FATE 1 antagonizes calcium‐ and drug‐induced apoptosis by uncoupling ER and mitochondria
2016; Springer Nature; Volume: 17; Issue: 9 Linguagem: Inglês
10.15252/embr.201541504
ISSN1469-3178
AutoresMabrouka Doghman, Veronica Granatiero, Silviu Sbiera, Iuliu Sbiera, Sandra Lacas‐Gervais, Frédéric Brau, Martin Faßnacht, Rosario Rizzuto, Enzo Lalli,
Tópico(s)ATP Synthase and ATPases Research
ResumoScientific Report11 July 2016free access Source DataTransparent process FATE1 antagonizes calcium- and drug-induced apoptosis by uncoupling ER and mitochondria Mabrouka Doghman-Bouguerra Institut de Pharmacologie Moléculaire et Cellulaire CNRS UMR 7275, Sophia Antipolis, Valbonne, France NEOGENEX CNRS International Associated Laboratory, Valbonne, France University of Nice – Sophia Antipolis, Valbonne, France Search for more papers by this author Veronica Granatiero Department of Biomedical Sciences, University of Padova, Padova, Italy CNR Neuroscience Institute, Padova, Italy Search for more papers by this author Silviu Sbiera Department of Internal Medicine I - Endocrine Unit, University Hospital, University of Würzburg, Würzburg, Germany Search for more papers by this author Iuliu Sbiera Department of Internal Medicine I - Endocrine Unit, University Hospital, University of Würzburg, Würzburg, Germany Search for more papers by this author Sandra Lacas-Gervais University of Nice – Sophia Antipolis, Valbonne, France Search for more papers by this author Frédéric Brau Institut de Pharmacologie Moléculaire et Cellulaire CNRS UMR 7275, Sophia Antipolis, Valbonne, France University of Nice – Sophia Antipolis, Valbonne, France Search for more papers by this author Martin Fassnacht Comprehensive Cancer Center Mainfranken, University of Würzburg, Würzburg, Germany Search for more papers by this author Rosario Rizzuto Department of Biomedical Sciences, University of Padova, Padova, Italy CNR Neuroscience Institute, Padova, Italy Search for more papers by this author Enzo Lalli Corresponding Author orcid.org/0000-0002-0584-5681 Institut de Pharmacologie Moléculaire et Cellulaire CNRS UMR 7275, Sophia Antipolis, Valbonne, France NEOGENEX CNRS International Associated Laboratory, Valbonne, France University of Nice – Sophia Antipolis, Valbonne, France Search for more papers by this author Mabrouka Doghman-Bouguerra Institut de Pharmacologie Moléculaire et Cellulaire CNRS UMR 7275, Sophia Antipolis, Valbonne, France NEOGENEX CNRS International Associated Laboratory, Valbonne, France University of Nice – Sophia Antipolis, Valbonne, France Search for more papers by this author Veronica Granatiero Department of Biomedical Sciences, University of Padova, Padova, Italy CNR Neuroscience Institute, Padova, Italy Search for more papers by this author Silviu Sbiera Department of Internal Medicine I - Endocrine Unit, University Hospital, University of Würzburg, Würzburg, Germany Search for more papers by this author Iuliu Sbiera Department of Internal Medicine I - Endocrine Unit, University Hospital, University of Würzburg, Würzburg, Germany Search for more papers by this author Sandra Lacas-Gervais University of Nice – Sophia Antipolis, Valbonne, France Search for more papers by this author Frédéric Brau Institut de Pharmacologie Moléculaire et Cellulaire CNRS UMR 7275, Sophia Antipolis, Valbonne, France University of Nice – Sophia Antipolis, Valbonne, France Search for more papers by this author Martin Fassnacht Comprehensive Cancer Center Mainfranken, University of Würzburg, Würzburg, Germany Search for more papers by this author Rosario Rizzuto Department of Biomedical Sciences, University of Padova, Padova, Italy CNR Neuroscience Institute, Padova, Italy Search for more papers by this author Enzo Lalli Corresponding Author orcid.org/0000-0002-0584-5681 Institut de Pharmacologie Moléculaire et Cellulaire CNRS UMR 7275, Sophia Antipolis, Valbonne, France NEOGENEX CNRS International Associated Laboratory, Valbonne, France University of Nice – Sophia Antipolis, Valbonne, France Search for more papers by this author Author Information Mabrouka Doghman-Bouguerra1,2,3, Veronica Granatiero4,5, Silviu Sbiera6, Iuliu Sbiera6, Sandra Lacas-Gervais3, Frédéric Brau1,3, Martin Fassnacht7, Rosario Rizzuto4,5 and Enzo Lalli 1,2,3 1Institut de Pharmacologie Moléculaire et Cellulaire CNRS UMR 7275, Sophia Antipolis, Valbonne, France 2NEOGENEX CNRS International Associated Laboratory, Valbonne, France 3University of Nice – Sophia Antipolis, Valbonne, France 4Department of Biomedical Sciences, University of Padova, Padova, Italy 5CNR Neuroscience Institute, Padova, Italy 6Department of Internal Medicine I - Endocrine Unit, University Hospital, University of Würzburg, Würzburg, Germany 7Comprehensive Cancer Center Mainfranken, University of Würzburg, Würzburg, Germany *Corresponding author. Tel: +33 (0)4 93 95 77 55; E-mail: [email protected] EMBO Rep (2016)17:1264-1280https://doi.org/10.15252/embr.201541504 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 Several stimuli induce programmed cell death by increasing Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria. Perturbation of this process has a special relevance in pathologies as cancer and neurodegenerative disorders. Mitochondrial Ca2+ uptake mainly takes place in correspondence of mitochondria-associated ER membranes (MAM), specialized contact sites between the two organelles. Here, we show the important role of FATE1, a cancer-testis antigen, in the regulation of ER–mitochondria distance and Ca2+ uptake by mitochondria. FATE1 is localized at the interface between ER and mitochondria, fractionating into MAM. FATE1 expression in adrenocortical carcinoma (ACC) cells under the control of the transcription factor SF-1 decreases ER–mitochondria contact and mitochondrial Ca2+ uptake, while its knockdown has an opposite effect. FATE1 also decreases sensitivity to mitochondrial Ca2+-dependent pro-apoptotic stimuli and to the chemotherapeutic drug mitotane. In patients with ACC, FATE1 expression in their tumor is inversely correlated with their overall survival. These results show that the ER–mitochondria uncoupling activity of FATE1 is harnessed by cancer cells to escape apoptotic death and resist the action of chemotherapeutic drugs. Synopsis This study shows that FATE1, a cancer-testis antigen, is localized at the interface between the ER and mitochondria where it modulates the coupling of the two organelles and apoptotic cell death. FATE1 is a cancer-testis antigen. FATE1 levels are a prognostic indicator in adrenocortical carcinoma patients. Introduction Cancer-testis antigens (CTAs) are a heterogeneous group of proteins characterized by their predominantly tissue-restricted expression in testis in physiological conditions and upregulation in one or more cancer types 12. The restricted expression of CTAs in neoplastic tissues, prevalently because of gene promoter demethylation, holds a potential for their use both as cancer biomarkers and as therapeutic targets in cancer treatment. However, even if many different CTAs have been identified, in general the molecular functions of those proteins are poorly understood 2. FATE1 is a gene expressed in fetal and adult testis mapped to Xq28 3 encoding for a protein identified as a CTA in hepatocellular carcinoma and gastric and colon cancer 4. We have previously demonstrated that steroidogenic factor-1 (SF-1), a transcription factor playing a key role in adrenal and gonadal development and in adrenocortical tumorigenesis 5, activates FATE1 expression in adrenocortical carcinoma (ACC) cells in a fashion dependent on its dosage 67. The 21-kDa protein encoded by the FATE1 gene is a member of the Miff protein family, whose founder is Mff (mitochondrial fission factor), a protein involved in the control of mitochondrial and peroxisomal fission 8. FATE1 bears similarity to Mff in its C-terminal domain, which is provided with a predicted transmembrane segment preceded by a coiled-coil region. However, it lacks a Mff-similar N-terminal domain that is essential for interaction of Mff with the dynamin-related GTPase Drp1, which operates mitochondrial fission 9. In normal tissues, FATE1 expression is mainly restricted to testis and adrenal gland 36, while it is overexpressed in a variety of cancers 410. Remarkably, FATE1 was identified as one of the genes whose silencing sensitizes a panel of non-small-cell lung cancer cell lines to toxicity from the chemotherapeutic drug paclitaxel 11. In the framework of our continuous effort to characterize novel SF-1 target genes and their role in adrenal tumorigenesis 6712, we set out to determine the cellular function of FATE1. Here, we show that FATE1 is localized in mitochondria-associated ER membranes (MAM) and is implicated in the regulation of Ca2+- and drug-dependent apoptosis in cancer cells by modulating ER–mitochondria distance. Results FATE1 localizes at the interface between ER and mitochondria The H295R/TR SF-1 ACC cell line we developed overexpresses SF-1 in a doxycycline (Dox)-dependent fashion 6. This cell line is a useful cellular model to study the SF-1-dependent phenotypes found in ACC 6712. Consistent with our previous results 6, FATE1 mRNA and protein expression was very low at the basal level in H295R/TR SF-1 cells and was strongly induced following Dox treatment (Fig 1A–C). Efficient knockdown of FATE1 was obtained by specific siRNA electroporation (Fig 1B). We also produced H295R-derived cell lines selectively expressing FATE1 (H295R/TR FATE1) or N-Flag FATE1 (H295R/TR N-Flag FATE1) in a Dox-dependent fashion (Fig 1A). No Dox-dependent FATE1 expression was detected in the parental H295R/TR cell line (Fig 1A). To define the subcellular localization of the FATE1 protein, we cotransfected Dox-treated H295R/TR SF-1 cells with fluorescent markers for ER, mitochondria, and Golgi. Our results show that endogenous FATE1 colocalizes with mitochondria and partially with ER, but not with Golgi (Fig 1D). The same results were obtained in HeLa cells transiently transfected with a FATE1 expression vector (Appendix Fig S1). Consistent with these results, FATE1 colocalizes with the mitochondrial marker HSP60 in H295R/TR SF-1 cells (Fig 1E). Mitochondrial localization of FATE1 was confirmed by immunoelectron microscopy, which showed that the protein is associated with the mitochondrial surface in H295R/TR N-Flag FATE1 cells (Fig 1F). Biochemical fractionation of Dox-treated H295R/TR N-Flag FATE1 cell extracts confirmed that FATE1 cosediments principally with the heavy membrane fraction (which contains crude mitochondria) and, in smaller amounts, with the light membrane fraction (which contains ER), while it is absent from the soluble cytosolic fraction (Fig 1G). Proteolysis experiments on the intact crude mitochondrial fraction confirmed that FATE1 was accessible to proteolytic digestion, in a manner independent of the presence of the detergent Triton X-100, similar to the outer mitochondrial membrane (OMM) proteins VDAC1 and TOM20. In contrast, the mitochondrial intermembrane space protein cytochrome c was only digested by protease after Triton X-100 extraction (Fig 1H). Alkaline extraction showed that FATE1 is membrane-anchored, similar to the OMM protein VDAC1 (Fig 1I). Altogether, these experiments show that FATE1 is an integral membrane protein facing the cytosol possessing two predicted coiled-coil domains (CC1 and CC2) and one C-terminal Mff-homology domain encompassing a transmembrane region (Fig 2A). Analysis of the FATE1 protein sequence did not reveal a potential mitochondrial targeting sequence. To identify the FATE1 domain(s) that direct(s) its localization, we generated a battery of protein mutants N-terminally fused with EGFP and assessed their subcellular localization in H295R/TR SF-1 cells stained with the mitochondrial marker TOM20. The C-terminal FATE1 domain (aa 125–183) is sufficient to target protein localization to mitochondria, while its isolated N-terminal domain (aa 1–124) has a prevalent nuclear localization, similar to the aa 1–162 construct (Fig 2B). Stretches of basic residues have been shown to be able to direct protein localization to mitochondria 13. Three amino acid stretches of this type were identified in the FATE1 C-terminal domain. Reduction of their net positive charge with mutation of basic residues into alanine, either single or in combination, attenuated mitochondrial-specific targeting of FATE1, with some mutants (RRR146-147-149AAA/RHR159-160-161AAA and RR138-139AA/RRR146-147-149AAA/RHR159-160-161AAA) being distributed throughout intracellular membranes, indicating that basic residues contribute but are not sufficient to address protein localization to mitochondria (Fig EV1 and Appendix Fig S1). Importantly, the L151D mutation, which is predicted to most severely affect the C-terminal coiled-coil structure (Appendix Fig S2), impaired mitochondrial localization of EGFP-FATE1, directing its localization to intracellular membranes (including ER and the nuclear membrane), similar to the EGFP fusion protein harboring only the FATE1 transmembrane domain (TMD) (Fig 2B and Appendix Fig S1). Increased colocalization of the FATE1 TMD and L151D mutants with the ER marker calreticulin and decreased colocalization with the mitochondrial marker HSP60 compared to wild-type FATE1 in H295R/TR SF-1 cells are shown in Fig EV2. Similar results were obtained in HeLa cells transfected to express EGFP-FATE1 deletions and mutant proteins (Fig EV2 and Appendix Fig S1). Subcellular fractionation of transfected HeLa cells showed results consistent with fluorescence analysis (Fig EV3). In particular, this method confirmed the loss of specific mitochondrial (heavy membrane) localization for the FATE1 TMD and L151D mutants. These results show that the C-terminal domain of the FATE1 protein directs its mitochondrial localization, with an essential role for CC2, and suggest that the FATE1 TMD is inserted in the ER membrane. Figure 1. FATE1 is associated with mitochondria in human ACC cells SF-1, FATE1 and β-tubulin protein levels shown in basal condition and after Dox treatment in H295R/TR, H295R/TR SF-1, H295R/TR FATE1, and H295R/TR N-Flag FATE1 cells. FATE1 mRNA expression is increased in H295R/TR SF-1 cells by Dox treatment. The efficiency of FATE1 knockdown by specific (siFATE1) vs. control (siC) siRNA nucleofection is shown (mean ± SEM; n = 4 with 3 replicates/experiment). Immunofluorescence showing endogenous FATE1 protein (red) induction of expression by Dox treatment of H295R/TR SF-1 cells. SF-1 (green), DAPI (blue). Scale bars, 10 μm. Subcellular localization of endogenous FATE1 (red) and transfected fluorescent markers for Golgi, ER, and mitochondria, respectively (green) in Dox-treated H295R/TR SF-1 cells. DAPI (blue). Scale bars, 10 μm. Subcellular localization of the endogenous FATE1 protein (green) in Dox-treated H295R/TR SF-1 cells costained with an antibody against the mitochondrial marker HSP60 (red). SF-1 (blue). Scale bar, 10 μm. Immunogold electron microscopy showing association of FATE1 with the mitochondrial outer surface in Dox-treated H295R/TR N-Flag FATE1 cells. Scale bar, 500 nm. Dox-treated H295R/TR N-Flag FATE1 cells were fractioned into nuclear (N), heavy membranes (HM), light membranes (LM), and cytosolic (C) fractions, and localization of SF-1, FATE1, ribosomal protein RPL7, VDAC1, and GAPDH was revealed by immunoblot. Effect of increasing concentrations (0, 1, 10 and 100 μg/ml) of proteinase K and 0.1% Triton X-100 (TX-100) treatment of the mitochondrial fraction from Dox-treated H295R/TR N-Flag FATE1 cells on VDAC1, FATE1, TOM20, and cytochrome c. FATE1 and VDAC1 are associated with the pellet (membrane; P) fraction after high-speed centrifugation of the alkaline-extracted mitochondrial fraction of Dox-treated H295R/TR SF-1 cells, while cytochrome c is found in the supernatant (S). Source data are available online for this figure. Source Data for Figure 1 [embr201541504-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Figure 2. Protein domains directing localization of FATE1 and its association with MAM Structure of the FATE1 protein. Two predicted coiled-coil domains (CC1 and CC2) and a C-terminal transmembrane region (TM) are present. Purple, Mff-homology region. The position of L151 is indicated. Subcellular localization of EGFP fusions of full-length (FL) FATE1 and its mutants: N-terminal (aa 1–124), C-terminal (aa 125–183), 1–162, transmembrane domain (aa 155–183), and L151D (green) transfected in H295R/TR SF-1 cells. Mitochondria were stained by anti-TOM20 antibody (red) and DNA by DAPI (blue). Scale bars, 10 μm. EMD and Flag-tagged FATE1, but not the other OMM protein TOM20, were coimmunoprecipitated from Dox-treated H295R/TR N-Flag FATE1 cells. Control immunoprecipitations were performed with anti-myc (αmyc) and non-immune rabbit IgG (IgG-R) antibodies. Confocal fluorescence microscopy showed colocalization of cytoplasmic EMD, as revealed by immunofluorescence (red), with ER, stained by ER-GFP BacMam (green). Scale bars, 10 μm. Mic60/mitofilin and Flag-tagged FATE1 were coimmunoprecipitated from Dox-treated H295R/TR N-Flag FATE1 cells. Control immunoprecipitations were performed with anti-myc (αmyc) antibody. Mitochondrial localization of Mic60/mitofilin (green) and FATE1 (red) in Dox-treated H295R/TR SF-1 cells. Scale bar, 10 μm. Left, triple-immunofluorescence labeling of endogenous FATE1 (green), ER labeled by calreticulin (red) and mitochondria labeled by HSP60 staining (blue) in Dox-treated H295R/TR SF-1 cells. One area showing close apposition of red, green and blue staining (white signals) is shown at higher magnification. Scale bar, 10 μm. Right, graph showing quantification of FATE1 signal in total mitochondria (white histogram) vs. ER–mitochondria contact sites (red histogram) (mean ± SEM; n = 13). **P < 0.01, Mann–Whitney test. H295R/TR SF-1 cells were fractioned into crude mitochondria (CM), endoplasmic reticulum (ER), purified mitochondria (PM) and mitochondria-associated membranes (MAM) fractions. H, total homogenate. The localization of SERCA2, SOAT1, VDAC1, GRP75, S1R, EMD, Mic60/mitofilin, FATE1, and TOM20 proteins in the different fractions was revealed by immunoblot. Source data are available online for this figure. Source Data for Figure 2 [embr201541504-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Subcellular localization of EGFP-FATE1 full-length proteins bearing mutations in C-terminal domain basic residues in transfected H295R/TR SF-1 cellsThe mitochondrial marker TOM20 is stained in red and DNA in blue with DAPI. Scale bars, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Subcellular localization of EGFP-FATE1 L151D and TMD proteins in transfected H295R/TR SF-1 and HeLa cellsHSP60 was stained as a mitochondrial marker and calreticulin as an ER marker. Manders' colocalization coefficient of both FATE1 mutants with HSP60 (red histograms) is significantly decreased compared to the full-length wild-type FATE1, while colocalization with calreticulin (pale blue histograms) is significantly increased (one-way ANOVA with Bonferroni's correction). *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Distribution of wild-type and mutant EGFP-FATE1 protein in subcellular fractions in transfected HeLa cells Immunoblot showing expression of the different EGFP-FATE1 proteins in whole-cell lysates. TMD, transmembrane domain (aa 155–183). Mut 9: RR138-139AA/RRR146-147-149AAA. Mut 10: RRR146-147-149AAA/RHR159-160-161AAA. Mut 11: RR138-139AA/RHR159-160-161AAA. Mut 12: RR138-139AA/RRR146-147-149AAA/RHR159-160-161AAA. Immunoblot showing the distribution of transfected EGFP-FATE1 proteins (G-F) and endogenous VDAC and ribosomal protein S6 in heavy membranes (HM), light membranes (LM), and insoluble cytosolic (IC) fractions. Source data are available online for this figure. Download figure Download PowerPoint FATE1 is a component of MAM and decreases ER–mitochondria contacts To identify proteins interacting with FATE1, we immunoaffinity purified FATE1-interacting proteins from Dox-treated H295R/TR N-Flag FATE1 cells and identified them by mass spectrometry (Appendix Table S1). Proteins copurified with FATE1 included proteins from the ER—chaperones and emerin (EMD)—and the mitochondrial protein Mic60/mitofilin. Databases from large-scale yeast two-hybrid screening campaigns also report FATE1–EMD interaction 1415. FATE1–EMD interaction was confirmed by coimmunoprecipitation, while EMD did not interact with the OMM protein TOM20 (Fig 2C). EMD is known to localize predominantly in the inner nuclear membrane and to have an important role in the organization of nuclear architecture 1617. In addition, previous reports showed that a sizeable pool of extranuclear EMD is localized in the ER 1819. We confirmed extranuclear EMD localization in the ER in H295R/TR N-Flag FATE1 cells (Fig 2D). We also confirmed specific interaction between FATE1 and Mic60/mitofilin in H295R/TR N-Flag FATE1 cells (Fig 2E). Mic60/mitofilin is a mitochondrial protein (Fig 2F) that controls cristae morphology 20 and is part of the conserved mitochondrial contact site (MICOS) complex, which has an essential role in the regulation of mitochondrial architecture, dynamics, and biogenesis 21222324. The interactions of FATE1 with both ER and mitochondrial proteins prompted us to investigate its implication in ER–mitochondria contacts, which take place at the level of MAM (mitochondria-associated membranes), sites of close contacts between those two organelles that have a crucial function in lipid metabolism and calcium signaling 2526. In crude mitochondrial extracts from Dox-treated H295R/TR N-Flag FATE1 cells, FATE1 migrates in a very high molecular weight (about 1 MDa) complex in a blue native gel (Appendix Fig S3). The method published by Wieckowski et al 27 is a robust procedure to fraction cell extracts into crude mitochondria, ER, pure mitochondria, and MAM. Using this method to fraction extracts from Dox-treated H295R/TR SF-1 cells, we could detect FATE1 in crude mitochondria, ER, and MAM but not in the pure mitochondrial fraction, consistent with the immunofluorescence experiments that indicate association of the FATE1 TMD with ER (Fig 2H). The efficacy of the fractionation procedure was confirmed by the presence of the known MAM components SERCA2, SOAT1, VDAC1, and Sigma-1 receptor (S1R) in the MAM fraction, while the OMM protein TOM20 was only found in the crude and pure mitochondrial fractions. Importantly, the FATE1-interacting proteins EMD and Mic60/mitofilin were also found in the MAM fraction, in addition to their ER and mitochondrial localization, respectively. The chaperone GRP75, which was also found to interact with FATE1 in our mass spectrometry analysis (Appendix Table S1), fractioned prevalently in the pure mitochondrial fraction, even if it was also present in MAM, consistent with previous reports 28. Consistent with the biochemical fractionation results, in confocal triple-immunofluorescence microscopy, endogenous FATE1 signal can be detected in close proximity of calreticulin (ER marker) and HSP60 (mitochondrial marker) in discrete regions inside Dox-treated H295R/TR SF-1 cells (Fig 2G, left). In these cells, the intensity of FATE1 staining is significantly enriched in correspondence of ER–mitochondria contact sites compared to the total mitochondrial surface (Fig 2G, right). Colocalization profile curves of the FATE1, ER, and mitochondria signals in the image shown in Fig 2G are shown in Appendix Fig S4. Equivalent results were obtained in Dox-treated H295R/TR N-Flag FATE1 cells (Appendix Fig S5). Consistent with its localization associated with ER–mitochondria contact sites, FATE1 expression in H295R/TR N-Flag FATE1 cells following Dox treatment had the effect of decreasing ER–mitochondria contacts compared to control cells, as shown by a decreased overlap of signals from ER and mitochondria fluorescent probes (Fig 3A) and by decreased signal of a novel split-GFP-based probe 29 (Fig 3B). Using transmission electron microscopy (TEM), we could confirm that both the number of ER–mitochondria contacts and the number of mitochondria displaying ER contact sites were reduced in Dox-treated H295R/TR N-Flag FATE1 compared to control cells (Fig 3C). Equivalent results were obtained in H295R/TR SF-1 cells (Fig 3D). In addition, Dox treatment of H295R/TR N-Flag FATE1 cells decreased mitochondrial fragmentation compared to control cells (Fig 3E and F), shifting the equilibrium of mitochondrial morphology toward a fused state, without altering mitochondrial membrane potential (Fig 3H). Consistent with these data, mitochondrial fragmentation was also decreased by Dox treatment in the H295R/TR SF-1 cell line (Fig 3G). Figure 3. Effects of FATE1 expression on ER–mitochondria contacts and mitochondrial shape H295R/TR N-Flag FATE1 cells were transfected with D1ER marker for ER (green) and mtRFP marker for mitochondria (red). Manders' coefficient for green-red signals colocalization in basal (white histograms) and Dox-treated (black histograms) cells is shown on the right (mean ± SEM; n = 12). ***P < 0.001, paired t-test. Scale bars, 10 μm. Effect of FATE1 expression on ER–mitochondria distance in H295R/TR N-Flag FATE1 cells measured using a split-GFP probe 29. Quantification is shown in the graph. White histogram, number of fluorescent objects/cell in basal conditions; black histogram, number of fluorescent objects/cell in Dox-treated cells (mean ± SEM; n = 6 with 90 cells analyzed in total). **P < 0.01, paired t-test. Scale bars, 5 μm. Transmission electron microscopy images of H295R/TR N-Flag-FATE1 cells cultured in basal conditions or treated with Dox. ER–mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n = 38 for basal and 47 for Dox-treated cells). ***P < 0.001, Mann–Whitney test. Transmission electron microscopy images of H295R/TR SF-1 cells cultured in basal conditions or treated with Dox. ER–mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n = 41 for basal and 42 for Dox-treated cells). ***P < 0.001, Mann–Whitney test. Mitochondrial shape (BacMam Mitochondria-RFP; in red) in H295R/TR N-Flag FATE1 cells shown by fluorescence confocal microscopy in basal conditions and after Dox treatment. FATE1 (green), DAPI (blue). A higher magnification of merged green and red signal is shown in the insets. Scale bars, 10 μm. Mitochondrial fragmentation index in H295R/TR N-Flag FATE1 cells after Dox treatment (red histogram) compared to cells cultured in basal conditions (white histogram) (mean ± SEM; n = 111 for basal and 101 for Dox-treated cells). ***P < 0.001, Mann–Whitney test. Mitochondrial fragmentation index in H295R/TR SF-1 cells after Dox treatment (green histogram) compared to cells cultured in basal conditions (white histogram) (mean ± SEM; n = 37 for basal and 49 for Dox-treated cells). ***P < 0.001, Mann–Whitney test. Mitochondrial membrane potential (∆Ψ) measured by TMRM fluorescence after Dox treatment of H295R/TR N-Flag FATE1 cells (red histogram) compared to cells cultured in basal conditions (white histogram) (mean ± SEM; n = 3 with 12 replicates/experiment). ns, not significant, Mann–Whitney test. Download figure Download PowerPoint FATE1 negatively affects Ca2+ transfer from the ER to mitochondria and steroid hormone production in ACC cells A major function of MAM is the facilitation of Ca2+ transfer from the ER to mitochondria, which, in turn, has a crucial role in the regulation of apoptosis 2526. To assess the impact of increased FATE1 expression on mitochondrial and cytosolic Ca2+ uptake, we used aequorin-based probes targeted to different subcellular compartments 30. In H295R/TR SF-1 cells, Dox treatment significantly decreased mitochondrial Ca2+ uptake after stimulation with ATP, while it had no effect on the parental cell line (H295R/TR). Mitochondrial Ca2+ concentration was also decreased in both Dox-treated H295R/TR N-Flag FATE1 and H295R/TR FATE1 cells compared to control cells (Fig 4A). No effect of SF-1 or FATE1 overexpression was detected on cytosolic Ca2+ concentration in any of those cell lines (Fig 4B), nor on Ca2+ concentration in the ER (Fig EV4A and B). FATE1 knockdown had the opposite effect on mitochondrial Ca2+ concentration in Dox-treated H295R/TR SF-1 cells, significantly increasing it while still having no effect on cytosolic Ca2+ concentration (Fig 4C). The effect of FATE1 expression on mitochondrial Ca2+ uptake is lost after treatment of digitonin-permeabilized Dox-treated H295R/TR N-Flag FATE1 cells with cyclopiazonic acid (CPA), a specific inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases (Fig EV4C and D). These data show that the effect on Ca2+ transfer into mitochondria triggered by FATE1 expression is MAM-dependent. On the other hand, the intracellular levels of proteins involved in ER–mitochondria Ca2+ signaling and having a role in the mitochondrial uptake machinery were similar before and after Do
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