MCOLN1 is a ROS sensor in lysosomes that regulates autophagy

Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes ‘host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca2+-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca2+ release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell.

R eactive oxygen species (ROS) are generated mainly as byproducts of mitochondrial respiration, and their cytosolic levels are tightly controlled by multiple anti-oxidant mechanisms 1 . A regulatory imbalance can result in elevated ROS levels and oxidative stress, which are believed to underlie a variety of metabolic and neurodegenerative diseases, as well as aging 1,2 . Although high levels of ROS may additionally cause severe oxidative damage of proteins and lipids, a moderate ROS increase may serve as a sufficient 'signal' to trigger autophagy and other cell-survival mechanisms 1,2 . Autophagy can target oxidized and damaged biomaterials selectively for lysosomal degradation 3 . Because unhealthy mitochondria may further augment ROS production, ROS-induced mitophagy is required for effective removal of excess ROS 1,4,5 . Hence, ROS and autophagy may constitute a negative feedback mechanism that mitigates oxidative stress and promotes cell survival.
Autophagy is a multi-step catabolic process that involves initiation (that is, phagophore formation), autophagosome biogenesis, lysosome biogenesis, autophagosome-lysosome fusion and lysosomal degradation 3,6 . ROS are known to induce autophagy, but the mechanisms underlying this induction are poorly understood 1,7 . ATG4, a cysteine protease and component of the cellular autophagy machinery, was recently identified as a direct target of ROS 8 . Oxidized ATG4 promotes lipidation of LC3 (a.k.a. ATG8), a process that is essential for autophagy initiation 8 . Because coping with prolonged oxidative stress may require sustained autophagy and sufficient lysosome supplies to achieve efficient autophagosome-lysosome fusion in an ongoing manner, it is hypothesized that the lysosome-participating steps of autophagy need to be upregulated in a coordinated manner by ROS signalling 1,6,7,9,10 . Indeed, given the essential role of lysosomes in autophagic clearance 6 , inadequate lysosomal function inevitably leads to metabolic and neurodegenerative diseases, even though autophagy induction is often elevated under these pathological conditions 3,6 .
Lysosomes are organelles that 'host' important nutrientsensitive molecules 11 . mTOR (the mechanistic target of rapamycin), described as the master regulator of growth, is a nutrient sensor that resides on lysosomal membranes 6,11 . Under starvation conditions, inhibition of mTOR results in a decrease in the phosphorylation of transcription factor (TF)EB, a master transcriptional regulator of both autophagy and lysosomal biogenesis [12][13][14][15] . Dephosphorylated TFEB proteins translocate rapidly to the nucleus from the cytosol and lysosomes, inducing or increasing the expression of a unique set of genes that are related specifically to autophagosome and lysosome biogenesis 13,15 . It is not yet known whether the mTOR-TFEB pathway regulates lysosome function in response to other cellular stresses.
Very recently, it was reported that TFEB-nuclear translocation can also be stimulated by lysosomal Ca 2 þ release and the Ca 2 þdependent phosphatase calcineurin 9 . Lysosomal Ca 2 þ release is believed to be mediated by mucolipin 1 (MCOLN1 (a member of the transient receptor potential channel family), a.k.a. TRPML1), a key Ca 2 þ -conducting channel on the lysosomal membrane that releases Ca 2 þ from the lumen into the cytosol 16 . However, it is unclear whether and how TRPML1 is activated by specific autophagy-inducing conditions, for example, oxidative stress and nutrient starvation.
Lysosomes are required for quality-control regulation of mitochondria, and oxidative stress is a common feature of lysosome storage diseases 16 . Recent studies suggest that mitochondria, the major source of endogenous ROS, are localized in close physical proximity to lysosomes 7,17 . Hence the lysosomal membrane is potentially an accessible and direct target of ROS signalling. Given that ROS reportedly regulate ion channels 18 , we hypothesize that lysosomal conductances, particularly through lysosomal Ca 2 þ channels such as TRPML1, may mediate ROS regulation of lysosomal function.
In the present study, we demonstrate a direct and specific activation of lysosomal TRPML1 channels by both exogenous oxidants and mitochondria-derived ROS in the endolysosomal patch-clamp recordings. This ROS-induced TRPML1 activation leads to lysosomal Ca 2 þ release, calcineurin-dependent TFEB-nuclear translocation and increases of LC3-II expression and autophagy. Genetic inactivation or pharmacological inhibition of TRPML1 impairs ROS-induced autophagy and blocks the clearance of damaged mitochondria and removal of excess ROS.
We next investigated the mechanisms of oxidant-induced TRPML1 activation. Specifically, we tested whether the effect is direct in HEK293 cells expressing TRPML1-4A, a surfaceexpressing mutant TRPML1 channel 26,27 . In the inside-out, but not whole-cell, configuration, both ChT and NaOCl activated I TRPML1-4A ( Fig. 1f and Supplementary Figs 7 and 8), suggesting that the action is from the cytoplasmic side. ChT induced a dramatic and sustained increase in the channel open probability (NPopen) of single TRPML1 currents, which had a single-channel conductance comparable to the ML-SA1-activated currents (Fig. 1f,g). In contrast, TRPML1 Va , a constitutively active mutant of TRPML1 (ref. 20), was insensitive to oxidants (Fig. 1j).
Endogenous ROS trigger lysosomal Ca 2 þ release via TRPML1. Because mitochondria are the primary source of endogenous ROS 1,4,5 , to evaluate the effect of endogenous ROS on TRPML1, we exposed cells to mitochondrial respiration inhibitors, for example, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and rotenone, which are commonly used to induce ROS production, mitochondrial damage and subsequent mitophagy 28,29 . Following 1 h exposure to CCCP (5-10 mM) or rotenone (10 mM), intracellular ROS levels increased significantly ( Fig. 2a and Supplementary Fig. 11), as reflected by visualization of CM-H2DCFDA, a ROS-sensitive fluorescent dye 29 . N-acetylcysteine (NAC), a commonly used membrane-permeable antioxidant 30 , abolished CCCP-or rotenone-induced increases in ROS levels (Fig. 2a,b and Supplementary Fig. 11). Because drugs like CCCP might produce ROS-independent effects due to mitochondrial depolarization/damage in addition to ROS production 28,29 , NAC sensitivity test was routinely performed in various cellular assays in the current study.
Remarkably, after CCCP pretreatment in TRPML1-expressing COS-1 cells, basal whole-endolysosome I TRPML1 increased significantly (Fig. 2a,b). Likewise, in non-transfected cells, ML-SA1-evoked endogenous I TRPML1 also increased ( Fig. 2c and Supplementary Fig. 12). The CCCP-induced increase in basal I TRPML1 , however, was largely abolished by NAC (Fig. 2a,b). In contrast, oxidant-insensitive I zTRPML1.1 was not affected by CCCP nor NAC pretreatment (Fig. 2b). Taken together, these results suggest that lysosomal TRPML1 is activated or sensitized by mitochondria-generated ROS in the cell. Next, we investigated the effect of CCCP-generated mitochondrial ROS on lysosomal Ca 2 þ release. In COS-1 cells transfected with EGFP-TRPML1, ML-SA1-evoked lysosomal Ca 2 þ release 10 was increased on CCCP (10 mM) pretreatment for 1 h (Fig. 2d-f). Under our experimental settings, CCCP, although known as a proton gradient de-coupler 28,29 , did not have a direct effect on I TRPML1 or lysosomal pH ( Supplementary Figs 13 and 14). Consistently, acute treatment of CCCP did not directly evoke lysosomal Ca 2 þ release in GCaMP7-TRPML1-overexpressing cells ( Supplementary Fig. 13). The constant Ca 2 þ release in CCCP-treated cells, presumably mediated by constitutive activity of TRPML1, is expected to continuously decrease Ca 2 þ stores in lysosomes. To test this possibility, glycyl-L-phenylalanine-betanaphthylamide (GPN), a lysosome-specific substrate that causes channel-independent 'leakage' of Ca 2 þ , was employed to probe lysosome Ca 2 þ stores 31 . In contrast to ML-SA1-induced release, GPN-induced Ca 2 þ release was reduced in CCCP-treated cells (Fig. 2e,f). Note that in most other lysosomal Ca 2 þ studies, the ML-SA1 and GPN responses are positively correlated 10,23 .
Together, these results are mostly consistent with an interpretation that in CCCP-treated cells, there is increased TRPML1-mediated Ca 2 þ release from lysosomes caused by high basal TRPML1 activity resulting from ROS elevation.
TRPML1 is specifically required for ROS-induced autophagy. CCCP-induced mitochondrial depolarization and/or ROS production is known to induce general autophagy, and mitophagy if there is extensive mitochondrial damage 5 . To investigate the role of TRPML1 in this process, we measured autophagic induction in HeLa cells stably expressing mRFP-GFP-LC3 (ref. 32). Because LC3-II is recruited specifically to phagophores and autophagosomes, and because of the pH sensitivity of the GFP signal, mRFP þ GFP þ and mRFP þ GFP À puncta indicate nonacidified autophagosomes and acidified autolysosomes, respectively 3 . Three-hour exposure to CCCP (5 mM) led to a dramatic increase in autophagosomes, and this increase could be prevented by 3-methyladenine, an inhibitor of autophagy induction 3 (Fig. 3a,b and Supplementary Fig. 15). Notably, under this experimental setting (that is, low dose of CCCP), there was only minimal initiation of mitophagy, assessed indirectly via the recruitment of PARKIN 4 ( Supplementary Fig. 16).
Consistently, CCCP treatment also dramatically increased endogenous LC3-II levels in human fibroblasts (Fig. 3d,e). All these effects of CCCP were abolished by NAC (Fig. 3a,b and Supplementary Fig. 17), suggesting that CCCP-induced autophagy is mediated exclusively by ROS. Consistent with the hypothesis that ROS mediated the CCCP effect, H 2 O 2 (100 mM, 3 h) treatment was sufficient to enhance autophagosome formation ( Supplementary Fig. 18). Collectively, these results demonstrate that, in agreement with previous studies 1,7 , CCCPmediated ROS generation induces autophagy.
Remarkably, CCCP-and H 2 O 2 -induced autophagosome formation was blocked by BAPTA-AM (membrane-permeable Ca 2 þ chelator 31 ) or ML-SI3 (Fig. 3a,b and Supplementary  Figs 17 and 18), suggesting that ROS induce autophagy via a Ca 2 þ -and TRPML1-dependent mechanism. Furthermore, CCCP and H 2 O 2 treatment failed to increase LC3-II levels in mucolipidosis IV patient-derived TRPML1 KO (ML-IV) fibroblasts (Fig. 3d,e and Supplementary Fig. 18), which exhibited high basal levels of LC3-II compared with WT cells (Supplementary Fig. 19). In both WT and ML-IV cells, LC3-II levels were readily increased by Torin 1, a potent inhibitor of mTOR that is commonly used to induce autophagy (Fig. 3d,e), suggesting that TRPML1 has a role specifically for ROS-mediated, but not for general autophagy induction. On the other hand, artificial activation of TRPML1 by the synthetic agonist ML-SA1, or the more potent agonists ML-SA3 and ML-SA5 (ref. 10), was sufficient to induce mRFP þ GFP þ LC3 puncta formation ( Fig. 3c and Supplementary Fig. 18), but these effects were insensitive to NAC treatment ( Fig. 3c and Supplementary  Fig. 20). Furthermore, the induction effect of ML-SA5 on   Fig. 16). Using JC-1 fluorescence dye 33 to monitor mitochondrial membrane potential, we found that such CCCP treatment resulted in rapid depolarization of mitochondria (data not shown). PARKIN proteins are known to be recruited specifically to the damaged mitochondria, which are then autophagocytosed and delivered to An increase in mitochondrial ROS (for example, by CCCP-mediated mitochondrial depolarization) may activate TRPML1 channels on the perimeter membranes of lysosomes, inducing lysosomal Ca 2 þ release of that activates calcineurin. Subsequently, Ca 2 þ -bound calcinurin dephosphorylates TFEB, which is otherwise kept in its phosphorylated form by the nutrient-sensitive lysosome-localized mTOR kinase 15 . Nucleus-localized TFEB then activates the transcription of a unique set of genes related to autophagy induction, autophagosome biogenesis and lysosome biogenesis. Lysosomal Ca 2 þ release may also directly promote lysosome reformation/biogenesis 9 . Subsequently, autophagy is promoted to facilitate clearance of damaged mitochondria and removal of excessive ROS. lysosomes for degradation 4 . In HeLa cells stably expressing mCherry-Parkin (PARKIN stable cells), PARKIN-positive puncta were increased significantly following CCCP treatment (Fig. 4a,b and Supplementary Fig. 21). Meanwhile, the increased LC3 puncta were often co-localized with PARKIN aggregates in GFP-LC3overexpressing PARKIN stable cells, suggestive of increases in mitophagy ( Supplementary Fig. 21). After CCCP washout, the majority of the PARKIN-positive puncta disappeared and most mitochondria returned to the repolarized state (Fig. 4a-d). The LC3-PARKIN co-localization, however, persisted during the recovery phase ( Supplementary Fig. 21), suggesting a continuous removal of damaged mitochondria through active mitophagy. In contrast, acute inhibition of TRPML1 with ML-SI3 or ML-SI4 during the CCCP treatment phase was sufficient to block the disappearance of PARKIN, even though ML-SI3 or ML-SI4 alone did not induce the prolonged accumulation of PARKIN (Fig. 4a,b and Supplementary Fig. 21). Accordingly, CCCP-induced LC3-PARKIN co-localization (mitophagy) was significantly suppressed by ML-SI3 (Supplementary Fig. 21). Furthermore, JC-1 recovery was also blocked in ML-IV fibroblasts, compared with WT fibroblasts (Fig. 4c,d). Taken together, these results suggest that TRPML1 is required for ROS-and mitophagy-dependent clearance of damaged mitochondria.
Ongoing basal level of mitophagy may protect mitochondria from damage 7 . Although low-dose (for example, 5 mM) CCCP did enhance LC3 accumulation, no significant PARKIN aggregation could be observed ( Supplementary Figs 16 and 23). Likewise, H 2 O 2 treatment (100 mM for 3 h) did not produce significant PARKIN aggregation ( Supplementary Fig. 22), although it did increase LC3 puncta formation and expression ( Supplementary  Fig. 18). However, in the presence of ML-SI3, low-dose CCCP treatment was sufficient to increase the number of PARKINpositive puncta, although neither treatment alone had obvious effects ( Fig. 4a and Supplementary Fig. 23). Hence, low levels of ROS may be sufficient to induce mitophagy, even though not detected experimentally by PARKIN recruitment. Taken together, these results suggest that TRPML1 may have a role in preventing the accumulation of damaged mitochondria. TRPML1 is required for efficient removal of excess ROS. ROSinduced mitophagy is hypothesized to remove excess ROS, preventing further oxidative damage to mitochondria 1,4,7 . As shown in Figs 2a and 4f, CCCP treatment increased ROS levels, which declined gradually on withdrawal of CCCP. However, in the presence of ML-SI3 or ML-SI4, the recovery of ROS levels was impeded (Fig. 4f,g). Furthermore, ROS levels were constitutively elevated in ML-IV cells, as well as in NPC (Niemann-Pick Type C) cells wherein TRPML1 is chronically inhibited 23 (Fig. 4e). Collectively, these results suggest that TRPML1 activation is required for ROS-induced ROS removal, a negative feedback mechanism that is used by cells to circumvent oxidative stress.
TRPML1 mediates ROS-induced TFEB activation. We next investigated the mechanisms by which TRPML1 activation leads to enhanced autophagy induction and mitophagic clearance. TFEB regulates biogenesis of both autophagosomes and lysosomes 34 . Recent evidence suggests that TRPML1 and TFEB may form a positive-feedback loop in regulating autophagy 9,10 . In HEK293 cells stably expressing mCherry-TFEB (TFEB stable cells), we found that a mild increase in ROS levels due to CCCP treatment (5 mM for 1 h) was sufficient to induce TFEB-nuclear translocation (nuclear to cytosol ratio increased from 0.54 ± 0.02 to 2.67±0.14 (mean±s.e.m., n ¼ 3 experiments for each treatment); see Fig. 5a,b). Likewise, endogenous TFEB in human fibroblasts was also activated (that is, underwent nuclear translocation) in response to CCCP administration in a dosedependent manner (Fig. 5g,h and Supplementary Fig. 24). The specificity of TFEB immunoreactivity was confirmed in TFEB KO HeLa cells (Supplementary Fig. 25) generated by the CRISPR/Cas9 system 35 . In addition, nuclear isolation analyses also demonstrated that CCCP treatment induced nuclear translocation of TFEB proteins (Fig. 5c,d) in TFEB stable cells. In both WT and PARKIN stable cells, CCCP (5-20 mM) activated TFEB ( Supplementary Fig. 26), even in the presence of NOX inhibitors, which block mitochondria-independent ROSgenerating NADPH oxidases 36 (Supplementary Fig. 27). Likewise, rotenone (10 mM for 2 h) and H 2 O 2 treatment (50 mM for 1 h) were also sufficient to induce TFEB translocation in WT HeLa cells ( Supplementary Figs 28 and 29). Remarkably, in all cases, TFEB-nuclear translocation, induced by CCCP, rotenone or H 2 O 2 , was largely blocked by NAC, BAPTA-AM or ML-SI3 (Fig. 5a-f and Supplementary Figs 23, 28 and 29).
ROS levels are reportedly elevated during starvation 8 , a condition that activates TFEB and promotes autophagy 9 . Indeed, short-term (4-6 h) starvation increased ROS levels and caused TFEB-nuclear translocation ( Supplementary Figs 30 and 31). However, starvation-induced TFEB activation was not blocked by NAC or its more potent derivative NACA, nor by ML-SI3 ( Supplementary Figs 30 and 31). These results suggest that starvation may induce TFEB activation via mechanisms that do not require TRPML1. Consistently, 4-h starvation induced TFEBnuclear translocation in both WT and ML-IV human fibroblasts ( Supplementary Fig. 31). Collectively, these results suggest that whereas ROS activation of TRPML1 is sufficient to activate TFEB, additional TRPML1-independent mechanisms may be responsible for starvation-induced TFEB activation.
CCCP-mediated TFEB translocation was largely abolished in ML-IV fibroblasts (Fig. 5g,h and Supplementary Fig. 24), as well as in WT cells treated with ML-SI3 or ML-SI4 (Fig. 5e-h). Furthermore, ML-SA5 alone was sufficient to cause TFEB-nuclear translocation in WT cells (Fig. 5i,j and Supplementary Fig. 32). This ML-SA5-induced TFEB translocation was sensitive to BAPTA-AM, but not NAC (Supplementary Fig. 32). Neither CCCP nor ML-SA5 affected the activity of mTOR, as reflected by levels of phosphorylated S6K, a primary mTOR substrate 11 ( Supplementary Fig. 33). On the other hand, Torin-1-induced TFEB-nuclear translocation, observed in both WT and ML-IV cells (Fig. 5g, h and Supplementary Fig. 24), was not sensitive to BAPTA-AM or ML-SI3 (Fig. 5a-f). Hence, TRPML1 and lysosomal Ca 2 þ appear to play a specific role in ROS-induced, but not the general mTOR-inhibition-mediated, autophagy. More strikingly, in PARKIN stable cells, when TFEB translocation was blocked by ML-SI3, obvious aggregation of PARKIN-positive puncta was observed even with a low-dose CCCP treatment ( Supplementary Fig. 23). Consistent with the reported role of calcineurin in TFEB activation 9 , we found that TFEB translocation induced by CCCP was also largely blocked by calcineurin inhibitors, FK506 and Cyclosporin A ( Supplementary  Fig. 34). Taken together, these results suggest that TRPML1dependent activation of TFEB plays a crucial role in ROS-induced autophagy and mitophagy.
ROS-induced lysosome biogenesis requires TRPML1. Because TFEB is a transcriptional regulator of lysosome biogenesis 11 , we next investigated the roles of ROS and TRPML1 in lysosome biogenesis. Increased expression of lysosomal housekeeping gene Lamp1 was employed as a read-out of lysosome biogenesis 10,16 . Upon CCCP (5 mM) treatment for 1 h, which produced a modest increase in ROS levels, expression of Lamp1 increased gradually in HeLa cells (Supplementary Fig. 35). Therefore, a brief ROS burst is sufficient to trigger lysosome biogenesis. However, the increase was largely attenuated by ML-SI3 (Fig. 6a,b) and also diminished in TFEB KO cells ( Supplementary Fig. 35), suggesting that TRPML1 is required for ROS-induced lysosome biogenesis through TFEB activation.

Discussion
In the current study, we identified the missing links between ROS and autophagy: lysosomal Ca 2 þ , TRPML1 and TFEB (see Fig. 6e). Our results suggest a model wherein an elevation of ROS levels (for example, due to mitochondrial damage) leads to TRPML1 activation and lysosomal Ca 2 þ release. Lysosomal Ca 2 þ , acting via calcineurin-mediated dephosphorylation 9 , induces TFEB-nuclear translocation, promoting both autophagosome biogenesis and lysosome biogenesis. Lysosomal Ca 2 þ may also directly regulate lysosome biogenesis and autophagosome-lysosome fusion 16 . The subsequent increase in autophagic flux may facilitate removal of damaged mitochondria and restoration of redox homeostasis. Hence TRPML1 may serve as a ROS sensor in the lysosome that regulates an autophagydependent negative-feedback mechanism essential for cellular redox homeostasis.
Although the connection between ROS and autophagy has been well-documented 1,7 , the mechanisms by which ROS promote autophagy are largely unknown. Several TFs, including NRF2 and FOXO3, are known to become active under oxidative stress conditions 7 . Recently, ATG4, a cytosolic negative regulator of LC3/ATG8, was identified as a direct ROS target that provides a rapid switch mechanism for autophagy induction 8 . TRPML1 is activated rapidly by exogenous oxidants in isolated lysosomes, or in excised patches containing TRPML1 re-directed to the plasma membrane. Hence, it is very likely that direct oxidation of TRPML1 occurs, and such oxidation favours the channel adopting a 'Va-like' constitutively-open state 20 . Because autophagy is a multi-step process 3 , there may be multiple ROS sensors involved in regulating autophagy. Notwithstanding, in our experimental paradigm, in which endogenous mitochondrial ROS are generated to induce autophagy, autophagy induction is almost completely blocked by Ca 2 þ chelators and TRPML1 inhibitors. The robustness of these results suggests that TRPML1 may play a pivotal role in autophagy regulation.
Our findings are consistent with the reported role of Ca 2 þ in autophagosome biogenesis 37 . However, in light of the observations that increasing autophagosome formation alone does not result in an increase in autophagic flux, ROS-dependent mechanisms that promote lysosome biogenesis and function must exist. Given the established roles of TRPML1 and TFEB in lysosomal trafficking and biogenesis 9,10,16 , our identification of TRPML1 as a lysosomal ROS sensor has revealed a unique ROS-regulated paradigm in which both initiation and maturation of autophagy are coordinated.
Lysosomal membranes are the focal sites of multiple nutrientsensitive kinases that regulate autophagy, including mTOR and AMPK 11 . It may not be coincidental that TRPML1, acting as a major autophagy-regulating ROS sensor, is also localized on the lysosomal membrane. TRPML1, a protein implicated in lysosome reformation and autophagosome-lysosome fusion, is upregulated in response to cellular stress, such as nutrient starvation 10,16 . TRPML1 and TFEB/TFE3 may form a positive-interaction loop to regulate lysosome function and autophagy 9,10,16 . TRPML1's direct role in stress-sensing makes TRPML1 and TFEB wellsituated to play important roles in lysosome adaptation to environmental cues 10 . However, TFEB can also be activated by TRPML1-independent pathways, such as by mTOR inhibition and PIKfyve inhibition 10 . Hence TRPML1 appears to be uniquely positioned to respond to certain stress pathways, including ROS signalling.
ROS regulation of TRPML1 may also affect other lysosomerelated functions. ROS can be generated within lysosomes when heavy metal ions (for example, iron) catalyse the Fenton reaction, thereby generating free radicals 38 . Indeed, TRPML1's iron permeability 39 and ROS sensitivity are well-suited to constitute a negative feedback mechanism to regulate redox homeostasis in the lysosome, preventing lysosomal membrane permeability and lysosomal cell death 16 . Likewise, ROS regulation of TRPML1 may also be involved in membrane repair and phagocytic killing of bacteria, both of which are known to involve oxidant signalling 40 .
Under physiological conditions, a mild increase in ROS levels may act as a 'survival' signal triggering autophagy for the 'quality control' purpose, whereas a large increase in ROS levels may produce severe oxidative damage and stress and serve as a 'death' signal 7 . Hence before the 'death threshold' is reached, ROS can signal both autophagy induction and lysosome biogenesis, promoting autophagic flux. Supra-threshold ROS, however, may cause lysosomal membrane permeability, lysosomal dysfunction and autophagic failure. For example, the excessive and sustained surge in ROS that occurs during cardiac ischemia-reperfusion may actually inhibit autophagy, contributing to cardiomyocyte death 41 . However, under pathological hyper-sensitive conditions, in which lysosome function and autophagy are not functioning appropriately, even a mild increase in ROS levels may cause oxidative stress. For example, when the TRPML1-TFEB pathway is inactive or compromised, as is seen in several lysosomal storage diseases, cells exhibit mitochondrial fragmentation, elevation of basal ROS levels and oxidative stress 16 . On the other hand, when TRPML1-TFEB activity is upregulated, oxidative stress is mitigated 9,10,16,42 . Hence, the TRPML1-TFEB pathway may represent a potential therapeutic target by which preemptive modulation of oxidative stress may alleviate symptoms in patients with lysosomal storage diseases and neurodegenerative diseases characterized by excess ROS.

Methods
Molecular biology. TRPML1 mutants were constructed with a site-directed mutagenesis kit (Qiagen) using mouse TRPML1 as the template. GCaMP7-TRPML1 was generated by inserting the full-length GCaMP7 between the HindIII and EcoRI sites at a pcDNA6-mTRPML1 construct 23 . The mCherry-PARKIN construct was provided by Dr Richard Youle through Addgene 4 . All constructs were confirmed by DNA sequencing.
Mammalian cell culture. COS-1 and HEK-293T were cultured in a 1:1 mixture of DMEM and Ham's F12 (DF12) media with 10% fetal bovine serum (FBS). HeLa and HAP1 cells were maintained in DMEM and IMDM, respectively, both with 10% FBS. Lipofectamine 2000 (Invitrogen) was used for the transfection of above cells. Human skin fibroblast cell lines from a mucolipidosis IV (TRPML1 KO) patient (clone GM02048) and a healthy control (clone GM05659) were obtained from the Coriell Institute for Medical Research (NJ, USA). Fibroblasts were transfected with a Neon electroporation kit (Invitrogen). Culture media were refreshed 18-24 h post-transfection, and cells were imaged 48 h post-transfection to allow sufficient recovery time following transfection.
Stable cell lines. The mCherry-PARKIN stable cell line was generated in HeLa cells under the selection of 500 mg l À 1 Geneticin (G418, Invitrogen). The mCherry-TFEB stable cell line was generated using the Flip-In T-Rex 293 cell line (Invitrogen) under blasticidin selection. GFP-mRFP-LC3 and GFP-TFEB stable cell lines were kindly provided by Drs David Rubinsztein 32 and Shawn M. Ferguson 13 , respectively. Unless otherwise indicated, all cell lines were maintained in DMEM medium supplemented with 10% Tet-free FBS at 37°C in a humidified 5% CO 2 incubator.
Confocal imaging. For TFEB and TFE3 immunofluorescence detection, cells were grown on glass coverslips and then fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 after treatments. The cells were then blocked with 1% bovine serum albumin in phosphate buffered saline (PBS). Endogenous TFEB and TFE3 were recognized by incubating cells with anti-TFEB (1:200; Cell Signaling Technology) or anti-TFE3 antibody (1:1,000 Sigma) at 4°C overnight. Cells were then washed four to five times with PBS and incubated with anti-rabbit secondary antibodies conjugated to Alexa Fluor 568 or 488 (Invitrogen) for 1 h. After three washes with PBS, coverslips were mounted on the slides with Fluoromount-G (Southern Biotech). Images were acquired with an Olympus Spinning-Disk Confocal microscope.
ROS and lysosomal pH imaging. ROS levels were detected with a CM-H2DCFDA dye assay (Invitrogen). Briefly, cells were incubated with 2.5-5 mM CM-H2DCFDA in the culture media without FBS at 37°C for 30 min, and then recovered in the complete media for 10 min before imaging. For non-quantitative estimation of lysosomal luminal pH, cells were incubated with 50 nM LysoTracker Red DND-99 (Invitrogen) in complete culture medium for 15 min before imaging. The fluorescence was visualized with a DP71 camera (Olympus) mounted on an Olympus IX-71 inverted microscope. Images were captured at 20 Â magnification with DPController software. The fluorescence intensity was quantified with the ImageJ software (NIH).
Mitochondrial membrane potential measurement. Human fibroblasts were incubated with 1 mM JC-1 (Invitrogen) in complete culture medium at 37°C for 30 min before imaging. The fluorescence was detected at 520 nm for J-monomer and 600 nm for J-aggregates (excitation wavelength ¼ 488 nm) by a Leica confocal microscope.
Whole-endolysosome electrophysiology. Isolated endolysosomes were subjected to whole-endolysosomal electrophysiology by a modified patch-clamp method 19,20 . Briefly, cells were treated with 1 mM vacuolin-1 overnight to selectively increase the size of late endosomes and lysosomes 44 . Enlarged vacuoles were released into the dish by mechanical disruption of the cell membrane with a fine-tip glass electrode. Unless otherwise indicated, vacuoles were bathed continuously in an internal (cytoplasmic) solution containing 140 mM K þ -Gluconate, 4 mM NaCl, 1 mM EGTA, 2 mM Na 2 -ATP, 2 mM MgCl 2 , 0.39 mM CaCl 2 , 0.1 mM GTP and 10 mM HEPES (pH adjusted with KOH to 7.2; free [Ca 2 þ ] i E100 nM). The pipette (luminal) solution contained 145 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, 10 mM MES and 10 mM glucose (pH adjusted to 4.6 with NaOH). The whole-endolysosome configuration was achieved as described previously 19 . In brief, after formation of a gigaseal between the patch pipette and an enlarged endolysosome, voltage steps of several hundred millivolts with a millisecond duration were applied to break into the vacuolar membrane 19 . All bath solutions were applied via a fast perfusion system that produced a complete solution exchange within a few seconds. Data were collected via an Axopatch 2A patchclamp amplifier, Digidata 1440 and processed with 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 in pCLAMP10 (Axon Instruments) and Origin 8.0 (OriginLab).