The mitochondrial Ca 2+ uptake regulator, MICU1, is involved in cold stress‐induced ferroptosis

Report6 April 2021free access Source DataTransparent process The mitochondrial Ca2+ uptake regulator, MICU1, is involved in cold stress-induced ferroptosis Toshitaka Nakamura Toshitaka Nakamura orcid.org/0000-0002-4248-2058 Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Motoyuki Ogawa Motoyuki Ogawa Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kazuki Kojima Kazuki Kojima Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Saki Takayanagi Saki Takayanagi Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Shunya Ishihara Shunya Ishihara Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kazuki Hattori Kazuki Hattori orcid.org/0000-0002-5592-4257 Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Isao Naguro Corresponding Author Isao Naguro [email protected] orcid.org/0000-0002-0338-8026 Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hidenori Ichijo Corresponding Author Hidenori Ichijo [email protected] orcid.org/0000-0002-5005-6438 Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Toshitaka Nakamura Toshitaka Nakamura orcid.org/0000-0002-4248-2058 Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Motoyuki Ogawa Motoyuki Ogawa Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kazuki Kojima Kazuki Kojima Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Saki Takayanagi Saki Takayanagi Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Shunya Ishihara Shunya Ishihara Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kazuki Hattori Kazuki Hattori orcid.org/0000-0002-5592-4257 Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Isao Naguro Corresponding Author Isao Naguro [email protected] orcid.org/0000-0002-0338-8026 Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hidenori Ichijo Corresponding Author Hidenori Ichijo [email protected] orcid.org/0000-0002-5005-6438 Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Author Information Toshitaka Nakamura1, Motoyuki Ogawa1, Kazuki Kojima1, Saki Takayanagi1, Shunya Ishihara1, Kazuki Hattori1, Isao Naguro *,1 and Hidenori Ichijo *,1 1Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan *Corresponding author. Tel: +81 3 5841 4858; E-mail: [email protected] *Corresponding author. Tel: +81 3 5841 4859; E-mail: [email protected] EMBO Reports (2021)22:e51532https://doi.org/10.15252/embr.202051532 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Ferroptosis has recently attracted much interest because of its relevance to human diseases such as cancer and ischemia-reperfusion injury. We have reported that prolonged severe cold stress induces lipid peroxidation-dependent ferroptosis, but the upstream mechanism remains unknown. Here, using genome-wide CRISPR screening, we found that a mitochondrial Ca2+ uptake regulator, mitochondrial calcium uptake 1 (MICU1), is required for generating lipid peroxide and subsequent ferroptosis under cold stress. Furthermore, the gatekeeping activity of MICU1 through mitochondrial calcium uniporter (MCU) is suggested to be indispensable for cold stress-induced ferroptosis. MICU1 is required for mitochondrial Ca2+ increase, hyperpolarization of the mitochondrial membrane potential (MMP), and subsequent lipid peroxidation under cold stress. Collectively, these findings suggest that the MICU1-dependent mitochondrial Ca2+ homeostasis-MMP hyperpolarization axis is involved in cold stress-induced lipid peroxidation and ferroptosis. SYNOPSIS This study identifies MICU1 as a key regulator for lipid peroxidation and subsequent ferroptosis under cold stress, suggesting that MICU1 can be a potential target for preventing cell death in organ preservation. Genome-wide CRISPR screening reveals that MICU1 is required for cold stress-induced ferroptosis. MICU1 deficiency attenuates lipid peroxidation via suppressing hyperpolarization of the mitochondrial membrane potential and mitochondrial Ca2 + uptake induced by cold stress. Intracellular Ca2 + increase, partly through TRPM8, plays important roles in cold stress-induced ferroptosis. Introduction Ferroptosis, an iron-dependent and lipid peroxidation-induced regulated necrosis, has been defined (Dixon et al, 2012) and has received much attention because of its relevance to various diseases, such as cancer, neurodegeneration, and ischemia-reperfusion injury (Angeli et al, 2017; Stockwell et al, 2017; Friedmann Angeli et al, 2019; Nakamura et al, 2019). Ferroptosis is caused by the accumulation of reactive oxygen species (ROS), particularly lipid ROS. In addition, it has recently been proposed that in cysteine deprivation-induced ferroptosis, the mitochondrial membrane potential (MMP) is hyperpolarized associated with lipid ROS accumulation (Gao et al, 2019). Upon lipid ROS accumulation, glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1, also known as AIFM2) play important roles in scavenging lipid ROS and preventing ferroptosis (Yang et al, 2014; Ingold et al, 2018; Bersuker et al, 2019; Doll et al, 2019). Therefore, inhibitors targeting GPX4 or FSP1 and reagents disrupting the cellular metabolism of their substrates, e.g., glutathione or coenzyme Q10, are known to induce ferroptosis (Dixon et al, 2012; Yang et al, 2014; Shimada et al, 2016; Doll et al, 2019). Without using these artificial inducers, we have recently reported that cold stress also induces lipid ROS-dependent ferroptosis (Hattori et al, 2017), but the molecular mechanisms by which cold stress leads to accumulated lipid ROS remain largely unknown. Changes in extracellular temperature affect various cellular functions, such as proteostasis, metabolism, and membrane fluidity. Cold stress-induced tissue damage during organ preservation is an example of clinical environment. Recently, some organ preservation methods have been developed, including a machine perfusion system (Jing et al, 2018) and preservation solution supplemented with iron chelators (Lautenschläger et al, 2018). However, there are few drugs targeting the molecules or mechanisms of cold stress-induced tissue damage. We have reported that severe cold stress drives the lipid ROS accumulation and subsequent activation of the ASK1-p38 pathway, which culminates in ferroptosis (Hattori et al, 2017). In this study, using genome-wide CRISPR screening, we identified mitochondrial calcium uptake 1 (MICU1), as one of the regulators of ferroptosis under cold stress. MICU1 has been characterized as an essential molecule for gatekeeping and activating mitochondrial calcium uniporter (MCU) during mitochondrial Ca2+ uptake (Perocchi et al, 2010; Mallilankaraman et al, 2012; Csordás et al, 2013; Kamer & Mootha, 2014; Patron et al, 2014). We found that some domains, which have been shown to be necessary for MICU1-dependent MCU activation, are also required for cold stress-induced lipid ROS accumulation and subsequent cell death. Moreover, as observed in typical ferroptosis induced by cysteine deprivation (Gao et al, 2019), MMP was hyperpolarized with mitochondrial Ca2+ influx under cold stress. Collectively, we demonstrate that MICU1-dependent mitochondrial Ca2+ uptake and the resultant MMP hyperpolarization facilitate lipid ROS accumulation, which plays a central role in cold stress-induced ferroptosis. Results and Discussion MICU1 deficiency suppresses cold stress-induced ferroptosis To identify the genes required for cold stress-induced ferroptosis, we performed a genome-wide CRISPR screening (Fig 1A) of A549 cells stably expressing Cas9 (Cas9-A549 cells). We chose A549 cells because cold stress-induced ferroptosis of A549 cells was readily reproducible and well characterized (Hattori et al, 2017). The cells were infected with an extremely low infection ratio (MOI = 0.08) with a lentivirus-based GeCKOv2 library (Sanjana et al, 2014; Shalem et al, 2014). The cells were divided into two groups after puromycin selection for 3 days; then, cold stress was applied for 30 h and repeated twice (for details, see methods). Then, the surviving cells were harvested, and genomic DNA was deep-sequenced to obtain reads counts for each sgRNA. The reads counts were analyzed by the MAGeCK program (Li et al, 2014). Evaluating by a beta score, a well-known ferroptosis inducer (ACSL4) and ferroptosis suppressors (GPX4, AIFM2, and SLC7A11) were found outside the ± 2 × SD range as genes required for cell death and cell survival, respectively (Fig EV1A). Figure 1. MICU1 deficiency suppresses cold stress-induced ferroptosis A. Schematic model showing the design of the screening. B. MICU1 was identified using the MAGeCK program with a robust ranking aggregation (RRA) score. C. Immunoblots of endogenous MICU1 for validating gene knockout. *Nonspecific signals. D. LDH assay was performed after 24 h of cold stress on ice. Data are presented as mean ± SEM; n = 3, biological replicates. ****P < 0.0001, ***P < 0.001 by one-way ANOVA followed by Dunnett’s multiple comparison test. E,F. Lipid peroxidation was measured by C11-BODIPY 581/591 after cold stress for 5 h. Representative plots are shown in (E), and the data (F) are presented as mean ± SEM; n = 4–6, biological replicates. ****P < 0.0001, *** P < 0.001 by two-way ANOVA followed by Tukey’s multiple comparison test Source data are available online for this figure. Source Data for Figure 1 [embr202051532-sup-0005-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Analysis of screening, and mitochondria itself is involved in cold stress-induced ferroptosis, related to Fig 1 Some genes are described as ferroptosis inducer or suppressors for the adequate evaluation of this screening. A well-known ferroptosis inducer, acyl-CoA synthetase long-chain family member 4 (ACSL4) and ferroptosis suppressors, ferroptosis suppressor 1 (FSP1 also known as AIFM2), glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11), are highlighted after analyzing the count data from CRISPR screening using the MAGeCK program. Red and blue genes are outside of the 2 × SD range and candidates of ferroptosis inducers and suppressors, respectively, which are enriched in MAGeCKFlute packages. Results of Gene Ontology (GO) analysis with biological process. After analysis of count data using MAGeCK, positive genes (cut-off of P < 0.01, see also Dataset EV1) were analyzed by GO analysis using clusterProfiler packages for R. Lipid peroxidation was measured by C11-BODIPY 581/591 after cold stress for 5 h. Representative density plot is presented as reported previously (Hattori et al, 2017). Deferoxamine (DFO, 200 µM) and ferrostatin-1 (Fer-1, 1 µM) were pretreated for 30 min before cold stress. Download figure Download PowerPoint Evaluated by a robust ranking aggregation (RRA) score using the MAGeCK program, the genes required for cold stress-induced cell death were determined on the basis of the requirements for a P < 0.01 (Dataset EV1). When these genes were analyzed by GO analysis (Yu et al, 2012), mitochondria-related genes were found to be highly enriched (Fig EV1B). Although a recent report suggests that the mitochondria play an important role in ferroptosis (Gao et al, 2019), the molecular mechanisms remained largely unknown. In terms of mitochondrial function and high RRA score, we were interested in MICU1 (Fig 1B). MICU1 was reported to be involved in apoptosis (Mallilankaraman et al, 2012; Csordás et al, 2013; Hall et al, 2014) but not necrosis. MICU1-knockout single clones were established by CRISPR/Cas9 knockout using two different sgRNAs (Fig 1C). Wild-type (WT) A549 cells and MICU1-knockout cells were exposed to sustained cold stress for 24 h, and cell death was measured by lactate dehydrogenase (LDH) release (Fig 1D). MICU1 deficiency attenuated cold-induced cell death. Moreover, MICU1 knockdown by siRNA also reduced the rate of cell death induced by cold stress (Fig EV2A and B). Click here to expand this figure. Figure EV2. MCU deficiency does NOT suppress cold stress-induced ferroptosis, related to Figures 1 and 2 Immunoblots of endogenous MICU1 or MCU signals after siRNAs transfection in the WT A549 cells. LDH release was measured after cold stress for 24 h. Data are presented as mean ± SEM; n = 4 (B), biological replicates, **P < 0.01, n.s.: not significant, one-way ANOVA followed by Dunnett’s test. Components of the MCU complex (MCU, MCUR1, MCUb (CCDC109B), MICU1, MICU2, MICU3, and EMRE (SMDT1)) are highlighted in red in the RRA score plot calculated by the MAGeCK program. Immunoblots of endogenous MCU signals after Cas9/sgRNA lentivirus infection in the WT A549 cells. LDH release was measured after cold stress for 24 h. Data are presented as mean ± SEM; n = 3, biological replicates, n.s.: not significant, one-way ANOVA followed by Dunnett’s test. Immunoblots of endogenous MICU1 or MCU signals after siRNAs transfection in the WT A549 cells and MICU1-KO cells. LDH release was measured after cold stress for 24 h. Data are presented as mean ± SEM; n = 3, biological replicates, ****P < 0.0001, *P < 0.05, one-way ANOVA followed by Tukey’s test. Immunoblots of endogenous and overexpressing MCU signals after MCU-HA lentivirus infection in the WT A549 cells. LDH release was measured after cold stress for 24 h. Data are presented as mean ± SEM; n = 3, biological replicates, n.s.: not significant, unpaired t-test. Schematic model for MICU1 and MCU expression and its effect on cell death induced by cold stress. LDH release was measured after cold stress for 16 h. Data are presented as mean ± SEM; n = 3, biological replicates, n.s.: not significant, one-way ANOVA followed by Dunnett’s test. MICU2 mRNA was quantified by RT–qPCR. Data are presented as mean ± SEM; n = 3, biological replicates, ****P < 0.0001, one-way ANOVA followed by Dunnett’s test. Download figure Download PowerPoint To investigate the requirement of MICU1 for lipid peroxidation, we used C11-BODIPY 581/591 to measure lipid peroxidation (Drummen et al, 2002). Lipid peroxidation was clearly increased in WT A549 cells after cold stress for 5 h, but not in the presence of iron chelator: deferoxamine (DFO), or a radical trapping antioxidant: ferrostatin-1 (Fer-1) as previously shown (Fig EV1C) (Hattori et al, 2017). The lipid peroxidation under cold stress was not observed in the MICU1-deficient A549 cells (Fig 1E and F). ASK1 is known to be activated by cold stress-dependent lipid peroxidation (Hattori et al, 2017). Cold stress-induced ASK1 activation was also suppressed in MICU1-deficient A549 cells (Fig EV4A). Together, these results suggest that MICU1 is necessary for the lipid peroxidation and subsequent cell death in the context of cold stress-induced ferroptosis. Click here to expand this figure. Figure EV4. ASK1 activity in various conditions and hyperpolarization of MMP under cold stress, related to Figs 1, 3 and 4 A. Immunoblots of endogenous phospho-ASK signals after cold stress for 5 h of the WT A549 cells, cloned MICU1-KO cells. B. Mitochondrial membrane potential (MMP) was measured by JC-1 after cold stress at the indicated time points. Quantification data for the WT A549 cells are presented as mean ± SEM; n = 4, biological replicates, *P < 0.05, one-way ANOVA followed by William’s test. C,D. Mitochondrial membrane potential was measured by JC-1 after cold stress for 5 h. Representative (C) and quantification data (D) are represented as mean ± SEM; n = 6, one-way ANOVA followed by Dunnett’s test. Delta MMP was shown by the MMP of the WT A549 or MICU1 KO cells subtracted from that of each control condition. E,F. Immunoblots of endogenous phospho-ASK signals after cold stress for 5 h of the WT A549 cells. Inhibitors (FCCP: 200 µM, Antimycin A: 50 µM, BAPTA: 10 µM, mitoQ: 0.5 µM) were used as pretreatments for 30 min. G. TRPM8 mRNA was quantified by RT–qPCR. Data are presented as mean ± SEM; n = 4, *P < 0.05, unpaired t-test. H. Immunoblots of endogenous phospho-ASK signals after cold stress for 5 h of the WT A549 cells or TRPM8-KD cells by siRNA transfection. Download figure Download PowerPoint DIME interaction and dimerization domains of MICU1 are necessary for cold stress-induced ferroptosis MICU1 plays an essential role in gatekeeping and activating MCU through its interaction with some components of the MCU complex (Fig 2A). MICU1 has two EF hand domains for sensing Ca2+ concentration (Perocchi et al, 2010; Mallilankaraman et al, 2012), the DIME interaction domain (DID) for interacting with the D-ring of the MCU pore to regulate mitochondrial Ca2+ uptake (Paillard et al, 2018), the homo- or heterodimerization domain of MICU1 for appropriate gatekeeping and MCU-MICU1 rearrangements (Patron et al, 2014; Petrungaro et al, 2015; Gottschalk et al, 2019), and the EMRE binding domain for tethering MICU1 to the MCU complex without affecting mitochondrial Ca2+ uptake (Sancak et al, 2013; Paillard et al, 2018). Figure 2. DIME interaction and dimerization domains of MICU1 are necessary for cold stress-induced ferroptosis A. Schematic model describing the domains of MICU1 and the brief structure of the MCU complex green points describing Ca2+ and red points describing mutants. B. Immunoblots of MICU1 and its mutants rescued by the lentivirus. Arrows indicate MICU1 or the dimer. * indicates the potential DTT-resistant dimer. C. LDH assay was performed after 16 h of cold stress on ice. Data are presented as mean ± SEM; n = 3, biological replicates. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, n.s.: not significant, by one-way ANOVA followed by Tukey’s multiple comparison test D,E. Lipid peroxidation was measured by C11-BODIPY 581/591 after cold stress for 5 h. Representative plots are shown in (D). Quantification data (E), whose raw value were normalized by the C11-BODIPY 581/591 of the WT A549 cells after cold stress application, are presented as mean ± SEM; n = 4, biological replicates. ****P < 0.0001, ***P < 0.001, **P < 0.01, n.s.: not significant, by one-way ANOVA followed by Tukey’s multiple comparison test. Source data are available online for this figure. Source Data for Figure 2 [embr202051532-sup-0006-SDataFig2.pdf] Download figure Download PowerPoint Thus, we next determined which domain of MICU1 was involved in cold stress-induced cell death. We preformed rescue experiments in the MICU1-KO A549 cells (MICU1 #1) by reconstituting various MICU1 mutants, including ∆EF (D231A, E242K, D421A, and E432K), ∆DID (438KQRLMRGL > AQALMAGL), ∆DIMER (C463A), and ∆EMRE (99KKKKR > AAAAA) (Fig 2B). First, we examined the effect of the reconstituted MICU1 mutants on cell death, which was determined by LDH release. Re-expression of MICU1 WT, ∆EF, and ∆EMRE but not ∆DID or ∆DIMER rescued cell death of MICU1 KO cells in cold stress, suggesting that the interaction of MICU1 with MCU and MICU1/2 would be required for cold stress-induced cell death (Fig 2C). Next, we examined the effect of these mutants on lipid peroxidation. Similar to the findings on cell death, the reintroduction of the MICU1 WT, ∆EF, and ∆EMRE rescued the cold stress-induced lipid peroxidation, but the reintroduction of ∆DID or ∆DIMER did not have this effect (Fig 2D and E). The C-terminus of MICU1 is required for MCU-MICU1 complex rearrangement and the subsequent regulations for MCU as a gatekeeper in mitochondrial Ca2+ uptake (Patron et al, 2014; Wang et al, 2014; Paillard et al, 2018; Gottschalk et al, 2019). ∆DID (K438A;R440A;R443A) and ∆DIMER (C463A) mutations are located in the C-terminus (Fig 2A) and lack MICU1-MCU/MICU2 interactions necessary for gatekeeping MCU complex. On the other hand, EF hand mutation of MICU1 impairs its Ca2+-dependent activating but not gatekeeping activity of MCU (Perocchi et al, 2010; Gottschalk et al, 2019). One of the interpretations of these data would be that the ability of MICU1 to interact with MCU and/or MICU2 and to serve as a gatekeeper for MCU may be necessary for cold stress-induced ferroptosis. Next, we examined the effect of MICU1 deficiency on Ca2+ concentration in mitochondria and cytosol under cold stress. Firstly, we tried measuring Ca2+ by imaging experiments using mt-pericam and CEPIA2mt for mitochondria Ca2+ (Nagai et al, 2001; Suzuki et al, 2014), and Fura-2 AM and Fura-8 AM for cytosol Ca2+. However, we could not construct a reliable measuring system, because we did not have an equipment to keep cold circumstances on confocal microscopy. Instead, we used Rhod-2 for mitochondria Ca2+ and by Cal-520 for cytosol Ca2+ in plate reader-based measuring system, because these Ca2+ probes showed better signal intensity. The mitochondrial Ca2+ concentration measured by Rhod-2 AM was increased after cold stress. The increased Rhod-2 signal at 1 h after cold stress was partially but significantly reduced in the MICU1-KO cells compared with the level in the WT cells, while the basal mitochondrial Ca2+ showed a slight increase without significant difference in MICU1-KO cells (Fig EV3, EV4, EV5). Cytosolic Ca2+ measured by Cal-520 was also increased after cold stress, and it was rather enhanced in MICU1-deficient cells, while the basal cytosolic Ca2+ showed a slight increase without significant difference in MICU1-KO cells (Fig EV3, EV4, EV5). Some reports show that mitochondrial Ca2+ uptake by MICU1 deficiency was reduced in high Ca2+ conditions or after Ca2+ mobilizing agonists treatment(Csordás et al, 2013; Liu et al, 2016). Thus, it seems that MICU1-deficient cells may decrease mitochondrial Ca2+ uptake from cytosol after cold stress. However, since Rhod-2 and Cal-520 may have some technical problems to measure precise Ca2+ concentration, further investigations may be required for precise conclusion. Click here to expand this figure. Figure EV3. Cold stress-induced Ca2+ increase in matrix and cytosol, related to Figs 1–4 A-F. Mitochondrial Ca2+ concentration (A-C) and cytosolic Ca2+ concentration (D-F) were monitored by Rhod-2 AM and Cal-520 AM, respectively, at every 15 min. Data (A,B, D and E) were normalized by data of before cold stress. The data before cold stress (C, F) and at 60 min (B, E) are presented as mean ± SEM; n = 6 (A-C), 5 (D-F), biological replicates, ***P < 0.001, *P < 0.05, n.s.: not significant, by one-way ANOVA followed by Dunnett’s multiple comparison test. G,H. Mitochondrial Ca2+ concentration was monitored by Rhod-2 AM at every 15 min. FCCP (200 µM) and antimycin A (50 µM) were used as pretreatments for 30 min. Data were normalized by data of DMSO before cold stress and the data at 60 min (H) are presented as mean ± SEM; n = 3, biological replicates, ****P < 0.0001, **P < 0.01, by one-way ANOVA followed by Dunnett’s multiple comparison test. I-L. Cytosolic Ca2+ concentration (I-J) and mitochondrial Ca2+ concentration (K-L) were monitored by Cal-520 AM and Rhod-2 AM, respectively, at every 15 min. BAPTA-AM (10 µM) were used as pretreatments for 30 min. Data were normalized by data of DMSO before cold stress, and the data at 60 min (J, L) are presented as mean ± SEM; n = 4, biological replicates, *P < 0.05, by paired T-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. MICU1 can be one of potential targets for cold-induced cell death, but MICU1 and cytosolic Ca2+ may not be involve in other ferroptotic cell death A. LDH release was measured after DMSO or erastin (10 µM) treatment with DMSO or BAPTA-AM (10 µM) for 24 h. Data are presented as mean ± SEM; n = 7, biological replicates, ****P < 0.0001, n.s.: not significant, one-way ANOVA followed by Bonferroni’s test. B,C. Cell viability was measured after erastin treatment as indicated concentrations for 24 hours in A549 cells and MICU1 #1/#2 KO A549 cells or Cas9-HT-1080 cells and Cas9-HT-1080 cells infected with lentivirus containing sgRNA of MICU1 #1/#2. Data are presented as mean ± SEM; n = 3 biological replicates. D. Immunoblots of MICU1 were present for knockout confirmation in Cas9-HT-1080 cells after lentivirus infection. E. Relative mRNA expression of MICU1 in normal human tissue was normalized by GAPDH mRNA expression. Data was obtained from Refex database. F. LDH release was measured after cold stress for 24 h in indicated cell lines. Data are presented as mean ± SEM; n = 4, biological replicates, **P < 0.01, *P < 0.05, one-way ANOVA followed by Dunnett’s test. G. Immunoblots of MICU1 were present for knockdown confirmation in HepG2 cells after siRNA transfection. H. LDH release was measured after cold stress for 24 h in indicated cell lines. Data are presented as mean ± SEM; n = 3, biological replicates, ****P < 0.0001, one-way ANOVA followed by Dunnett’s test. I. Immunoblots of MICU1 were present for knockdown confirmation in HEK293A cells after siRNA transfection. Download figure Download PowerPoint MCU and MICU2 are not necessary for cold stress-induced ferroptosis Although MICU1-dependent regulation of MCU appeared to be important for cold stress-induced ferroptosis, other components of the MCU complex were not enriched in our screening (Fig EV2C). Moreover, the MCU deficiency induced by CRISPR/Cas9 or siRNA could not suppress cell death (Fig EV2A, B, D and E). Interestingly, when MCU was silenced by siRNA in the MICU1-KO cells, the MICU1-KO-induced suppression of cell death was abrogated (Fig EV2F and G). These data indicate that MCU deficiency per se did not suppress cold stress-induced ferroptosis but could cancel the effect of MICU1 deficiency. This may be because the complete MCU deficiency may activate alternative Ca2+ uptake pathways in cold stress or MICU1 has MCU-independent functions. These hypotheses may be supported by some reports indicating that MCU-knockout mice are viable (Pan et al, 2013), and that MCU-independent Ca2+ uptake is also induced by ryanodine receptors, TRPC3, UCP2/3, and LETM1 (Trenker et al, 2007; Jiang et al, 2009; Ryu et al, 2010; Feng et al, 2013). Moreover, MICU1 controls cristae structure in a MCU-independent manner (Gottschalk et al, 2019; Tomar et al, 2019). Thus, the cell death mediated by MICU1/MCU double deficiency might be caused by the mitochondrial Ca2+ regulation in a MCU complex-independent manner. Another hypothesis is that activation of MCU rather suppresses cell death via buffering cytosolic Ca2+ as previously discussed (Marchi et al, 2020). To investigate this point, we overexpressed MCU in A549 WT cells, but there was no effect on cell death (Fig EV2, EV3, EV4, EV5). In addition, the cytosolic Ca2+ buffering hypothesis seems inconsistent, because MICU1 deficiency rather increased cytosolic Ca2+ but suppressed cold stress-induced ferroptosis (Fig EV3, EV4, EV5). Loss of MICU1 can increase MCU-dependent heavy metals uptake such as Fe2+ (Wettmarshausen et al, 2018). Considering that Fe2+ is required for ferroptosis, it may be possible that loss of MICU1 can reduce cytosolic Fe2+ through MCU-dependent permeability. However, if heavy metals increase in mitochondria, it would generally result in cell death (Wettmarshausen et al, 2018), inconsistent with our observed phenotype. Nonetheless, we tried to measure cytosolic Fe2+ in MICU1 deficiency using the specific Fe2+ probe (Hirayama et al, 2017), but the probe appeared to be affected by cold temperature and we could not properly measure the Fe2+ concentration (Appendix Fig S1B). In addition, MICU2 plays an opposing role to MICU1 on MCU in histamine-induced mitochondrial Ca2+ influx (Patron et al, 2014). We thus investigated the role of MICU2 in cold stress-induced ferroptosis. MICU2 depletion by siRNAs resulted in neither promotion nor inhibition of cell death (Fig EV2K and L). Since MICU2 silencing is reported to reduce MICU1’s gatekeeping activity (Patron et al, 2014), our results suggest the cold stress-induced ferroptosis may be regulated not only by the gatekeeping activity of MICU1. Altog