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

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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 dy- namics. 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
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