A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB

Abstract

The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB-/- cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation- and stress-induced nuclear translocation of TFEB. These data indicate that the lysosome senses its content and regulates its own biogenesis by a lysosome-to-nucleus signalling mechanism that involves TFEB and mTOR.

Figure 1

Lysosomal stress induces TFEB nuclear translocation. (A) Immunofluorescence of HEK-293T cells that express TFEB–3 × FLAG, subjected to the indicated treatments and stained with antibodies against FLAG and the lysosomal marker LAMP2. The FLAG and LAMP2 channels are in green and red, respectively, in the merge. DAPI (blue) is included in the merge. Scale bars represent 10 μm. (B) Quantification of the number of cells with nuclear TFEB–3 × FLAG in the four conditions in (A). Each value represents mean±s.d. from three independent fields with N=300. (C) Immunofluorescence of HEK-293T cells treated as indicated and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μm. (D) Immunoblotting of proteins extracted from HeLa cells that express TFEB–3 × FLAG treated with DMSO, chloroquine (CQ) or SalA, subjected to nuclear/cytosolic fractionation and blotted with antibody against FLAG to detect TFEB. H3 and tubulin were used as nuclear and cytosolic markers, respectively. Blots are representative of triplicate experiments.

Figure 2

mTORC1 regulates TFEB. (A) Lysosomal stress inhibits mTOR signalling. Immunoblotting of protein extracts isolated from HeLa cells treated overnight as indicated. Membranes were probed with antibodies against p-T202/Y204-ERK1/2, ERK1/2, p-T389-S6K, and S6K to measure ERK and mTORC1 activities. (B) Torin 1 induces TFEB dephosphorylation and nuclear translocation. FLAG immunoblotting of cytosolic and nuclear fractions isolated from TFEB–3 × FLAG HeLa cells starved in amino acid-free media and subsequently stimulated as indicated for at least 3 h. Correct subcellular fractionation was verified with H3 and tubulin antibodies. (C) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB–GFP HeLa cells were seeded in 96-well plates, cultured for 12 h, and then treated with the indicated concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2. After 3 h at 37°C, cells were processed and images were acquired using the OPERA automated confocal microscope (Perkin-Elmer). Scale bars represent 30 μm. (D) Dose–response curves of the effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB–GFP HeLa cells were seeded in 384-well plates, cultured for 12 h, and treated with 10 different concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2 ranging from 2.54 nM to 50 μM. The graph shows the percentage of nuclear translocation at the different concentrations of each compound (in log of the concentration). The EC50 for each compound was calculated using Prism software (see Materials and methods for details). (E) Immunofluorescence of HEK-293T cells treated with DMSO or Torin 1 and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μM. (F) Rag GTPase knockdown induces TFEB nuclear translocation. HeLa cells stably expressing TFEB–3 × FLAG were infected with lentiviruses encoding Short hairpin (Sh-) RNAs targeting luciferase (control) or RagC and RagD mRNAs. In all samples, 96 h post infection, cells were left untreated (N=normal media), starved (S=starved media) or treated with Torin 1 (T=Torin 1) for 4 h and then subjected to nuclear/cytosolic fractionation. TFEB localization was detected with a FLAG antibody, whereas tubulin and H3 were used as controls for the cytosolic and nuclear fraction, respectively; levels of S6K phosphorylation were used to test RagC and RagD knockdown efficiency. (G) Loss of mTORC2 does not affect TFEB phosphorylation. Mouse embryonic fibroblasts (MEFs) isolated from Sin1−/− or control embryos (E14.5) were infected with a retrovirus encoding TFEB–3 × FLAG; 48 h post infection, cells were treated with Torin 1 (T) for 4 h where indicated, subjected to nuclear/cytosolic fractionation and immunoblotted for FLAG, tubulin, and H3. (H) Binding of TFEB to mTORC1. HEK-293T cells that express TFEB–3 × FLAG were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR, the mTORC1 subunit raptor and the mTORC2 components rictor and Sin1. FLAG–Rap2A served as negative control.

Figure 3

mTORC1 phosphorylates TFEB at serine 142 (S142). (A) Torin 1 induces S142 dephosphorylation. HeLa cells were treated as indicated and total and nuclear extracts were probed with a TFEB p-S142 phosphoantibody and with anti-FLAG antibody. Disappearance of TFEB S142 phosphorylation upon starvation or Torin 1 treatment correlates with accumulation of TFEB in the nuclear fraction. (B) mTORC1 in-vitro kinase assays. Highly purified FLAG–S6K1, TFEB–3 × FLAG, or TFEB^S142A–3 × FLAG were incubated with radiolabelled ATP without kinase, with purified mTORC1 or with mTORC1+Torin 1, and analysed by autoradiography. The lower panel shows a FLAG immunoblot of the substrates. (C) Schematic representation of TFEB protein structure with the predicted mTORC1 phosphorylation sites and their conservation among vertebrates (for mTORC1 phophosite prediction see Material and methods). Numbering is according to human isoform 1. (D) Sequence conservation scores of the phosphosites and quantitative agreement between mTOR consensus motif and the sequence around the phosphosites of TFEB. (E) S142 and S211 regulate TFEB localization. FLAG immunostaining (red) of HeLa cells expressing serine-to-alanine mutated versions of TFEB–3 × FLAG. Nuclei were stained with DAPI (blue). Values are means of five fields containing at least 50 transfected cells. Student's t-test (unpaired) ^***P<0.001. Scale bars represent 30 μm.

Figure 4

mTORC1 binds and phosphorylates TFEB on the lysosomal surface. (A) Spinning disk confocal image of a MEF cell that co-expresses TFEB–GFP and mRFP–Rab7 (green and red in the merge, respectively). (B) Time-lapse of TFEB- and Rab7-positive lysosomes from the boxed region in (A). Time intervals are in seconds. (C) Time-lapse analysis of Torin 1 treatment in a MEF cell expressing TFEB–GFP. Arrow indicates the time of Torin 1 addition. Yellow arrowheads indicate Torin 1-induced lysosomal accumulation of TFEB–GFP. Time intervals are in minutes. (D) Immunofluorescence of HEK-293T cells expressing TFEB–3 × FLAG, treated with DMSO (top) or Torin1 (bottom) and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). (E) FRAP analysis of TFEB–GFP-positive lysosomes from control MEFs (blue) or MEFs treated with Torin 1 (red). Each data point represents mean±s.d. from five independent spots. (F) Time-lapse of photobleaching and fluorescence recovery of TFEB–GFP-positive lysosomes from control-treated MEFs (top) or MEFs treated with Torin 1 (bottom). Red arrowheads indicate time of photobleaching. Time intervals are in seconds. (G) Torin 1 increases binding of TFEB to mTORC1. HEK-293T cells that express TFEB–3 × FLAG along with HAGST-Rap2A or HAGST-Rags^DN were treated with vehicle or with Torin1, lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR and raptor. FLAG–Metap2 served as negative control. (H) Immunofluorescence of HEK-293T cells that express TFEB–3 × FLAG along with Rap2A (top) or the Rags^DN mutants (bottom), treated with Torin 1 and stained with antibodies against FLAG and LAMP2 (green and red in the merge, respectively; DAPI is in blue). In all images, scale bars represent 10 μm.

Figure 5

Rag GTPases control TFEB subcellular localization. (A–C) Immunofluorescence of HEK-293T cells that express TFEB–3 × FLAG along with a control GTPase or the indicated Rag mutants. Cells were deprived of amino acids (top) or deprived and then stimulated (bottom) for the indicated times and stained for FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). (D) Quantification of the number of cells with nuclear TFEB from each condition in (A–C). (E, F) Immunofluorescence of HEK-293T cells that express TFEB–3 × FLAG along with Rap2A (E) or the Rags^CA mutants (F), subjected to the indicated treatments and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue). (G) Quantification of the number of cells with nuclear TFEB from DMSO- and CQ-treated fields in (E) and (F). In all fields, scale bars represent 10 μm. In all histograms, each value represents mean±s.d. from three independent fields with N=300.

Figure 6

The lysosome regulates gene expression via TFEB. (A) Chloroquine treatment inhibits mTORC1 activity in primary hepatocytes. Primary hepatocytes isolated from 2-month-old Tcfebflox/flox (control) and Tcfebflox/flox;Alb-Cre(Tcfeb−/−) mice were left untreated, or treated overnight with Torin 1, U0126, or Chloroquine. Subsequently, cells were lysed and protein extracts were immunoblotted with the indicated antibodies. (B, C) TFEB mediates the transcriptional response to chloroquine and Torin 1. Quantitative PCR (qPCR) of TFEB target genes in primary hepatocytes from control (flox/flox) and Tcfeb−/− mice. Cells were treated with Chloroquine (left) or Torin 1 (right). The graphs show the relative increased expression in the treated versus the corresponding untreated samples. Values represent means±s.d. of three independent hepatocyte preparations (three mice/genotype). Student's t-test (two tailed) ^*P-value ⩽0.05.

Figure 7

Model of lysosomal sensing and lysosome-to-nucleus signalling by TFEB and mTOR. (A) (Left) Under full nutrients and in the absence of lysosomal stress, the complex formed by v-ATPase, Ragulator, and Rag GTPases is in the active state and recruits mTORC1 to the lysosomal surface, where mTORC1 becomes activated. At the lysosome, mTORC1 binds and phosphorylates TFEB, which cycles between the cytoplasm and the lysosomal surface. Phosphorylation by mTORC1 maintains TFEB in the cytoplasm and prevents it from translocating to the nucleus. (Right) Starvation, v-ATPase inhibition, or lysosomal stress switch the Rags off, leading to mTORC1 detachment from the lysosome and to its inactivation. TFEB can no longer be phosphorylated and translocates to the nucleus, where it activates gene expression programs that boost lysosomal function and autophagy. (B) Side-by-side diagrams of a healthy cell and a starved/stressed cell, showing the respective distribution of mTORC1 and TFEB in relationship to lysosomes, cytoplasm, and nucleus.