Abstract
Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)—a master transcriptional regulator of lysosomal biogenesis and autophagy—is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.
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Data availability
Data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
Change history
04 October 2022
A Correction to this paper has been published: https://doi.org/10.1038/s41556-022-01017-4
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Acknowledgements
We thank M. Lazarou (Monash University) for providing us with ATG8s-deficient HeLa cells. S.N. is supported by AMED-PRIME (grant no. 17gm6110003h0001), JSPS KAKENHI (grant no. 17H05064), the Senri Life Science Foundation, Takeda Science Foundation, Nakajima Foundation, MSD Life Science Foundation, Astellas Foundation for Research on Metabolic Disorders and Mochida Memorial Foundation for Medical and Pharmaceutical Research. T.Y. is supported by JST CREST (grant no. JPMJCR17H6), AMED (grant no. JP19gm5010001), Takeda Science Foundation, JSPS A3 Foresight Program and an HFSP research grant. This work was also supported by grants from the Italian Telethon Foundation (grant no. TGM16CB6), MIUR FIRB (grant no. RBAP11Z3YA; A.B.), European Research Council (advanced investigator grant no. 694282; LYSOSOMICS; A.B.), Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.) (A.B.), US National Institutes of Health (grant no. R01-NS078072; A.B.), the Huffington foundation (A.B.), European Regional Development Fund—POR Campania FESR 2014/2020 (A.B.), MIUR PRIN (grant no. 2017YF9FBS; G.N.), University of Naples ‘Federico II’ STAR L1 2018 (G.N.) and Associazione Italiana per la Ricerca sul Cancro MFAG 2019, grant no. 23538 (G.N.).
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S.N., S.M., G.N., A.B., Y.T. and T.Y. wrote the manuscript with input from the other authors. S.N. and T.Y. supervised the project. S.N., S.S. and T.M. generated the stable cell lines and performed calcium imaging. S.N., S.S., S.M., A.K., M.M., T.S., S.A., A.E., G.N. and A.Tokumura conducted western blotting and immunohistochemistry using cell culture. S.N, S.M., S.S. and T.N.-H. obtained and analysed the transmission electron microscopy images. S.M., J.N., Y.T. and A.Takahashi conducted the mouse experiments. K.Y., M.H., M.Y., Y. Okada, T.N., Y.T., Y.I., H.X. and M.K. analysed data and provided intellectual support. S.N., M.S., Y. Oe, T. F. and S.T. generated the KO cells and plasmid constructs. A.B. generated the TFEB construct and TFEB flox mice.
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S.N. and T.Y. have applied for a patent for the assay related to this work (patent application no. 2020-63351). T.Y. is founder of AutoPhagyGO. A.B. is a co-founder of CASMA Therapeutics, Inc.
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Extended data
Extended Data Fig. 1 TFEB is essential for lysosomal homeostasis after the injury.
(a) Representative immunoblots confirming efficient reduction of ATG16L1 and TFEB protein levels by indicated siRNA knockdown (representative of 3 independent experiments). Actin was used as a loading control. (b) Representative immunoblots confirming absence of TFEB protein in established TFEB-KO cells (representative of 3 independent experiments). (c) LLOMe treatment and subsequent chase did not affect the cell viability of WT and TFEB-KO HeLa cells. Cells were treated with 1 mM LLOMe for 1 h and then subjected to cell viability assay at the indicated time point after LLOMe washout. Bars represent mean ± s.d. (n = 3 biologically independent experiments). (d) Transmission electron microscopy (TEM) observation of ultrastructural changes of lysosomes in WT and TFEB-KO cells after LLOMe treatment (representative of 2 independent experiments). Cells were treated with 1 mM LLOMe for 1 h and then fixed at the indicated time point after LLOMe washout. Lysosomes showed enlarged and disorganized morphology soon after LLOMe treatment. After 3 h wash-off by DMEM, the morphology of lysosomes appeared to be normal round shape in WT cells, while it showed still distorted shape in TFEB-KO cells. Scale bars, 500 nm. Uncropped blots and statistical source data are provided in Source Data Extended Data Fig. 1.
Extended Data Fig. 2 Lysosomal damage, but not starvation, induces TFEB nuclear translocation in an ATG conjugation system-dependent manner.
(a, b) Representative immunoblots showing TFEB, phospho S6K, total S6K, phospho GSKαβ, total GSKβ, LC3 (a) and actin in WT and several KO HeLa cells (3 independent experiments for both a and b). TFEB dephosphorylation (downshift) was observed in all KO cells under EBSS starvation (b), whereas TFEB dephosphorylation was partly impaired in ATG7-KO and ATG16L1-KO cells under LLOMe treatment (a). Samples were collected after EBSS starvation (4 h), or LLOMe treatment (3 h washout after 1 h LLOMe treatment). (c, d) Representative immunoblots showing TFEB expression in WT and ATG3-KO (c) or ATG5-KO (d) HeLa cells under control conditions, EBSS starvation (4 h), or LLOMe treatment (3 h washout after 1 h LLOMe treatment) (n = 3 independent experiments for both c and d). TFEB dephosphorylation (downshift) induced by LLOMe but not EBSS was impaired in ATG3-KO and ATG5-KO cells. (e) Representative immunoblots showing LC3 in WT and several ATG conjugation-deficient HeLa cells which we generated in this study treated with or without Bafilomycin A (3 independent experiments). Note that lipidated LC3-II form was not present in these ATG conjugation-deficient cells. Uncropped blots are provided in Source Data Extended Data Fig. 2.
Extended Data Fig. 3 Requirement of Atg8 paralogues for TFEB nuclear localization during lysosomal damage.
(a) MEF cell lines stably expressing TFEB::mNeonGreen (green) were treated with or without LLOMe (3 h washout after 1 h LLOMe treatment). (b) Quantification of nuclear/cytoplasmic ratio of TFEB::mNeonGreen under each condition. More than 100 cells were analysed per condition by cell profiler and the experiments were repeated three times. Bars represent mean ± s.d. (n = 3 biologically independent experiments, one-way ANOVA with Tukey’s test, *P = 0.0166) Scale bars, 50 μm. Statistical source data are provided in Source Data Extended Data Fig. 3.
Extended Data Fig. 4 Transfection of Effectene-coated beads induces TFEB nuclear localization in an ATG conjugation system-dependent manner.
(a) Representative immunofluorescence images showing TFEB::mNeonGreen and Gal-3 counterstained by DAPI. Transfection of Effectene-coated beads damaged endosomes or lysosomes, whereas transfection with PEI-coated beads did not. Note that cells harbouring the damage visualized by Gal-3 exhibited TFEB nuclear translocation (open arrowheads). DIC images showed that transfected beads were internalized in cells. (b) Ratio of nuclear to cytoplasmic TFEB::mNeonGreen in WT and ATG7-KO cells transfected with either Effectene- or PEI-coated beads. More than 100 cells were analysed per condition by cell profiler and the experiments were repeated three times. Bars represent mean ± s.d. (n = 3 biologically independent experiments, one-way ANOVA with Tukey’s test, **P = 0.001 for WT PEI versus effectene, P = 0.9711 for ATG7-KO PEI versus effectene) Scale bars, 50 μm. Statistical source data are provided in Source Data Extended Data Fig. 4.
Extended Data Fig. 5 Calcium efflux from lysosomes triggers TFEB nuclear translocation in an ATG conjugation system-dependent manner.
(a) Representative immunoblots showing TFEB expression in WT and ATG3-KO HeLa cells treated with DMSO (control) or ML-SA1 for 1 h (3 independent experiments). In ATG3-KO cells, TFEB activation (downshift) was defective after ML-SA1 treatment. (b) The Ca2+ chelator BAPTA-AM reduced TFEB::mNeonGreen nuclear localization induced by ML-SA1. (c) Ratio of nuclear to cytoplasmic TFEB::mNeonGreen in WT HeLa cells treated with indicated drugs. Bars represent mean ± s.d. (n = 3 biologically independent experiments, one-way ANOVA with Tukey’s test, *P = 0.0435 for DMSO control versus ML-SA1, P = 0.216 for BAPTA control versus ML-SA1). (d) Representative images showing that knockdown of PPP3CB did not abolish TFEB::mNeonGreen nuclear translocation after LLOMe treatment (3 h after 1 h LLOMe treatment) (3 independent experiments). (e) Representative blots for TFEB and PPP3CB (3 independent experiments). Knockdown of PPP3CB did not interfere with TFEB dephosphorylation. (f) Representative blots for TFEB and PPP3CB (3 independent experiments). Double knockdown of PPP3CB and PPP3CA did not largely affect TFEB status by LLOMe treatment. (g) qRT-PCR confirmed PPP3CA transcripts were significantly reduced by PPP3CA knockdown. Two different siRNA (#1 and #2) for PPP3CA were used. Bars represent mean ± s.d. (n = 3 biologically independent experiments, one-way ANOVA with Tukey’s test, ****P < 0.0001 for siLuc versus siPPP3CA#1 or #2). Scale bars, 50 μm (b and d). Uncropped blots and statistical source data are provided in Source Data Extended Data Fig. 5.
Extended Data Fig. 6 TRPML1 interacts with ATG8s.
(a) Co-immunoprecipitation experiments revealed that TRPML1::HA interacted with endogenous LC3A, LC3B, GABARAP, GABARAPL1 and GABARAPL2 (representative of 3 independent experiments). (b) Co-immunoprecipitation experiments revealed that TRPML1::HA interacted with 3x FLAG::LC3C (representative of 3 independent experiments). (c) TRPML1::HA overexpression increased LC3 dots. Most of LC3 dots(magenta) were colocalized on lysosomes in TRPML1::HA overexpressed cells. (d) Quantification of numbers of LC3 dots per cells with HA or TRPML1::HA overexpressed cells. Bars represent mean ± s.d. (n = 3 biologically independent experiments, two-tailed unpaired t-test, *P = 0.0262). (e) Representative immunoblots confirming efficient reduction of TSG101 and ALIX protein levels by indicated siRNA knockdown (3 independent experiments). Scale bars, 50 μm (c). Uncropped blots and statistical source data are provided in Source Data Extended Data Fig. 6.
Extended Data Fig. 7 CaOx crystals cause lysosomal damage in mouse kidneys.
(a) Representative images of cultured PTECs stably expressing GFP-galectin 3 under the indicated conditions (n = 3 animals in each group). (b) Levels of plasma UN and creatinine after oxalate administration (n = 6 animals for 0 h, n = 3 animals for 2, 6, 12, 24 and 48 h). (c) Representative images of Pizzolato staining on the kidney sections of mice 24 h after oxalate administration. CaOx crystals are shown within the PTECs (arrows) as well as in the tubular lumen. (d) Quantification of positive area after Pizzolato staining is shown (n = 2 animals for 12 h; n = 3 animals for 0, 2, 6, 24 and 48 h). (e–j) WT mice were assessed 24 h after vehicle or oxalate injection (75 mg/kg) (n = 3 animals in each group). Representative images of PAS staining (e), immunostaining for LAMP1 (f), Galectin-3 and LAMP1 (g), Galectin-3 and LRP2/MEGALIN (h, left), Galectin-3 and THP (h, middle), Galectin-3 and AQP2 (h, right), and TFEB (j) on the kidney sections. Kidney sections were counterstained with hematoxylin (f) or DAPI (g and h). (i) Electron micrographs of PTECs. Arrowheads indicate elongating isolation membrane along the surface of the lysosome (n = 3 animals). Mt, mitochondria; asterisk: lysosome. Original magnification: ×100 (c, left); ×200 (e, left; f, left; j, left); ×400 (c, middle; e, middle; j, right);×1000 (c, right; e, right; f, right). Scale bars, 20 μm (a, g and h), 50 μm (c, e, f and j), 500 nm (i). Uncropped blots and statistical source data are provided in Source Data Extended Data Fig. 7.
Extended Data Fig. 8 Lysosomal damage induces TFEB nuclear translocation in an ATG5-dependent manner in PTECs.
(a) Representative immunofluorescence images of mNeonGreen-tagged TFEB in ATG5-negative or ATG5-positive PTECs counterstained with DAPI (blue). Non-treated control samples and samples 1 h after 1 mM LLOMe treatment (1 h) are shown (3 independent experiments). (b, c) Representative images of PAS staining (a) and electron micrographs (b) of the kidneys in control and PTEC-specific Tfeb-deficient mice [n = 6 animals (a) and n = 3 animals (b) in each group]. Mt, mitochondria; asterisk, lysosome. Scale bars, 20 μm (a), 50 μm (b), 500 nm (c).
Extended Data Fig. 9 The severity of kidney injury is comparable between control and TFEB-KO mice at 6 h after oxalate administration.
(a–f) Images of Pizzolato staining (a), quantification of positive area (b), PAS staining (c), PAS injury score (d), levels of plasma UN and creatinine (e), mRNA expression levels of kidney injury marker genes (f), TUNEL staining (g), and numbers of TUNEL-positive cells (h) are shown. TfebF/F and TfebF/F;KAP-Cre mice were subjected to oxalate injection (75 mg/kg) and verified after 6 h (a–f, n = 3 animals in each group) and 48 h (g and h, TfebF/F, n = 15 animals; TfebF/F;KAP-Cre, n = 9 animals). two-tailed unpaired t-test, P = * 0.0285 (h). Bars: means ± s.d. Scale bars, 50 μm (a, c and g). Statistical source data are provided in Source Data Extended Data Fig. 9.
Extended Data Fig. 10 Mitochondrial morphological changes during the lysosomal damage response.
(a) TfebF/F and TfebF/F;KAP-Cre mice were subjected to oxalate injection (75 mg/kg) and verified after 48 h (3 animals in each group). Representative images of electron microscopy are shown. Mt, mitochondria. (b) Transmission electron microscopy (TEM) observation of ultrastructural changes of mitochondria in WT and TFEB-KO HeLa cells after LLOMe treatment (representative of 2 independent experiments). Cells were treated with 1 mM LLOMe for 1 h and then fixed for TEM imaging at the indicated time point after LLOMe washout. Fragmented mitochondria was observed soon after LLOMe treatment (0 h) both in WT and TFEB- KO cells. The fragmentation seems to be recovered after 3h LLOMe washout. In TFEB -KO cells, the fragmented mitochondria was observed even in control cells. (c) Representative images showing mitochondrial morphological changes immunostained by TOMM20 antibody (green) during the lysosomal damage (3 independent experiments). Mitochondrial fragmentation occurred after 1 h LLOMe treatment, while interestingly, during the continuous LLOMe treatment for 3 h mitochondrial morphology recovered to normal. These morphological changes occurred in WT, FIP200-KO, ATG7-KO and TFEB-KO cells, indicating that these changes depend neither on the function of autophagy nor TFEB. (d) Representative immunoblots showing the change of complex III core subunit 1 protein level in indicated cells during lysosomal damage response (3 independent experiments). Mitophagy was not induced by 1 mM LLOMe treatment for 1 h or 3 h. (e) Wild type mice were subjected to oxalate injection (75 mg/kg) and verified after 48 h (n = 3 animals in each group). Representative images of electron microscopic analysis revealed that contents of lysosome-like structures were observed within the tubular lumen, indicating the presence of lysosomal exocytosis. N, nucleus; TL, tubular lumen; arrows, contents of lysosome-like structures. Scale bars, 1 μm (a and e), 500 nm (b), 50 μm (c). Uncropped blots are provided in Source Data Extended Data Fig. 10.
Supplementary information
Supplementary Video 1
Effect of 50 μM ML-SA1 on the cytosolic Ca2+ levels of Fura-2-loaded WT HeLa cells. Images were acquired every 3 s for 15 min and played back at a rate of 30 frames s−1. Warmer colours represent the Fura-2 fluorescence ratio, which is proportional to the Ca2+ concentration. Biologically independent experiments were repeated three times with similar results.
Supplementary Video 2
Fura-2 calcium imaging in ATG7-KO HeLa cells. Effect of 50 μM ML-SA1 on the cytosolic Ca2+ levels of Fura-2-loaded ATG7-KO HeLa cells. Images were acquired every 3 s for 15 min and played back at rate of 30 frames s−1. Warmer colours represent the Fura-2 fluorescence ratio, which is proportional to the Ca2+ concentration. Biologically independent experiments were repeated three times with similar results.
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Nakamura, S., Shigeyama, S., Minami, S. et al. LC3 lipidation is essential for TFEB activation during the lysosomal damage response to kidney injury. Nat Cell Biol 22, 1252–1263 (2020). https://doi.org/10.1038/s41556-020-00583-9
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DOI: https://doi.org/10.1038/s41556-020-00583-9
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