Fig 1.
Direct activation of lysosomal TRPML1 channels by Rap.
(A) Whole-endolysosome recording configuration. Pipette (luminal) solution was standard Tyrode’s solution with the pH adjusted to 4.6 to mimic the lysosomal lumen. Bath (internal) solution was a K+-based solution (140 mM K+-gluconate). Inward currents indicate cations flowing out. (B) Representative time course of whole-endolysosome TRPML1-mediated currents (ITRPML1, open circles, at −120 mV) activated by bath application of Rap (in μM: 1, 2, 5, 10, 20, 50). ITRPML1 was recorded from an enlarged vacuole isolated from EGFP-TRPML1–transfected COS1 cells. Currents were elicited by repeated voltage ramps (−120 to +120 mV; 200 ms) with a 4-s interstep interval. (C) Representative ITRPML1 by 2 μM, 5 μM, 10 μM, and 20 μM Rap (time points as in panel B). Partial voltage protocol is shown (holding potential, 0 mV). (D) Dose-dependent activation of TRPML1 by Rap. (E) Rap-evoked ITRPML1 was blocked by coapplication of ML-SI3, a synthetic inhibitor of TRPML1. (F) Constitutively active ITRPML1-Va was not affected by Rap. (G) Rap evoked endogenous ITRPML1 in WT parietal cells. (H) No Rap-induced ITRPML1 was detected in TRPML1 KO parietal cells. (I) Whole-endolysosome ITRPML2 was activated by Rap in mCherry-TRPML2–transfected COS1 cells. (J) Rap did not activate ITRPML3. (K) Rap did not produce activation of whole-endolysosome ITPC2 in EGFP-TPC2–transfected COS1 cells. (L) Summary of Rap effects on TRPML1, 2, and 3, and TPC2. Data are presented as mean ± SEM. Dashed line indicates 1 (no change in current). Only representative data are shown in (E–K). The individual data underlying (D) and (L) can be found in S1 Data. COS1, CV-1 in Origin Simian-1; EC50, half maximal effective concentration; EGFP, enhanced green fluorescent protein; KO, knockout; mCherry, a monomeric red fluorescent protein; ML, TRPML; ML-SA1, TRPML1 synthetic agonist 1; ML-SI3, TRPML1 synthetic inhibitor 3; Rap, rapamycin; TPC2, two-pore channel 2; TRPML1, transient receptor potential channel mucolipin 1; WT, wild type.
Fig 2.
Rap and rapalogs activate TRPML1 in an mTOR-independent manner.
(A) Effect of Torin-1 (10 μM), a potent ATP-competitive mTOR inhibitor, on ITRPML1. (B) Tem (10 μM) and Eve (10 μM) stimulation of ITRPML1. (C) No effects of Defo (10 μM), Zota (10 μM), and Seco-Rap (a Rap metabolite, 10 μM) on ITRPML1 measured at −120 mV. (D) Summary of differential effects of rapalogs on ITRPML1. Data are presented as mean ± SEM. (E) Rap and rapalogs inhibited mTOR activity, which was assayed by phosphorylation of the mTOR substrate S6K at Thr 389. (F) Rap activated ITRPML1 in cells transfected with WT mTOR (left) and a kinase-dead mTORD2357E mutant (right). (G) mTOR mutants did not alter Rap sensitivity of ITRPML1. Data are presented as mean ± SEM. (H) Rap activated ITRPML1 in both WT (left) and TSC2 KO (mTOR constitutively active, right) MEF cells. Inset shows the lack of TSC2 proteins in the TSC2 KO. (I) Rap effects on ITRPML1 in RagA and B KO (mTOR deficient, right) MEF cells. Inset shows the lack of RagA proteins in the RagA and B KO. (J) Rap activated larger endogenous ITRPML1 in p18 KO (right) compared with WT (left) HEK293 cells. Inset shows the lack of p18 proteins in the p18 KO. Note that in p18 KO cells, endogenous TFEB was localized in the nucleus, presumably due to mTOR deficiency (see S2K Fig), which in turn increased ITRPML1, because TRPML1 is the one of major target genes of TFEB [10]. Only representative data are presented in A–C, F, and H–J. The individual data underlying D and G can be found in S1 Data. CTRL, control; Defo, deforolimus; Eve, everolimus; HEK293, human embryonic kidney 293 cell; KO, knockout; MEF, mouse embryonic fibroblast; mTOR, mechanistic target of rapamycin; p18, late endosomal/lysosomal adaptor, MAPK and mTOR activator 1 (LAMTOR1); Rag, Ras-related GTP-binding protein; Rap, rapamycin; Seco, seco-rapamycin; S6K, S6 kinase; Tem, temsirolimus; TFEB, transcription factor EB; Thr 389, threonine 389; TRPML1, transient receptor potential channel mucolipin 1; TSC2, tuberous sclerosis complex 2; WT, wild type; Zota, zotarolimus.
Fig 3.
Rap and rapalogs bind TRPML1 in vitro.
(A) Lack of FK506 effect on ITRPML1. Representative ITRPML1 was shown. (B) Rap bound to immuno-purified EGFP-TRPML1 immobilized on Pro-A biosensors in a dose-dependent manner. (C) Dose-dependent Tem-TRPML1 binding. (D) Weak or nonspecific binding of Zota to TRPML1. (E) Weak or nonspecific binding of FK506 to TRPML1. Panels B–E show representative binding activity from at least 4 independent experiments. (F) Dose-dependent Rap- and rapalog-TRPML1 binding. To avoid the interference of other Rap-targeting proteins, e.g., mTOR, we subtracted Rap binding activity in nontransfected HEK293 cells from that in EGFP-TRPML1–overexpressing cells. Data are presented as mean ± SEM (n = 4–6 independent experiments), and the individual data can be found in S1 Data. a.u., arbitrary unit; EGFP, enhanced green fluorescent protein; FK506, tacrolimus; HEK293, human embryonic kidney 293; mTOR, mechanistic target of rapamycin; Pro-A, protein A; Rap, rapamycin; Tem, temsirolimus; TRPML1, transient receptor potential channel mucolipin 1; Zota, zotarolimus.
Fig 4.
Rap and rapalogs induce TRPML1- and Ca2+-dependent TFEB nuclear translocation in TRPML1-overexpressing cells.
(A) Rap (5 μM) and Tem (5 μM) induced TFEB nuclear translocation in TFEB-GFP stable cells overexpressing mCherry-TRPML1 (asterisks). TFEB nuclear translocation was not seen with Zota (5 μM). Scale bar = 10 μm. (B) Summary of rapalog effects on TFEB nuclear translocation. (C) Blockade of Tem-induced TFEB translocation by ML-SI3 (10 μM). Scale bar = 10 μm. (D) Quantification of ML-SI3 effect. (E) BAPTA-AM (5 μM, 1 h pretreatment) blocked Tem-induced TFEB nuclear translocation. (F) Tem (5 μM) induced TFEB nuclear translocation in TFEB-GFP stable cells overexpressing mCherry-TRPML2. Quantification is shown in the right panel. (G) The effects of Tem (5 μM, 2 h) and ML-SA1 (5 μM, 2 h) on TFEB nuclear translocation in TFEB-GFP stable cells that were transfected with mCherry-TRPML3. Data are quantified in the left panel. mCherry-positive cells are indicated by asterisks. Scale bar = 10 μm. Data shown in B and D–G were obtained from 30 to 40 cells from at least 3 independent experiments and are presented as mean ± SEM. The individual data supporting B and D–G can be found in S1 Data. ***P < 0.001, one-way ANOVA. BAPTA-AM, 1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis (acetoxymethyl ester); CTRL, control; Cyt, cytoplasm; Defo, deforolimus; Eve, everolimus; GFP, green fluorescent protein; mCh, mCherry; mCherry, monomeric red fluorescent protein; ML1, TRPML1; ML-SA1, TRPML1 synthetic agonist 1; ML-SI3, TRPML1 synthetic inhibitor 3; Nuc, nuclear; O/E, overexpression; Rap, rapamycin; Seco, seco-rapamycin; Tem, temsirolimus; TFEB, transcription factor EB; ML1/TRPML1, transient receptor potential channel mucolipin 1; Zota, zotarolimus.
Fig 5.
Tem activates the endogenous TRPML1-TFEB pathway.
(A) Tem (10 μM, 9 h) induced TFEB (green) nuclear translocation in WT but not ML1−/− fibroblasts. TFEB nuclear translocation was inhibited by coapplication of ML-SI3 (10 μM). Nuclei were labelled with DAPI (red, pseudo-color). Scale bar = 10 μm. (B) Summary of Tem effects on TFEB nuclear translocation in WT and ML1−/− human fibroblasts. (C) Dose-dependent and time-dependent effects of Tem on TFEB translocation. (D) The effects of Tem (10 μM, 6 h) on cells derived from human disease tissues, e.g., ML1−/−, NPC, HD, and DMD. (E) Quantification of Tem effects shown in (D). Data shown in B, C, and E were obtained from more than 40 cells from at least 3 independent experiments. (F) The effects of Tem (10 μM, 16 h) on mRNA expression levels of TRPML1, CTSD, and LAMP1 (n = 3–5 independent experiments). (G) The effects of Tem (10 μM, 16 h) on TFEB activity, measured using a 4X-CLEAR luciferase reporter (n = 4 independent experiments); Torin-1 (1 μM, 16 h) was used as a positive control. Data shown in B, C,and E–G are presented as mean ± SEM, and the individual data can be found in S1 Data. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. CTRL, control; CTSD, cathepsin D; Cyt, cytoplasm; DMD, Duchenne Muscular Dystrophy; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; HD, Huntington disease; LAMP1, lysosome-associated membrane protein 1; ML1−/−, Mucolipidosis IV; ML-SI3, TRPML1 synthetic inhibitor 3; NPC, Niemann-Pick type C; Nuc, nuclear; Tem, temsirolimus; TFEB, transcription factor EB; TRPML1, transient receptor potential channel mucolipin 1; WT, wild type; 4X-CLEAR, four CLEAR elements (GTCACGTGAC) in tandem derived from LAMP1 promoter + HTK.
Fig 6.
Tem increases autophagic flux through TRPML1.
(A) Tem (5 μM, 3 h) induced TFEB (green) nuclear translocation in TRPML1 stable cell lines (TRPML1 HEK Tet-On) upon Dox (1 μg/ml, overnight) induction. GPN (200 μM, 2 h) was used as a positive control due to its consistent activation on TFEB in HEK cells [37]. Nuclei were labelled with DAPI (red, pseudo-color). Scale bar = 10 μm. (B) Tem (10 μM, 9 h) dramatically increased LC3-II levels in Dox-induced cells, which was blocked by ML-SI3 (10 μM). (C) Summary of Tem effects on LC3-II levels (normalized with GAPDH expression). (D) Dose-dependent effects of Tem (0.5, 1, 2.5, 5, and 10 μM; 9 h treatment) on LC3-II expression levels in Dox-induced TRPML1 stable cells. (E) Quantification of dose-dependent Tem effects shown in D. (F) Tem (10 μM, 9 h) elevated LC3-II levels in WT but not ML1−/− human fibroblasts. Baf-A1 treatment increased LC3-II levels in both WT and ML1−/− cells. Tem effects in WT cells were blocked by ML-SI3. (G) Quantification of Tem effects on LC3-II levels in fibroblasts. Data shown in C, E, and G were obtained from at least 3 independent experiments and are presented as mean ± SEM. The individual data of C, E, and G can be found in S1 Data. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. Baf-A1, Bafilomycin A1; CTRL, control; Dox, doxycycline; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GPN, Glycyl-l-phenylalanine 2-naphthylamide; HEK, human embryonic kidney 293 cells; LC3-II, microtubule-associated proteins 1A/1B light chain 3B-II; ML−/−, Mucolipidosis IV; ML-SI3, TRPML1 synthetic inhibitor 3; Tem, temsirolimus; Tet-On, Tetracycline-On; TFEB, transcription factor EB; TRPML1, transient receptor potential channel mucolipin 1; WT, wild type.
Fig 7.
A working model of Rap stimulation of cellular clearance via the TRPML1-Ca2+-TFEB pathway.
Rap effects are sensitive to TRPML1 expression levels in “Rap-insensitive” cells. When TRPML1 expression is low, mTOR is in an active state in which it phosphorylates and inactivates TFEB via cytosolic retention. Rap inhibition of mTOR is insufficient to cause TFEB nuclear translocation. In “Rap-sensitive” cells, in which the Rap-TRPML1-TFEB pathway is sensitized, or stressed cells with up-regulated TRPML1, Rap binds and activates TRPML1 channels, inducing substantial lysosomal Ca2+ release. Increases in perilysosomal Ca2+ levels activate Cn, causing TFEB translocation from the cytosol to the nucleus. Activated TFEB then promotes the expression of autophagic and lysosomal genes, enhancing the autophagic-lysosomal degradation pathway and cellular clearance. Cn, calcineurin; mTOR, mechanistic target of rapamycin; Rap, rapamycin; TFEB, transcription factor EB; TRPML1, transient receptor potential channel mucolipin 1.