Mitochondria-Lysosome Contacts Regulate Mitochondrial Ca2+ Dynamics Via Lysosomal TRPML1

Mitochondria-lysosome contacts regulate mitochondrial Ca2+ dynamics via lysosomal TRPML1

Wesley Penga, Yvette C. Wonga, and Dimitri Krainca,1

aDepartment of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611

Edited by Dejian Ren, Department of Biology, University of Pennsylvania, Philadelphia, PA, and accepted by Editorial Board Member David E. Clapham June 23, 2020 (received for review February 24, 2020) Mitochondria and lysosomes are critical for cellular homeostasis, and degeneration, and developmental delay (33, 47–49), and which has dysfunction of both organelles has been implicated in numerous been associated with various lysosomal and mitochondrial aber- diseases. Recently, interorganelle contacts between mitochondria and rations (45, 46, 50–54). However, whether TRPML1-mediated lysosomes were identified and found to regulate mitochondrial dy- lysosomal calcium release modulates mitochondrial calcium dynamics. However, whether mitochondria–lysosome contacts serve ad- namics via mitochondria–lysosome contact sites, and the role of ditional functions by facilitating the direct transfer of metabolites or mitochondria–lysosome contact site dysfunction in the patho- ions between the two organelles has not been elucidated. Here, using physiology of lysosomal storage disorders such as MLIV, has not high spatial and temporal resolution live-cell microscopy, we identified previously been studied. – a role for mitochondria lysosome contacts in regulating mitochondrial Using live-cell high spatial and temporal resolution microscopy, calcium dynamics through the lysosomal calcium efflux channel, tran- we show that TRPML1 lysosomal calcium release mediates the sient receptor potential mucolipin 1 (TRPML1). Lysosomal calcium re- direct transfer of calcium into mitochondria. Calcium transfer from leasebyTRPML1promotescalciumtransfer to mitochondria, which lysosomes to mitochondria is modulated by mitochondria–lysosome was mediated by tethering of mitochondria–lysosome contact sites. contact site tethering and is modulated by the outer and inner Moreover, mitochondrial calcium uptake at mitochondria–lysosome contact sites was modulated by the outer and inner mitochondrial mitochondrial membrane proteins, voltage-dependent anion chan- membrane channels, voltage-dependent anion channel 1 and the mi- nel 1 (VDAC1) and mitochondrial calcium uniporter (MCU), re- tochondrial calcium uniporter, respectively. Since loss of TRPML1 func- spectively. Importantly, MLIV patient fibroblasts with loss of – tion results in the lysosomal storage disorder mucolipidosis type IV TRPML1 function exhibit disrupted mitochondria lysosome contact (MLIV), we examined MLIV patient fibroblasts and found both altered site dynamics and contact-dependent calcium transfer, suggesting a mitochondria–lysosome contact dynamics and defective contact- potential contribution of dysregulated mitochondria–lysosome con- dependent mitochondrial calcium uptake. Thus, our work highlights tact site dynamics in lysosomal storage disorders. Our results thus mitochondria–lysosome contacts as key contributors to interorganelle elucidate an additional mechanism for regulating intracellular cal- calcium dynamics and their potential role in the pathophysiology of cium dynamics via mitochondria–lysosome contact sites, which are disorders characterized by dysfunctional mitochondria or lysosomes. further implicated in disease pathophysiology.

mitochondria–lysosome contacts; interorganelle membrane contact sites | Significance lysosomal storage disorder | TRPML1 | calcium Mitochondria and lysosomes are critical for cellular homeostasis nterorganelle contact sites have become increasingly appreci- and defects in both organelles are observed in several diseases. Iated as essential regulators of cellular homeostasis. Contact Recently, contact sites between mitochondria and lysosomes sites, which form dynamically between two distinct organelles in were identified and found to modulate mitochondrial dynamics. close proximity, have been shown to have a variety of functions, However, whether mitochondria–lysosome contacts have addi- including the ability to act as platforms for the direct transfer of tional functions is unknown. Here, we identify a function of ions, such as calcium (1–6). Recently, interorganelle contact sites mitochondria–lysosome contacts in facilitating the direct trans- between mitochondria and lysosomes were characterized, re- fer of calcium from lysosomes to mitochondria. Transfer of cal- vealing a novel mechanism of cross-talk between the two or- cium at mitochondria–lysosome contacts is mediated by the ganelles (7–18). Interestingly, both mitochondria and lysosomes lysosomal channel TRPML1 and is disrupted in mucolipidosis are also important players in cellular homeostasis, including in- type IV, a lysosomal storage disorder caused by loss-of-function tracellular calcium dynamics (19–22), and a number of diseases mutations in TRPML1. Calcium transfer from lysosomes to mi- presenting with mitochondrial and lysosomal dysfunction also tochondria at mitochondria–lysosome contacts thus presents an exhibit dysregulation of cellular calcium (23–30). Although the additional mechanism of intracellular calcium regulation that calcium dynamics of mitochondria and lysosomes have previ- may further contribute to various disorders. ously been studied individually or in relation to other organelles (1–5, 31, 32), whether mitochondria and lysosomes can interact Author contributions: W.P., Y.C.W., and D.K. designed research; W.P. performed research; directly to modulate their calcium states has not been elucidated. W.P. analyzed data; and W.P., Y.C.W., and D.K. wrote the paper. Mitochondria–lysosome contacts may thus enable the direct Competing interest statement: D.K. is the Founder and Scientific Advisory Board Chair of Lysosomal Therapeutics Inc. and Vanqua Bio. D.K. serves on the scientific advisory boards transfer of calcium between lysosomes and mitochondria and of The Silverstein Foundation, Intellia Therapeutics, and Prevail Therapeutics, and is a function as an additional pathway in regulating intracellular Venture Partner at OrbiMed. calcium homeostasis. This article is a PNAS Direct Submission. D.R. is a guest editor invited by the Transient receptor potential mucolipin 1 (TRPML1) is a lyso- Editorial Board. somal/late-endosomal cation channel that mediates lysosomal Published under the PNAS license. calcium efflux (33–38) and function (39–44), and dysfunction in Data deposition: Data are available at the Open Science Framework, https://osf.io/wf83s/. TRPML1 has been associated with several mitochondrial defects 1To whom correspondence may be addressed. Email: [email protected]. (45, 46). In addition, loss-of-function mutations in TRPML1 cause This article contains supporting information online at https://www.pnas.org/lookup/suppl/ mucolipidosis type IV (MLIV), an autosomal recessive lysosomal doi:10.1073/pnas.2003236117/-/DCSupplemental. storage disorder characterized by psychomotor retardation, retinal First published July 23, 2020.

19266–19275 | PNAS | August 11, 2020 | vol. 117 | no. 32 www.pnas.org/cgi/doi/10.1073/pnas.2003236117 Downloaded by guest on September 25, 2021 Results TRPML1 Activation Preferentially Increases Mitochondrial Calcium at – TRPML1-Mediated Lysosomal Calcium Efflux Leads to Mitochondrial Mitochondria Lysosome Contacts. Because activation of lysosomal Calcium Influx. To evaluate whether lysosomal TRPML1 calcium calcium release via TRPML1 led to increased mitochondrial cal- efflux modulated mitochondrial calcium (Fig. 1A), we used live-cell cium, we next evaluated whether this increase in mitochondrial confocal microscopy at high spatial and temporal resolution to calcium preferentially occurred at mitochondria–lysosome contact image mitochondrial calcium dynamics using the mitochondria- sites. We found that stable mitochondria–lysosome contacts dy- targeted genetically encoded calcium sensor Mito-R-GECO1 (55) namically formed in wild-type HeLa cells, defined as lysosomes (Fig. 1B). We first verified correct localization of Mito-R-GECO1, remaining tethered to mitochondria for over 10 s (Fig. 2A), as re- which was found to localize to the mitochondrial matrix as dem- cently described (8, 9). To assess whether TRPML1 mediated the onstrated by colocalization with the mitochondrial matrix-targeted direct transfer of calcium at mitochondria–lysosome contacts, we BFP-mito (SI Appendix,Fig.S1A). Next, mitochondrial calcium analyzed the calcium dynamics of mitochondria that were either in responses were measured in wild-type HeLa cells upon activation of contact or not in contact with lysosomes upon TRPML1 activation TRPML1 lysosomal calcium release with the TRPML1 agonist ML- (Fig. 2B, SI Appendix,Fig.S2A,andMovie S2). Mitochondria stably SA1 (54). Following treatment with ML-SA1, total mitochondrial in contact with lysosomes (>10 s) had a significantly higher increase calcium was significantly increased (Fig. 1 C and D and Movie S1). in calcium after TRPML1 activation, compared to mitochondria Compared to control cells, cells treated with ML-SA1 showed a not in contact with lysosomes (Fig. 2 B and C). This preferential sustained elevation in mitochondrial calcium (Fig. 1D)andasig- increase in mitochondrial calcium at mitochondria–lysosome con- nificant increase in maximum mitochondrial calcium, mean mito- tacts was observed in multiple cell types, including fibroblasts and chondrial calcium, and mitochondrial calcium at multiple time HCT116 cells, which similarly showed a mitochondria–lysosome points (Fig. 1 E–G). These results were further validated by acti- contact-dependent increase in mitochondrial calcium upon activa- vation of TRPML1 with an additional small-molecule agonist, tion of TRPML1 lysosomal calcium release (SI Appendix,Fig. MK6-83 (56) (SI Appendix,Fig.S1B–F), as well as with its physi- S2 B–D and Movies S3 and S4). ological activator, PI(3,5)P2 (57) (SI Appendix,Fig.S1G–J), both of We then investigated whether directly modulating mitochondria which resulted in a sustained increase in mitochondrial calcium. –lysosome contact sites could increase the transfer of lysosomal Thus, lysosomal TRPML1 calcium efflux robustly modulates mito- calcium into mitochondria. We previously showed that mitochondria chondrial calcium dynamics by increasing calcium influx into the –lysosome contact site untethering is regulated by the activity of the mitochondrial matrix. mitochondrial-localized Rab7 GTPase-activating protein (TBC1D15) CELL BIOLOGY A B

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Fig. 1. TRPML1-mediated lysosomal calcium efflux leads to mitochondrial calcium influx. (A) Model of activation of lysosomal calcium release by TRPML1 agonist, ML-SA1, resulting in mitochondrial calcium influx at mitochondria–lysosome contacts. (B) Experimental design for the assessment of mitochondrial calcium responses (ΔF/F) to TRPML1 activation in live cells. (C and D) Mitochondrial calcium response in live HeLa cells expressing mitochondrial-matrix targeted calcium sensor, Mito-R-GECO1, in response to TRPML1 activation with ML-SA1 (31.25 μM) (yellow arrow) or control treatment (white arrow) at t = 0s with representative time-lapse confocal images (C, n = 23 cells for ML-SA1, n = 20 cells for control) and mitochondrial calcium traces (ΔF/F) (D, n = 23 cells for ML-SA1, n = 20 cells for control). (Scale bars, 10 μm; 1 μm in zoom images.) (E–G) Quantification of maximum mitochondrial calcium response (E), mean mitochondrial calcium response (F), and mitochondrial calcium response at 30, 60, 90, and 120 s (G) after TRPML1 activation with ML-SA1 (31.25 μM) or control treatment from confocal time-lapse images in C (n = 23 cells for ML-SA1, n = 20 cells for control). Data are means ± SEM (***P < 0.001, ****P < 0.0001, unpaired two-tailed t test).

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Fig. 2. TRPML1 activation preferentially increases mitochondrial calcium at mitochondria–lysosome contacts. (A) Representative time-lapse confocal images showing stable mitochondria–lysosome contact site tethering (white arrows) in live HeLa cells expressing lysosomal marker, lamp1-mGFP, and mitochondrial- matrix targeted calcium sensor, Mito-R-GECO1. (Scale bar, 1 μm.) (B) Representative time-lapse confocal images of increase in mitochondrial calcium at t = 30 s following TRPML1 activation with ML-SA1 (31.25 μM) in mitochondria in contact with lysosomes (Lower, yellow arrow) versus those not in contact with lysosomes (Upper, white arrow) in live HeLa cells expressing mitochondrial matrix calcium sensor, Mito-R-GECO1, and lysosomal marker, lamp1-mGFP (n = 100 events from 20 cells for each condition). (Scale bars, 1 μm.) (C) Quantification of mitochondrial calcium responses from mitochondria in contact and not in contact with lysosomes following TRPML1 activation with ML-SA1 (31.25 μM) from confocal time-lapse images (from B)(n = 100 events from 20 cells for each condition). (D and E) Mitochondrial calcium response (ΔF/F) in live wild-type or TBC1D15 knockout (KO) HCT116 cells expressing mitochondrial-matrix targeted calcium sensor, Mito-R-GECO1, following TRPML1 activation with ML-SA1 (31.25 μM) at t = 0 s with representative time-lapse confocal images (D, n = 18 cells for each condition) and mitochondrial calcium traces (ΔF/F) (E, n = 18 cells for each condition). (Scale bars, 5 μm.) (F–H) Quantification of maximum mitochondrial calcium response (F), mean mitochondrial calcium response (G), and mitochondrial calcium response at 30 and 60 s (H) after treatment with ML- SA1 (31.25 μM) in live wild-type or TBC1D15 KO HCT116 cells from confocal time-lapse images (from D)(n = 18 cells for each condition). Data are means ± SEM (*P < 0.05, **P < 0.01, ****P < 0.0001, unpaired two-tailed t test).

driving Rab7 GTP hydrolysis on lysosomes/late endosomes, and We further confirmed that mitochondrial uptake of lysosomal consequently that TBC1D15 knockout significantly prolonged calcium was not dependent on the endoplasmic reticulum (ER) mitochondria–lysosome contact site tethering (8). To further (31, 32), as blocking ER calcium release using an inositol 1,4,5- investigate whether mitochondrial calcium dynamics could be triphosphate receptor (IP3R) antagonist (Xestospongin-C, pre- regulated at mitochondria–lysosome contacts, we compared treatment for 20 min) (SI Appendix, Fig. S3 A–D) or ryanodine SI Ap- HCT116 wild-type with TALEN-generated HCT116 TBC1D15 receptor antagonist (DHBP, pretreatment for 10 min) ( pendix E–G knockout cells (58), as TBC1D15 knockout cells have signifi- , Fig. S3 ) did not prevent an increase in mitochon- cantly increased mitochondria–lysosome contact tethering du- drial calcium (Mito-R-GECO1) upon TRPML1 activation. Similarly, neither blocking store-operated calcium entry with a ration (8). Upon TRPML1 activation with ML-SA1, TBC1D15 stromal interaction molecule 1 (STIM1) inhibitor (SKF-96365, knockout cells showed a significantly greater increase in total pretreatment for 10 min) (SI Appendix, Fig. S3 H–J) nor de- D mitochondrial calcium compared to wild-type cells (Fig. 2 pleting ER calcium using a SERCA (sarco/endoplasmic reticu- E and ), as well as significantly increased maximum mitochon- lum Ca2+-ATPase) pump inhibitor (thapsigargin, pretreatment drial calcium, mean mitochondrial calcium, and mitochondrial for 10 min) (SI Appendix, Fig. S3 K–M) altered mitochondrial calcium at multiple time points (Fig. 2 F–H). Thus, directly calcium increase (Mito-R-GECO1) upon TRPML1 activation. modulating mitochondria–lysosome contact site tethering is suf- TRPML1-mediated mitochondrial calcium influx was also unal- ficient to increase lysosomal calcium transfer into mitochondria. tered upon chelation of cytosolic calcium (BAPTA-AM, pretreatment

19268 | www.pnas.org/cgi/doi/10.1073/pnas.2003236117 Peng et al. Downloaded by guest on September 25, 2021 for 20 min), further suggesting that calcium transfer primarily dominant-negative TRPML1 showed a significant reduction in occurs at mitochondria–lysosome contacts (SI Appendix, Fig. S4). mitochondrial calcium influx compared to wild-type TRPML1- Altogether, these findings indicate that TRPML1-mediated cal- expressing cells (Fig. 3 A and B). In addition, expression of the cium influx into mitochondria occurs preferentially at mitochondria dominant-negative TRPML1 mutant significantly reduced the –lysosome contacts and, furthermore, can be directly regulated by maximum mitochondrial calcium, mean mitochondrial calcium, modulating mitochondria–lysosome contact tethering machinery. and mitochondrial calcium at multiple time points (Fig. 3 C–E). These results thus suggest that TRPML1 activity is important for Lysosomal TRPML1 Specifically Modulates Mitochondrial Calcium and modulating mitochondrial calcium dynamics. Mitochondria–Lysosome Contact Dynamics. To further demonstrate In order to investigate the role of TRPML1 at contact sites, we that lysosomal TRPML1 activity modulates mitochondrial cal- analyzed whether TRPML1 specifically modulated mitochon- cium, we expressed either wild-type TRPML1 (TRPML1 WT- drial calcium at mitochondria–lysosome contact sites. HeLa cells Halo) or the dominant-negative, nonconducting TRPML1 pore expressing wild-type TRPML1 displayed a significantly higher mutant (TRPML1 D471K-Halo) in HeLa cells and examined increase in mitochondrial calcium after TRPML1 activation for mitochondrial calcium dynamics (Mito-R-GECO1) upon ML- mitochondria that were in contact with lysosomes, compared to SA1 treatment. We first confirmed that both wild-type TRPML1 mitochondria not in contact with lysosomes (Fig. 3F). In con- and dominant-negative TRPML1 were localized to the lyso- trast, this difference in contact-dependent calcium transfer was somal/late-endosomal compartment as evidenced by colocaliza- entirely abolished in cells expressing the dominant-negative tion with the lysosomal membrane marker, BFP-lysosomes (SI TRPML1 mutant (Fig. 3F). To probe downstream effects of Appendix, Fig. S5 A and B), and that both wild-type and calcium transfer at mitochondria–lysosome contacts, we investi- dominant-negative TRPML1 were expressed at similar levels (SI gated whether TRPML1-mediated mitochondrial calcium influx pro- Appendix, Fig. S5C). Upon ML-SA1 treatment, cells expressing moted mitochondrial permeability transition pore (mPTP) opening,

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Fig. 3. Lysosomal TRPML1 specifically modulates mitochondrial calcium and mitochondria–lysosome contact dynamics. (A and B) Mitochondrial calcium response in live HeLa cells expressing mitochondrial-matrix targeted calcium sensor, Mito-R-GECO1, and either wild-type or dominant-negative (D471K) TRPML1 mutant, in response to TRPML1 activation with ML-SA1 (31.25 μM) at t = 0 s with representative time-lapse confocal images (A, n = 20 cells for each condition) and mitochondrial calcium traces (ΔF/F) (B, n = 20 cells for each condition). (Scale bars, 10 μm.) (C–E) Quantification of maximum mitochondrial calcium response (C), mean mitochondrial calcium response (D), and mitochondrial calcium response at 30, 60, 90, and 120 s (E) after TRPML1 activation with ML-SA1 (31.25 μM) in live HeLa cells expressing TRPML1 wild-type or TRPML1 D471K mutant from confocal time-lapse images (from A)(n = 20 cells for each condition). (F) Quantification of mitochondrial calcium responses of mitochondria in contact and not in contact with lysosomes following TRPML1 activation with ML-SA1 (31.25 μM) in live HeLa cells expressing mitochondrial matrix calcium sensor, Mito-R-GECO1, lysosomal marker, lamp1-mGFP, and either wild- type (TRPML1 WT-pHcRed) or dominant-negative (TRPML1 D471-472K-pHcRed) TRPML1 mutant (n = 100 events from 20 cells for each condition). (G–I) Quantification of percentage of lysosomes contacting mitochondria (for >10 s; G, n = 10 cells for each condition) and minimum duration of mitochondria–lysosome contacts with corresponding histogram (H and I, n = 70 events from 10 cells for each condition) in live HeLa cells expressing mitochondrial outer membrane marker, Tom20-mEmerald, and lysosomal marker, BFP-lysosomes along with either wild-type (TRPML1 WT-pHcRed) or dominant- negative (TRPML1 D471-472K-pHcRed) TRPML1 mutant. Data are means ± SEM (*P < 0.05, **P < 0.01, ****P < 0.0001, ns, not significant, unpaired two-tailed t test).

Peng et al. PNAS | August 11, 2020 | vol. 117 | no. 32 | 19269 Downloaded by guest on September 25, 2021 which can be assessed by the quenching of mitochondrial-localized The MCU is the major transporter of calcium across the inner calcein in the presence of CoCl2 (SI Appendix,Fig.S6A). In mitochondrial membrane into the mitochondrial matrix (68, 69). contrast to ionomycin treatment, which induced mPTP opening To evaluate the role of the MCU in uptake of lysosomal calcium and rapid quenching of calcein, neither the TRPML1 agonist ML- across the inner mitochondrial membrane, we expressed either SA1 nor vehicle control reduced mitochondrial calcein fluores- wild-type MCU or MCU mutant (E264A), which disrupts cal- cence (SI Appendix, Fig. S6 B and C), suggesting that TRPML1- cium uptake (69, 70), and first confirmed that their expression mediated mitochondrial calcium influx does not induce sustained levels were similar (SI Appendix, Fig. S7G). We then examined mPTP opening. We also investigated whether TRPML1-mediated mitochondrial calcium dynamics (Mito-R-GECO1) in these cells mitochondrial calcium influx activated apoptotic pathways via in response to TRPML1 activation. Compared to cells expressing endogenous cytochrome C staining (SI Appendix,Fig.S6D). Rel- wild-type MCU, cells expressing mutant MCU had reduced total ative to staurosporine, which induced release of cytochrome C mitochondrial calcium increase upon TPRML1 activation from the mitochondria into the cytosol, we did not observe sig- (Fig. 4F and SI Appendix, Fig. S7E), as well as significantly lower nificant changes in mitochondrial distribution of cytochrome C in maximum mitochondrial calcium, mean mitochondrial calcium, cells treated with TRPML1 agonist ML-SA1 or vehicle control at and mitochondrial calcium at multiple time points after ML-SA1 multiple time points (SI Appendix,Fig.S6E–G). treatment (Fig. 4 G–I). Importantly, the MCU was also impor- In addition to modulating contact-dependent calcium transfer, tant for mitochondria–lysosome contact-dependent calcium – we found that TRPML1 modulated mitochondria lysosome con- transfer. Wild-type MCU-expressing cells showed significant tact site dynamics. In cells expressing the dominant-negative differences in calcium influx for mitochondria in contact with TRPML1 mutant, there was both a higher percentage of lyso- lysosomes compared to mitochondria not in contact, while ex- G somes in stable contact with mitochondria (Fig. 3 ) and a sig- pression of the MCU mutant (E264A) completely abolished this – nificant increase in minimum duration of mitochondria lysosome difference (Fig. 4J). These results indicate that MCU on the H I contacts (Fig. 3 and ). We further verified whether TRPML1 inner mitochondrial membrane modulates mitochondrial cal- – localized preferentially to mitochondria lysosome contacts in cells cium dynamics at mitochondria–lysosome contact sites. expressing HA-tagged TRPML1 and an HA-mCherry nanobody. SI Ap- TRPML1-HA puncta marked a subset of contact sites ( Loss of TRPML1 Function in MLIV Patient Fibroblasts Disrupts Mitochondria pendix A ,Fig.S7 ) and TRPML1-HA localization at the contact –Lysosome Contact and Calcium Dynamics. Loss-of-function mutations SI sites was significantly greater than expected by random chance ( in TRPML1 cause the autosomal recessive lysosomal storage dis- Appendix B ,Fig.S7 ). These findings highlight a role for TRPML1 order, MLIV (47, 48), which has been associated with both lyso- – in regulating mitochondria lysosome contact site dynamics and somal (50–54) and mitochondrial aberrations (45, 46). Given that contact-dependent calcium transfer into mitochondria. we found TRPML1 to be important for the regulation of mitochondrial calcium dynamics via direct transfer of calcium at VDAC1 and MCU Mediate Mitochondrial Uptake of Lysosomal Calcium mitochondria–lysosome contact sites, we evaluated whether MLIV at Mitochondria–Lysosome Contact Sites. Having shown that patient fibroblasts had defective mitochondrial calcium dynamics TRPML1-mediated lysosomal calcium release led to increased due to loss of TRPML1 function. We treated fibroblasts from mitochondrial calcium at mitochondria–lysosome contacts, we MLIV patients and age-matched healthy controls with ML-SA1 to next sought to identify the mitochondrial components promoting activate TRPML1 and examined mitochondrial calcium dynamics uptake of lysosomal calcium. VDACs on the outer mitochondrial (Mito-R-GECO1). While control fibroblasts showed a significant membrane have been implicated in mitochondrial calcium uptake (59–64) and specifically, VDAC1 was previously identified increase in total mitochondrial calcium upon TRPML1 activation, this was reduced in multiple MLIV patient fibroblast lines as a potential interactor of lysosomal TRPML1 (65). To first A confirm the interaction of TRPML1 with VDAC1, we conducted (Fig. 5 ). Consistent with these findings, maximum mitochondrial coimmunoprecipitation experiments in cells expressing TRPML1 calcium, mean mitochondrial calcium, and mitochondrial calcium at 30 s was also significantly decreased in MLIV patient lines -GFP and found that TRPML1 interacted with endogenous B–D VDAC1 (SI Appendix, Fig. S7C), but not endogenous VDAC2 or (Fig. 5 ). Moreover, MLIV patient fibroblasts showed defects VDAC3 (SI Appendix, Fig. S7C). We next investigated whether in contact-dependent calcium transfer. In control fibroblasts, mi- VDAC1 is important for the mitochondrial uptake of lysosomal tochondria in contact with lysosomes showed a significantly higher calcium by assessing mitochondrial calcium dynamics upon increase in calcium influx following TRPML1 activation compared TRPML1 activation in cells expressing either wild-type human to those not in contact with lysosomes. In contrast, there was no VDAC1 or a VDAC1 mutant (E73Q) with a single amino acid difference in calcium influx between mitochondria in and not in E substitution in a putative calcium-binding site (66, 67). In re- contact with lysosomes in MLIV fibroblasts (Fig. 5 ). sponse to TRPML1 activation with ML-SA1, cells expressing the In addition to changes in contact-dependent mitochondrial VDAC1 mutant showed significantly lower increase in mito- calcium responses, MLIV fibroblasts also displayed abnormal chondrial calcium (Mito-R-GECO1) (Fig. 4A and SI Appendix, mitochondria–lysosome contact dynamics. Interestingly, MLIV Fig. S7D), as well as significantly decreased maximum mito- fibroblasts had a significantly increased percentage of lysosomes chondrial calcium, mean mitochondrial calcium, and mitochon- in stable contact (>10 s) with mitochondria compared to control drial calcium at multiple time points compared to wild-type cells fibroblasts (Fig. 5F) and the duration of mitochondria–lysosome (Fig. 4 B–D). After verifying that wild-type and mutant VDAC1 contact tethering was also significantly prolonged (Fig. 5G). were expressed at similar levels (SI Appendix, Fig. S7F), we next Together, these data suggest that loss of TRPML1 function in assessed whether VDAC1 regulated calcium transfer preferentially at MLIV may contribute to disease pathogenesis by dysregulating mitochondria–lysosome contact sites. Mitochondria in contact with mitochondrial calcium dynamics at contact sites and additionally lysosomes had significantly elevated calcium influx after TRPML1 disrupting mitochondria–lysosome contact tethering dynamics. activation, compared to mitochondria not in contact with lysosomes We thus propose a model in which TRPML1-mediated lyso- in cells expressing wild-type VDAC1. In contrast, there was no dif- somal calcium efflux results in mitochondrial calcium influx ference in mitochondrial calcium response between mitochondria in preferentially at mitochondria–lysosome contacts through the and not in contact with lysosomes in VDAC1 mutant-expressing cells mitochondrial channels VDAC1 and the MCU, and that calcium (Fig. 4E). These findings thus suggest that VDAC1 on the outer transfer at mitochondria–lysosome contact sites is consequently mitochondrial membrane serves as a mediator of mitochondrial up- disrupted in the lysosomal storage disorder MLIV due to take of lysosomal calcium at mitochondria–lysosome contacts. loss-of-function TRPML1 mutations (Fig. 5H).

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Fig. 4. VDAC1 and the MCU modulate mitochondrial uptake of lysosomal calcium at mitochondria–lysosome contact sites. (A) Mitochondrial calcium response (ΔF/F) in live HeLa cells expressing mitochondrial-matrix targeted calcium sensor, Mito-R-GECO1, and either wild-type or mutant (E73Q) VDAC1 in response to TRPML1 activation with ML-SA1 (31.25 μM) at t = 0s(n = 22 cells for each condition). (B–D) Quantification of maximum mitochondrial calcium response (B), mean mitochondrial calcium response (C), and mitochondrial calcium response at 30, 60, 90, and 120 s (D) after TRPML1 activation with ML-SA1 (31.25 μM) in live HeLa cells expressing VDAC1 WT or VDAC1 E73Q mutant from confocal time-lapse images (from A)(n = 22 cells for each condition). (E) Quantification of mitochondrial calcium responses of mitochondria in contact and not in contact with lysosomes following TRPML1 activation with ML-SA1 (31.25 μM) in live HeLa cells expressing mitochondrial matrix calcium sensor, Mito-R-GECO1, lysosomal marker, lamp1-mGFP, and either wild-type or mutant (E73Q) VDAC1 (n = 100 events from 20 cells for each condition). (F) Mitochondrial calcium response (ΔF/F) in live HeLa cells expressing mitochondrial-matrix targeted calcium sensor, Mito-R-GECO1, and either wild-type or mutant (E264A) MCU in response to TRPML1 activation with ML-SA1 (31.25 μM) at t = 0s(n = 20 cells for each condition). (G–I) Quantification of maximum mitochondrial calcium response (G), mean mitochondrial calcium response (H), and mitochondrial calcium response at 30, 60, 90, and 120 s (I) after TRPML1 activation with ML-SA1 (31.25 μM) in live HeLa cells expressing MCU WT or MCU E264A mutant from confocal time-lapse images (from F)(n = 20 cells for each condition). (J) Quantification of mitochondrial calcium responses of mitochondria in contact and not in contact with lysosomes following TRPML1 activation with ML-SA1 (31.25 μM) in live HeLa cells expressing mitochondrial matrix calcium sensor, Mito-R-GECO1, lysosomal marker, lamp1-mGFP, and either wild-type or mutant (E264A) MCU (n = 100 events from 20 cells for each condition). Data are means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, unpaired two-tailed t test).

Discussion sites. TRPML1-mediated increase in mitochondrial calcium is We identified a role of mitochondria–lysosome contact sites in further modulated by VDAC1 and the MCU on the outer and modulating intracellular calcium dynamics, whereby TRPML1- inner mitochondrial membranes, respectively. Importantly, we mediated lysosomal calcium efflux leads to mitochondrial cal- show that mitochondrial calcium dynamics are disrupted in the cium influx preferentially at mitochondria–lysosome contact lysosomal storage disorder MLIV, which results from loss of

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0.5 50 10 % of Lysosomes in Contact % of Lysosomes with Mitochondria (> 10 sec) (normalized to Not in Contact) 0.0 0 Minimum Duration of Contact (s) 0 Con #1 Con #2 Con #3 MLIV #1 MLIV #2 MLIV #3

Con Con#1 Con#2 #3 MLIVMLIV #1MLIV #2 #3 Con Con#1 Con#2MLIV #3MLIV #1MLIV #2 #3

Fig. 5. Loss of TRPML1 function in MLIV patient fibroblasts disrupts mitochondria–lysosome contact and calcium dynamics. (A) Mitochondrial calcium response (ΔF/F) in fibroblasts from MLIV patients (MLIV #1, MLIV #2, MLIV #3) or age-matched healthy controls (Con #1, Con #2, Con #3) expressing mitochondrial-matrix–targeted calcium sensor, Mito-R-GECO1 in response to TRPML1 activation with ML-SA1 (31.25 μM) at t = 0s(n = 20 cells for each condition). (B–D) Quantification of maximum mitochondrial calcium response (B), mean mitochondrial calcium response (C), and mitochondrial calcium response at 30 s (D) after TRPML1 activation with ML-SA1 (31.25 μM) from fibroblasts from MLIV patients and controls (from A)(n = 20 cells for each condition). (E) Quantification of mitochondrial calcium responses of mitochondria in contact and not in contact with lysosomes following TRPML1 activation with ML-SA1 (31.25 μM) in fibroblasts from MLIV patients and controls expressing mitochondrial matrix calcium sensor, Mito-R-GECO1, and lysosomal marker, lamp1-mGFP (n = 100 events from 20 cells for each condition). (F and G) Quantification of percentage of lysosomes contacting mitochondria (for >10 s; F, n = 10 cells for each condition) and duration of mitochondria–lysosome contacts (G, n = 70 events from 10 cells for each condition) in fibroblasts from MLIV patients and controls expressing mitochondrial outer membrane marker, Tom20-mApple, and lysosomal marker, lamp1-mGFP. (H) Model of the regulation of calcium dynamics at mitochondria–lysosome contacts showing calcium transfer from lysosomes to mitochondria via TRPML1 (lysosome), VDAC1 (outer mitochondrial membrane), and MCU (inner mitochondrial membrane) leading to increased mitochondrial calcium in healthy cells (Left). In the lysosomal storage disorder MLIV, loss-of-function TRPML1 mutations lead to dysregulation of mitochondrial calcium dynamics (Right). Data are means ± SEM [*P < 0.05, ***P < 0.001, ****P < 0.0001, ns, not significant, one-way ANOVA with Tukey’s post hoc test (B, C, D, F, and G), unpaired two-tailed t test (E)].

TRPML1 function. We additionally find that altered mito- Our work further establishes the growing importance of chondrial calcium dynamics in MLIV are dependent on interorganelle contact sites in the regulation of cellular homeo- mitochondria–lysosome contacts, providing further evidence stasis. Defects in interorganelle contact sites have been impli- for the convergence of lysosomal and mitochondrial dysfunc- cated in multiple human diseases, including lysosomal storage tion in disease. disorders (6), peroxisomal diseases (71), and neurodegenerative

19272 | www.pnas.org/cgi/doi/10.1073/pnas.2003236117 Peng et al. Downloaded by guest on September 25, 2021 disorders (27, 72–75). Recently, mitochondria–lysosome contact Indeed, prolonged mitochondria-lysosome contacts have also been sites have been shown to be an important regulator of mito- observed in other disorders affecting lysosomal/late endosomal chondrial and lysosomal cross-talk independent of lysosomal genes including Charcot-Marie Tooth type 2B (27). It would be degradation of mitochondria (8, 9, 11–18), and to be involved in important in future studies to elucidate whether altered organelle regulating mitochondrial fission and intermitochondrial contact contacts in MLIV contribute to previously observed mitochondrial untethering (8, 16, 27), as well as the transfer of metabolites, phenotypes and whether MLIV mutations in TRPML1 alter addi- such as cholesterol (10). Here, we find that mitochondria– tional mitochondrial functions, such as ATP production and mito- lysosome contact sites further play a key role in regulating cal- chondrial fission/fusion dynamics in a contact-dependent manner. cium transfer between these two organelles, which is disrupted in Given that many critical mitochondrial functions are regulated by a lysosomal storage disorder. Uncovering the diverse functions of calcium (83), it is possible that dysregulation of mitochondrial cal- – mitochondria lysosome contact sites shall inform our under- cium, in conjunction with decreased lysosomal function, potentiates standing of how contact sites contribute to both physiological defects in mitochondrial metabolism and dynamics, which may and pathophysiological states. consequently contribute to downstream phenotypes, such as mito- Our findings also expand upon the previously described physi- chondrial fragmentation in MLIV disease pathogenesis. ological roles of TRPML1. TRPML1 is a nonselective cation – In addition to its role in MLIV, TRPML1 has also been im- channel that mediates lysosomal calcium efflux (33 38) and regu- plicated in various neurological and lysosomal storage diseases lates various lysosomal functions, including lysosomal exocytosis, (19, 90–93). Several disease models have shown down-regulation membrane trafficking, and lysosomal biogenesis (39, 41, 43, 56, of TRPML1, which impairs lysosomal function and promotes 76–78). Our data suggest that in addition to regulating lysosomal accumulation of toxic proteins (94, 95). Other studies have dynamics and function, TRPML1 directly impacts mitochondrial reported misregulation of TRPML1 activity due to alterations in homeostasis by modulating mitochondrial calcium dynamics via lipids or lysosomal pH (83, 89). TRPML1 activity is highly reg- mitochondria–lysosome contact sites. Mitochondria–lysosome ulated by specific lipids, including its endogenous activator contacts may act as platforms to provide localized pockets of high calcium concentration required for influx into mitochondria and, PI(3,5)P2 (57), which has been suggested to be misregulated in Charcot-Marie Tooth disease and amyotrophic lateral sclerosis moreover, contact-dependent transfer of calcium from lysosomes – to mitochondria may serve as a mechanism to spatially regulate (96 98), and sphingomyelins and cholesterol, which when accu- calcium transfer to a subset of mitochondria to facilitate down- mulated in Niemann-Pick type C, impair TRPML1-mediated stream, calcium-dependent mitochondrial functions, including ox- lysosomal calcium release (54). Moreover, TRPML1 activity is idative phosphorylation, motility, and reactive oxygen species likely regulated upstream by lysosomal pH as it has been shown CELL BIOLOGY (ROS) signaling (79–83). Indeed, mitochondrial function has also that TRPML1-mediated calcium dyshomeostasis and autophagic been shown to reciprocally regulate TRPML1 as increased mito- defects are rescued by restoration of lysosomal pH, but not chondrial ROS potentiates TRPML1 activity (77). Further studies calcium, in an Alzheimer’s model (19). While these previous investigating how TRPML1-mediated mitochondrial calcium influx studies predominantly describe the role of TRPML1 in regulat- modulates mitochondrial structure, dynamics, and function will ing lysosomal function in disease (43, 54, 76, 92), our findings provide additional insights into the direct communication between suggest that TRPML1 may also contribute to disease patho- lysosomes and mitochondria. genesis by modulating mitochondrial calcium dynamics. Altered In our study, we also identified VDAC1 on the outer mitochon- TRPML1-mediated calcium transfer at mitochondria–lysosome drial membrane and the MCU on the inner mitochondrial membrane contact sites and subsequent dysregulation of mitochondrial as mediators of mitochondrial calcium influx at mitochondria calcium dynamics may be an additional contributory mechanism –lysosome contact sites. While our finding that TRPML1 preferen- to pathophysiology. Indeed, many of these diseases share cellular tially interacts with VDAC1 and not other VDAC isoforms is con- hallmarks including mitochondrial and lysosomal dysfunction sistent with previous studies (65), it is possible that the other VDAC and calcium dyshomeostasis (23–26). Importantly, TRPML1 has isoforms, which have also been implicated in mitochondrial calcium recently emerged as a potential therapeutic target for the uptake (62–64), may play a role in contact-dependent calcium treatment of neurodegenerative and lysosomal storage disorders. transfer in other cell types. In addition, while we observed that Studies suggest that TRPML1 agonists may act multimodally by mutant MCU impaired mitochondrial calcium uptake following activating various lysosomal pathways, including autophagy and TRPML1-mediated lysosomal calcium release, it did not completely lysosomal exocytosis (43, 54, 92). Thus, our results further abolish mitochondrial uptake at contact sites. Thus, our results sug- highlight a potential role for therapeutically targeting TRPML1 gest that there may be alternative, MCU-independent mechanisms of in modulating mitochondrial calcium dynamics in disease. – calcium influx (83) into the mitochondrial matrix at mitochondria In summary, our work shows that lysosomes can directly lysosome contacts. Indeed, prior studies have proposed additional transfer calcium to mitochondria at mitochondria–lysosome con- – transporters mediating mitochondrial matrix calcium influx (84 87), tacts, thereby further supporting the emerging role for both or- which may also play a role in the uptake of lysosomal calcium. ganelles as critical players in modulating calcium dynamics Of clinical relevance, loss-of-function mutations in TRPML1 (20–22) and elucidating an additional pathway by which intracel- cause the autosomal-recessive lysosomal storage disorder MLIV, lular calcium can be regulated. A broader understanding of the which is characterized by psychomotor retardation, retinal de- – mechanisms underlying intracellular calcium regulation will ulti- generation, and neurodevelopmental delay (33, 47 49). Although mately inform our understanding of the role of mitochondrial and the pathophysiology of MLIV remains unclear, various cellular lysosomal cross-talk in both cellular homeostasis and disease. phenotypes, including defective lysosomal biogenesis, altered lysosomal pH, impaired autophagy, and mitochondrial fragmenta- Materials and Methods tion have been described (43, 45, 46, 50, 76, 77, 88, 89). Notably, Detailed materials and methods are included in SI Appendix, Materials our results demonstrate that MLIV is also associated with defec- and Methods. tive mitochondria–lysosome contact dynamics and contact- dependent calcium transfer. As increasing mitochondria-lysosome Cell Culture and Transfection. HeLa, HEK293, and HCT116 cells and fibroblasts

contact duration in healthy cells increases contact-dependent cal- were cultured according to standard procedures at 37 °C in a 5% CO2 in- cium transfer, it is possible that prolonged mitochondria-lysosome cubator. Cells were transfected with either X-tremeGENE HP DNA transfec- contacts in MLIV compensate for reduced calcium transfer from tion reagent (Roche; XTGHP-RO) or Lipofectamine LTX with PLUS reagent lysosomes to mitochondria due to loss of TRPML1 function. (Invitrogen; 15338100).

Peng et al. PNAS | August 11, 2020 | vol. 117 | no. 32 | 19273 Downloaded by guest on September 25, 2021 Live-Cell Confocal Microscopy. All confocal images were acquired on a Nikon Data Availability. All data that support the findings of this study are included A1R laser scanning confocal microscopy with GaAsp detectors using a Plan Apo in the article or are available at the Open Science Framework, https://osf.io/ λ 100× 1.45 NA oil-immersion objective (Nikon) using NIS-Elements (Nikon). wf83s/. Live cells were imaged in a temperature-controlled chamber (37 °C) at 5% CO2. – – Dual color videos were acquired as consecutive green red or blue green im- ACKNOWLEDGMENTS. We thank all members of the D.K. laboratory for ages. For calcium imaging, growth media was replaced with Krebs-Ringer advice. All imaging was performed at the Northwestern University Center solution (without calcium) (Boston BioProducts; BSS-255) immediately prior for Advanced Microscopy, supported by National Cancer Institute CCSG P30 to imaging and live cells were imaged at one frame every 1 to 5 s. CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. HCT116 wild-type and TBC1D15 knockout cells were gifts from R. Youle. This Image Analysis. Calcium responses for total mitochondria and contact-dependent work was supported by the following: NIH NINDS (National Institute of mitochondrial calcium responses were assessed using ΔF/F0 analysis. Analysis of Neurological Disorders and Stroke) and NIA (National Institute of Aging) mitochondria–lysosome contact dynamics were analyzed as previously described grants F30 AG066333 (to W.P.), K99 NS109252 (to Y.C.W.), and R01 NS076054 (8). All image analysis was conducted using ImageJ and NIS-Elements (Nikon). and R37NS096241 (to D.K.).

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