was extracted from cells using RNeasy Plus Mini Kit (Qiagen). Reverse transcription was performed using QuantiTect Rev Transcription Kit (Qiagen). Real-time quantitative PCR with reverse transcription (qRT–PCR) was carried out using the LightCycler System 2.0 (Roche Applied Science). HPRT was used for qRT-PCR as a reference gene. The parameters of real-time qRT-PCR amplification were according to Roche recommendations. The following primers were used in this study. Mouse primers: PGC1a; forward (fw) 50 -gaatcaagccactacagacaccg-30 , reverse (rev) 50 -catccctcttgagccttt cgtg-30 , MCOLN1; fw: 50 -gcgcctatgacaccatcaa-30 , rev: 50 -tatcctggactgctcgat-30 ; ATP6V0D1; fw: 50 -gcatctcagagcaggaccttga-30 , rev: 50 -ggataggacacatggcatcagc-30 , ATP6V1H; fw: 50 -gttgctgctcacgatgttggag-30 , rev: 50 -tgtagcgaacctgctggtcttc-30 , CTSF; fw: 50 -acgcctatgcagccataaag-30 , rev: 50 -cttttgccatctgtgctgag-30 , CTSB; fw: 50 -tt agcgctctcacttccactacc-30 , rev: 50 -tgcttgctaccttcctctggtta-30 , TPP1; fw: 50 -aagccagg cctacatagtcaga-30 , rev: 50 -ccaagtgcttcctgcagtttaga-30 , DPP7; fw: 50 -cgccagcaat actgtctggatac-30 , rev: 50 -aaatgatgttgctggctgcttta-30 , WIPI; fw: 50 -acaggacgcctgaac ttctc-30 , rev: 50 -cggcagtttctggatcgt-30 , TcFEB; fw: 50 -gcgagagctaacagatgctga-30 , rev: 50 -ccggtcattgatgttgaacc-30 , HPRT; fw: 50 -caagcttgctggtgaaaagg-30 , rev: 50 -gtcaaggg catatccaacaac-30 , PPP3R1; fw: 50 -tgcctgagttacagcagaacc-30 , rev: 50 -tcgcctttgaca ctgaactg-30 , ATG14; fw: 50 -gcttcgaaggtcacacatcc-30 , rev: 50 -cttgaggtcatggcactgtc-30 , PPP3CA; fw: 50 -atcccaagttgtcgacgacc-30 , rev: 50 -acactttcttccagcctgcc-30 . Human primers; PGC1a; fw: 50 -catgcaaatcacaatcacagg-30 , rev: 50 -ttgtggct tttgctgttgac-30 , MCOLN1; fw: 50 -gagtgggtgcgacaagtttc-30 , rev: 50 -tgttctcttcccgga atgtc-30 , ATP6V0E1; fw: 50 -cattgtgatgagcgtgttctgg-30 , rev: 50 -aactccccggttaggaccct ta-30 , ATP6V1H; fw: 50 -ggaagtgtcagatgatcccca-30 , rev: 50 -ccgtttgcctcgtggataat-30 , CTSF; fw: 50 -acagaggaggagttccgcacta-30 , rev: 50 -gcttgcttcatcttgttgcca-30 , TFEB; fw: 50 -caaggccaatgacctggac-30 , rev: 50 -agctccctggacttttgcag-30 , HPRT; fw: 50 -tggcgtc gtgattagtgatg-30 , rev: 50 -aacaccctttccaaatcctca-30 , CTSD; fw: 50 -cttcgacaacctgatgca gc-30 , rev: 50 -tacttggagtctgtgccacc-30 , PPP3CA; fw: 50 -gctgccctgatgaaccaac-30 , rev: 50 -gcaggtggttctttgaatcgg-30 , DPP7; fw: 50 -gattcggaggaacctgagtg-30 , rev: 50 -cggaagca ggatcttctgg-30 , CTSB; fw: 50 -agtggagaatggcacacccta-30 , rev: 50 -aagagccattgtcaccc ca-30 , TPP1; fw: 50 -gatcccagctctcctcaatac-30 , rev: 50 -gccatttttgcaccgtgtg-30 , NEU1; fw: 50 -tgaagtgtttgcccctggac-30 , rev: 50 -aggcaccatgatcatcgctg-30 .

NATURE CELL BIOLOGY


METHODS

DOI: 10.1038/ncb3114 High-content nuclear translocation assay. HeLaTFEB–GFP cells were seeded in 96- or 384-well plates, incubated for 12 h, and treated as indicated in the text, washed, fixed and stained with DAPI. For the acquisition of the images, at least 5 images fields were acquired per well of the 96/384-well plate by using confocal automated microscopy (Opera high-content system; Perkin-Elmer). A dedicated script was developed to perform the analysis of TFEB localization on the different images (Harmony and Acapella software; Perkin-Elmer). The script calculates the ratio value resulting from the average intensity of nuclear TFEB–GFP fluorescence divided by the average of the cytosolic intensity of TFEB–GFP fluorescence. The results were normalized using negative (RPMI medium, or otherwise indicated) and positive (starvation, or otherwise indicated) control samples in the same plate54. At least two independent experiments, and up to 3,000 individual cells per treatment from at least two independent wells, are routinely analysed. P values were calculated on the basis of mean values from independent wells. The data are represented by the percentage of nuclear translocation versus the indicated control using Excel (Microsoft) or Prism software (GraphPad software). The TFEB–GFP nuclear translocation analyses of different populations of transfected cells (detected by immunofluorescence of tagged-plasmids) were performed by a modified script that calculates the ratio value resulting from the average intensity of nuclear TFEB–GFP fluorescence divided by the average of the cytosolic intensity of TFEB–GFP fluorescence on both positive-transfected and negative-transfected cells. High-content LC3 autophagosome assay. Cells were seeded in 96-well plates, incubated for 12 h, and treated as indicated in the text, washed, fixed in icecold methanol, and immunofluorescence against LC3 (anti-LC3, Novus Biologicals, 1:400) was performed. After washing, the 96-well plate was analysed using confocal automated microscopy (Opera high content system; Perkin-Elmer). A dedicated script was developed to perform the analysis of the number of LC3 puncta on at least five different images per well (Harmony software; Perkin-Elmer).

Time-lapse. HeLaTFEB–GFP cells were reverse transfected and plated in Mattek glassbottomed dishes. Time-lapse movies were acquired for 45 min. One frame was acquired roughly every 20 s with lasers set at 30% power or below. All imaging was performed with a ×60 Plan Apo oil immersion lens using a Nikon Eclipse Ti spinning disc microscope, and images were annotated and 3D reconstructions made using the NIS Elements 4.20 software.

EGFP–2×FYVE assay. HeLa cells were reverse transfected with PPP3CB/R1 or scramble control siRNA using RNAiMax. Forty-eight hours later the cells were transfected with the GFP–2×FYVE construct55 using Lipofectamine LTX and Plus (Invitrogen), and 24 h later serum starved and fixed with 4% PFA in PBS. Imaging was performed with a ×63 PlanApochromat NA 1.4 DIC oil immersion objective on an LSM700 confocal microscope (Zeiss). Image analysis (spot count per cell, minimum of 15 cells counted per treatment group) was performed using ImageJ.

intensity at 488 nm (F488 ) was recorded with the spinning disc confocal imaging system, which consisted of an Olympus IX81 inverted microscope, a ×60 or ×100 objective (Olympus), a CSU-X1 scanner (Yokogawa), an iXon EMCCD camera (Andor), and MetaMorph Advanced Imaging acquisition software v.7.7.8.0 (Molecular Devices). Live imaging of ML1-GCaMP3 was performed on a heated stage.

Ratiometric GCaMP3 Ca2+ imaging in HeLa cells. HeLa cells were grown on 24 mm coverslips and transfected with plasmid encoding cytosolic-localized GCaMP6s or the perilysosomal-localized ML1-GCaMP3 calcium probes. After 24 or 48 h, the coverslips were placed in 1 ml of complete L-15 medium and imaging was performed on a Zeiss Axiovert 200 microscope equipped with a ×40/1.3 N.A. NeoFluar objective. Excitation was performed with a Deltaram V high-speed monochromator (Photon Technology International) equipped with a 75 W Xenon Arc lamp. Images were captured with a high-sensitivity Evolve 512 Delta EMCCD (Photometrics). The system is controlled by Metamorph 7.5 and was assembled by Crisel Instruments. To be truly quantitative, we took advantage of the isosbestic point in the GCaMP excitation spectrum: we experimentally determined in living cells that exciting GCaMP at 410 nm leads to fluorescence emission that is not Ca2+ dependent. As a consequence, the ratio between 474 and 410 nm excitation wavelengths is proportional to [Ca2+ ] and independent of probe expression. HeLa cells were thus alternately illuminated at 474 and 410 nm and fluorescence was collected through a 515/30 nm band-pass filter (Semrock). For GCaMP6s experiments, the exposure time was set to 50 ms at 474 nm and to 150 ms at 410 nm, to account for the low quantum yield at the latter wavelength. For the ML1-GCaMP3 experiments, the exposure time was set to 300 ms at 474 nm and to 400 ms at 410 nm. Each field was acquired for 20 min (1 frame per second) under continuous perfusion with the indicated solutions. Analysis was performed with the Fiji distribution of ImageJ. Both images were background-corrected frame by frame by subtracting mean pixel values of a cell-free region of interest. Data are presented as the mean ± s.e.m. Aequorin measurements. For the measurements of [Ca2+]cyt, HeLa cells grown on 13 mm round glass coverslips at 50% confluence were transfected with the cytosolic (cytAeq) probe. The coverslip with the cells was incubated at 37 ◦ C with 5 µM coelenterazine for 1–2 h in L-15 medium (Life Technologies) supplemented with 10% FBS, and then transferred to the perfusion chamber. Aequorin measurements were carried out in either complete L-15 or HBSS (supplemented with CaCl2 and MgCl2 , Life Technologies), as indicated. Agonists and other drugs were added to the same medium, as specified in the text. The experiments were terminated by lysing the cells with 100 µM digitonin in a hypotonic Ca2+ -rich solution (10 mM CaCl2 in H2 O), thus discharging the remaining aequorin pool. The light signal was collected and calibrated into [Ca2+ ] values by an algorithm based on the Ca2+ response curve of aequorin at physiological conditions of pH, [Mg2+ ] and ionic strength, as previously described56. Data are presented as mean ± s.d. of the histamine-induced peak in [Ca2+ ].

Endolysosomal electrophysiology. Endolysosomal electrophysiology was performed in isolated endolysosomes using a modified patch-clamp method35–37. Cos-1 cells were transfected using Lipofectamine 2000 (Invitrogen) with TFEBS211A fused to mCherry. To increase the size of isolated endolysosomes, cells were treated with 1 µM vacuolin-1, a lipid-soluble polycyclic triazine that can selectively increase the size of endosomes and lysosomes. Whole-endolysosome recordings were performed on isolated enlarged endolysosomes. In brief, a patch pipette (electrode) was pressed against a cell and quickly pulled away to slice the cell membrane35–37. Enlarged endolysosomes were released into a dish. TFEB-S211A-positive vacuoles were identified by monitoring mCherry fluorescence. The bath (internal/cytoplasmic) solution contained 140 mM K+ gluconate, 4 mM NaCl, 1 mM EGTA, 2 mM Na2 -ATP, 2 mM MgCl2 , 0.39 mM CaCl2 , 0.1 mM GTP and 10 mM HEPES (pH adjusted with KOH to 7.2; free [Ca2+ ]i approximately 100 nM). The pipette (luminal) solution was a pH 4.6 standard extracellular solution (modified Tyrode’s) with 145 mM NaCl, 5 mM KCl, 2 mM CaCl2 , 1 mM MgCl2 , 20 mM HEPES and 10 mM glucose (pH adjusted with NaOH). All bath solutions were applied by means of a fast perfusion system to achieve a complete solution exchange within a few seconds. Data were collected using an Axopatch 2A patch-clamp amplifier, Digidata 1440, and pClamp 10.0 software (Axon Instruments). Whole-endolysosome currents were digitized at 10 kHz and filtered at 2 kHz. All experiments were conducted at room temperature (21–23 ◦ C), and all recordings were analysed using pCLAMP10 (Axon Instruments) and Origin 8.0 (OriginLab). ML-SA1 was purchased from Princeton BioMolecular Research.

GCaMP3 Ca2+ imaging in GCaMP3–ML1-HEK293 cells. GCaMP3 Ca2+ imaging was performed in HEK cells that stably express GCaMP3–ML1, which are lysosome-targeted genetically encoded Ca2+ sensors36. The fluorescence

Animals and in vivo transfection experiments. Animals were handled by specialized personnel under the control of inspectors of the Veterinary Service of the Local Sanitary Service (ASL 16 - Padova), the local officers of the Ministry of Health. All procedures are specified in the projects approved by the Italian Ministero Salute, Ufficio VI (authorization numbers C65). All experiments were performed on adult 2-month-old male CD1 mice (three mice per group, 28–32 g). In vivo transfection experiments were performed by intramuscular injection of expression plasmids in tibialis anterior muscle followed by electroporation as previously described57. Eight days 8 after transfection mice were exercised on a treadmill. Mice performed concentric exercise on a treadmill (LE 8710 Panlab Technology 2B, Biological Instruments), with a 10◦ incline, until they were exhausted and then were euthanized.

Histology and fluorescence microscopy. Immunofluorescence staining was performed on cryosections as previously described24 and then monitored with a fluorescence microscope. For nuclear localization studies, cryosections were stained with mouse-anti-dystrophin-1 IgG2a antibodies (1:200; Novocastra) and Hoechst to identify the subsarcolemmal position of myonuclei.

Generation of the lysosomal/autophagic gene list. The complete gene list includes 322 members (Supplementary Table 1): 98 lysosomal genes described previously41,58; 9 newly identified genes co-localizing with the lysosomal marker LAMP2 (ref. 58); the master regulator of lysosomal biogenesis and autophagy, TFEB; all of the known lysosomal and non-lysosomal protein coding genes with a role in the different LSDs selected by using the Online Mendelian Inheritance in Man59,60 (OMIM); a subset of lysosomal and autophagy genes predicted by different bioinformatics tools. In detail, three different bioinformatics tools were used: AmiGO (ref. 61), Netview62 and uniPROT (ref. 63).

NATURE CELL BIOLOGY


METHODS

DOI: 10.1038/ncb3114

Microarray data processing. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus64 (GEO) and are accessible through GEO Series accession numbers GSE56671 (PPP3R1 WT Starved versus Normal medium) and GSE56672 (PPP3R1−/− starved versus normal medium). Statistical comparisons were made using analysis of variance (ANOVA). As the threshold for statistical significance we used false discovery rate (FDR) < 0.05 followed by an additional threshold (P value < 0.05).

Data analysis. Data are presented as the mean ± s.d. t-test statistical comparisons

were made using ANOVA. A P value < 0.05 was considered statistically significant. No statistical method was used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments. 52. Plyte, S. et al. Identification and characterization of a novel nuclear factor of activated T-cells-1 isoform expressed in mouse brain. J. Biol. Chem. 276, 14350–14358 (2001). 53. Daniele, T., Di Tullio, G., Santoro, M., Turacchio, G. & De Matteis, M. A. ARAP1 regulates EGF receptor trafficking and signalling. Traffic 9, 2221–2235 (2008). 54. Sittampalam, G. S. et al. (eds) in Assay Guidance Manual [Internet] (Eli Lilly Company and the National Center for Advancing Translational Sciences, 2004).

55. Pattni, K., Jepson, M., Stenmark, H. & Banting, G. A PtdIns(3)P-specific probe cycles on and off host cell membranes during Salmonella invasion of mammalian cells. Curr. Biol. 11, 1636–1642 (2001). 56. Granatiero, V., Patron, M., Tosatto, A., Merli, G. & Rizzuto, R. The use of aequorin and its variants for Ca2+ measurements. Cold Spring Harb. Protoc. 2014, 9–16 (2014). 57. Sandri, M. et al. Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117, 399–412 (2004). 58. Schroder, B. A., Wrocklage, C., Hasilik, A. & Saftig, P. The proteome of lysosomes. Proteomics 10, 4053–4076 (2010). 59. Amberger, J. et al. McKusick’s Online Mendelian Inheritance in Man (OMIM). Nucleic Acids Res. 37, D793–D796 (2009) (Database issue). 60. McKusick, V. A. Mendelian Inheritance in Man and its online version, OMIM. Am. J. Hum. Genet. 80, 588–604 (2007). 61. Carbon, S. et al. AmiGO: online access to ontology and annotation data. Bioinformatics 25, 288–289 (2009). 62. Belcastro, V. et al. Transcriptional gene network inference from a massive dataset elucidates transcriptome organization and gene function. Nucleic Acids Res. 39, 8677–8688 (2011). 63. Magrane, M. & U. Consortium, UniProt Knowledgebase: a hub of integrated protein data. Database: J. Biol. Databases Curation 2011 bar009 (2011). 64. Edgar, R., Domrachev, M. & Lash, A. E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 (2002).

NATURE CELL BIOLOGY


S U P P L E M E N TA R Y I N F O R M AT I O N DOI: 10.1038/ncb3114 Medina_Supplementary Figure 1

78 p= 0,

p= 0,

00

01

8

p= 0,

00

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Ratio Nuc/Cyt NFAT-GFP

1

22

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3 hour 1 hour

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thapsigargin

(3 V

ST

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ΔCaN HeLa cells

NFAT-GFP ΔCaN

1

0.5

0

N

FA TG

HeLa cells

p=0.012

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ΔCaN

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 (300nM, 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.

P

+

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N +Δ FA C T-G aN F

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h)

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ΔC

h)

aN

b

DCaN overexpression in fed conditions also induced NFAT-GFP mobility downshift (right blot) (n=3 independent experiments). (c) The overexpression of HA-tagged dominant positive calcineurin (DCaN) in HeLa cells transfected with NFAT-GFP induces NFAT nuclear translocation. DCaN 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 10mm. Source data are provided in Supplementary table 4.

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S U P P L E M E N TA R Y I N F O R M AT I O N

Medina_ Supplementary Figure 2

b

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is C TFE aN B C (1 (1 aM /3 /1 0) G (1 ) ST /3 ) (1 /3 ) H is C TFE aN B C aM H is H TFE is - B H TFE +C is a - B N H TFE +C is a -T B+ M FE C B aN +G +C ST aM

input

97-

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fed STV

)

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FP

a

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 3hr). 2 mg of lysates were immunoprecipitated with anti-GFP antibodies or rabbit IgG as a control and immunoblotted with the indicated

antibodies. 50 mg 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.

2

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S U P P L E M E N TA R Y I N F O R M AT I O N Medina_Supplementary Figure 3 PLA signal

b

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PARP1 tubulin

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tubulin

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 1µM ERtracker (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, 10µm. (c) Cell lysates (50 mg/sample) were analyzed by SDSPAGE and immunobloting with anti-TFEB (Cell Signaling). b-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

+/+

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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, 10µm. (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 10mm. (f) Nuclear fractions from cells treated as indicated for 3h, 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 3h, was assessed by immunoblotting analysis of mTOR substrate P70S6K. n=2 independent experiments. Source data are provided in Supplementary table 4.




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Medina_Supplementary Figure 4

b

TFEB Nuc/Cyt Ratio

2.5 2 1.5

NT

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MICU1

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fed

STV

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 (1hr), 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.

4

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a STV (h) 70

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0 0.5 1

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TFEB

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+siCaN

p=0.0003

p=0.0001 p=0.003 p=0.002 p=0.008

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% nuclear TFEB vs. DMSO

100

15' 30' 60' 120' 180'

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, 50mg 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

fed+SF-51

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 knockdown 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.




a

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TFEB target genes

TFEB target genes

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c

P G M C1 C a A OL TP N 6 1 A V0 TP D 6V 1 C 1H TS C F TS B TP P D 1 P P 7 W IP I P P P 3R 1

fold-change (MEFs)

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STV+SCRMBL (STV+siPPP3CB/R1) vs STV 10

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*

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E C B TS A C TS C F LN PG 3 C 1a W IP D I AT PP P6 7 AT V0D P6 1 V1 H N E AT U G 9B

0

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 TFEBmediated 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

Lysosomal calcium regulates autophagy.

Calcineurin signaling regulates neural induction through antagonizing the BMP pathway.

Hyperoside regulates the level of thymic stromal lymphopoietin through intracellular calcium signalling.

STIM via calcineurin-NFAT signalling.

ASMase regulates autophagy and lysosomal membrane permeabilization and its inhibition prevents early stage non-alcoholic steatohepatitis.

The KSR2-calcineurin complex regulates STIM1-ORAI1 dynamics and store-operated calcium entry (SOCE).

Neuronal-Targeted TFEB Accelerates Lysosomal Degradation of APP, Reducing Aβ Generation and Amyloid Plaque Pathogenesis.

RIP1 negatively regulates basal autophagic flux through TFEB to control sensitivity to apoptosis.

Strigolactone regulates shoot development through a core signalling pathway.

LAMTOR2 regulates dendritic cell homeostasis through FLT3-dependent mTOR signalling.

Lysosomal proteins in cell death and autophagy.

Curcumin targets the TFEB-lysosome pathway for induction of autophagy.

Phagocytosis Enhances Lysosomal and Bactericidal Properties by Activating the Transcription Factor TFEB.

MicroRNA-130a regulates autophagy of endothelial progenitor cells through Runx3.

Oxidant-induced autophagy and ferritin degradation contribute to epithelial-mesenchymal transition through lysosomal iron.

Oncogenic H-Ras up-regulates acid β-hexosaminidase by a mechanism dependent on the autophagy regulator TFEB.

Autophagy regulates the stability of sialin, a lysosomal sialic acid transporter.

TFE3 regulates whole-body energy metabolism in cooperation with TFEB.

Calcineurin regulates progressive motility activation of Rhinella (Bufo) arenarum sperm through dephosphorylation of PKC substrates.

Muscle-specific RING finger 1 negatively regulates pathological cardiac hypertrophy through downregulation of calcineurin A.

PKA regulates calcineurin function through the phosphorylation of RCAN1: identification of a novel phosphorylation site.

The autophagy-lysosomal system in subarachnoid haemorrhage.

mTOR signalling pathway.

Citreoviridin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human Liver HepG2 Cells.