Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development
2021; Springer Nature; Volume: 22; Issue: 9 Linguagem: Inglês
10.15252/embr.202051954
ISSN1469-3178
AutoresSergi Casellas‐Díaz, Raquel Larramona‐Arcas, Guillem Riqué‐Pujol, Paula Tena‐Morraja, Claudia Müller‐Sánchez, Marc Segarra‐Mondejar, Aleix Gavaldà‐Navarro, Francesc Villarroya, Manuel Reina, Ofelia M. Martínez-Estrada, Francesc X. Soriano,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle23 July 2021Open Access Transparent process Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development Sergi Casellas-Díaz Sergi Casellas-Díaz Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Raquel Larramona-Arcas Raquel Larramona-Arcas orcid.org/0000-0001-8427-8007 Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Guillem Riqué-Pujol Guillem Riqué-Pujol Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Paula Tena-Morraja Paula Tena-Morraja orcid.org/0000-0002-7617-3392 Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Claudia Müller-Sánchez Claudia Müller-Sánchez orcid.org/0000-0002-3654-2683 Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Search for more papers by this author Marc Segarra-Mondejar Marc Segarra-Mondejar Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Aleix Gavaldà-Navarro Aleix Gavaldà-Navarro Department of Biochemistry and Molecular Biomedicine, University of Barcelona, Barcelona, Spain Institute of Biomedicine, University of Barcelona, Barcelona, Spain CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Madrid, Spain Search for more papers by this author Francesc Villarroya Francesc Villarroya orcid.org/0000-0003-1266-9142 Department of Biochemistry and Molecular Biomedicine, University of Barcelona, Barcelona, Spain Institute of Biomedicine, University of Barcelona, Barcelona, Spain CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Madrid, Spain Search for more papers by this author Manuel Reina Manuel Reina Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Search for more papers by this author Ofelia M Martínez-Estrada Ofelia M Martínez-Estrada Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Biomedicine, University of Barcelona, Barcelona, Spain Search for more papers by this author Francesc X Soriano Corresponding Author Francesc X Soriano [email protected] orcid.org/0000-0003-1678-7162 Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Sergi Casellas-Díaz Sergi Casellas-Díaz Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Raquel Larramona-Arcas Raquel Larramona-Arcas orcid.org/0000-0001-8427-8007 Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Guillem Riqué-Pujol Guillem Riqué-Pujol Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Paula Tena-Morraja Paula Tena-Morraja orcid.org/0000-0002-7617-3392 Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Claudia Müller-Sánchez Claudia Müller-Sánchez orcid.org/0000-0002-3654-2683 Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Search for more papers by this author Marc Segarra-Mondejar Marc Segarra-Mondejar Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Aleix Gavaldà-Navarro Aleix Gavaldà-Navarro Department of Biochemistry and Molecular Biomedicine, University of Barcelona, Barcelona, Spain Institute of Biomedicine, University of Barcelona, Barcelona, Spain CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Madrid, Spain Search for more papers by this author Francesc Villarroya Francesc Villarroya orcid.org/0000-0003-1266-9142 Department of Biochemistry and Molecular Biomedicine, University of Barcelona, Barcelona, Spain Institute of Biomedicine, University of Barcelona, Barcelona, Spain CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Madrid, Spain Search for more papers by this author Manuel Reina Manuel Reina Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Search for more papers by this author Ofelia M Martínez-Estrada Ofelia M Martínez-Estrada Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Biomedicine, University of Barcelona, Barcelona, Spain Search for more papers by this author Francesc X Soriano Corresponding Author Francesc X Soriano [email protected] orcid.org/0000-0003-1678-7162 Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain Search for more papers by this author Author Information Sergi Casellas-Díaz1,2, Raquel Larramona-Arcas1,2, Guillem Riqué-Pujol1,2, Paula Tena-Morraja1,2, Claudia Müller-Sánchez1, Marc Segarra-Mondejar1,2, Aleix Gavaldà-Navarro3,4,5, Francesc Villarroya3,4,5, Manuel Reina1, Ofelia M Martínez-Estrada1,4 and Francesc X Soriano *,1,2 1Department of Cell Biology, Physiology and Immunology, Celltec-UB, University of Barcelona, Barcelona, Spain 2Institute of Neurosciences, University of Barcelona, Barcelona, Spain 3Department of Biochemistry and Molecular Biomedicine, University of Barcelona, Barcelona, Spain 4Institute of Biomedicine, University of Barcelona, Barcelona, Spain 5CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Madrid, Spain **Corresponding author. Tel: +34 934021535; E-mail: [email protected] EMBO Reports (2021)22:e51954https://doi.org/10.15252/embr.202051954 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 Mfn2 is a mitochondrial fusion protein with bioenergetic functions implicated in the pathophysiology of neuronal and metabolic disorders. Understanding the bioenergetic mechanism of Mfn2 may aid in designing therapeutic approaches for these disorders. Here we show using endoplasmic reticulum (ER) or mitochondria-targeted Mfn2 that Mfn2 stimulation of the mitochondrial metabolism requires its localization in the ER, which is independent of its fusion function. ER-located Mfn2 interacts with mitochondrial Mfn1/2 to tether the ER and mitochondria together, allowing Ca2+ transfer from the ER to mitochondria to enhance mitochondrial bioenergetics. The physiological relevance of these findings is shown during neurite outgrowth, when there is an increase in Mfn2-dependent ER-mitochondria contact that is necessary for correct neuronal arbor growth. Reduced neuritic growth in Mfn2 KO neurons is recovered by the expression of ER-targeted Mfn2 or an artificial ER-mitochondria tether, indicating that manipulation of ER-mitochondria contacts could be used to treat pathologic conditions involving Mfn2. Synopsis The bioenergetic role of Mfn2 relies on its localization in the ER. Mfn2 localization to mitochondria is dispensable in the presence of mitochondrial Mfn1, which tethers ER and mitochondria, and facilitates Ca2+ transfer. Mfn2 regulates metabolism independent of its mitochondrial fusion function. Mfn2 localization in the ER is necessary to maintain mitochondrial bioenergetics. Mfn2 is an ER-mitochondria tether whose depletion alters ER and mitochondrial Ca2+ levels. Restoration of ER-mitochondria contacts corrects altered Ca2+ homeostasis and bioenergetics defects in Mfn2 KO cells. Mfn2-mediated ER-mitochondria tethering is necessary for proper neurite outgrowth. Introduction Mitochondria are dynamic organelles whose morphology continuously changes through fusion and fission events. This dynamism affects mitochondrial bioenergetic properties, among other functions (Liesa & Shirihai, 2013; Schrepfer & Scorrano, 2016). Mfn1 and Mfn2 are two proteins from the large GTPase family that mediate outer mitochondrial membrane fusion. Despite their high degree of homology, there are functions that Mfn1 and Mfn2 do not share, as evidenced in the phenotypes of Mfn1 and Mfn2 KO mice (Chen et al, 2003; Chen et al, 2007). Mfn1 has been shown to be more efficient at mediating mitochondrial fusion; meanwhile, Mfn2 plays a more prominent role regulating mitochondrial metabolism (Bach et al, 2003; Chen et al, 2003; Eura et al, 2003; Ishihara et al, 2004; Pich et al, 2005). The mechanism through which Mfn2 has a greater impact on mitochondrial energetics than Mfn1 does is not fully understood, although several mechanisms have been proposed. Mfn2 has been reported to regulate the levels of proteins in the OXPHOS system (Pich et al, 2005) and the formation of supercomplexes (Segales et al, 2013). It has also been found to be necessary for coenzyme Q biosynthesis (Mourier et al, 2015). The finding that a small fraction of Mfn2, but not Mfn1, resides in the endoplasmic reticulum (ER) where it interacts, both homo- and heterotipically, with mitochondria-located Mfns and tethers the two organelles together (de Brito & Scorrano, 2008) has also suggested a mechanism by which Mfn2 could regulate mitochondrial metabolism via modulation of Ca2+ transfer from the ER to mitochondria (Chen et al, 2012; Seidlmayer et al, 2019). The ER and mitochondria establish functional contact in the area called mitochondria-associated membrane (MAM). The thickness of ER-mitochondria contact ranges 10–80 nm, with the distance between the organelles being what determines MAM function (Giacomello & Pellegrini, 2016; Herrera-Cruz & Simmen, 2017). The three most studied functions of the MAM are as follows: (i) lipid synthesis. Most of the enzymes involved in lipid biosynthesis are localized in the ER membrane, but some are located in the mitochondrial membrane. Thus, it is necessary to transfer different lipid intermediates from one organelle to other in order to complete the biosynthetic pathway (Vance, 2014). (ii) Mitochondrial fission. It has been observed that ER tubules mark sites of mitochondrial division (Friedman et al, 2011). (iii) Regulation of Ca2+ homeostasis. The two main organelles responsible for maintaining the Ca2+ homeostasis are the ER and mitochondria. These two organelles exchange Ca2+ which affects their respective functionality. Mitochondrial Ca2+ uptake is produced through the mitochondrial calcium uniporter (Baughman et al, 2011; De Stefani et al, 2011) (MCU); but given its low Ca2+ affinity, this takes place in microdomains with high Ca2+ concentrations in the proximity of the Ca2+-releasing channels in the ER (Rizzuto et al, 1993; Rizzuto et al, 1998). That is to say, it occurs in the regions of close proximity between the ER and mitochondria. The optimal separation between ER and mitochondria to generate microdomains with high enough Ca2+ concentrations to facilitate Ca2+ uptake by MCU is 12–24 nm (Giacomello & Pellegrini, 2016). Mitochondrial Ca2+ regulates the activity of dehydrogenases of the tricarboxylic acid cycle (TCA) and promotes dephosphorylation, hence activation of pyruvate dehydrogenase (Balaban, 2009). Thus, Ca2+ uptake by mitochondria following its release from ER is necessary to maintain cellular bioenergetics (Cárdenas et al, 2010). However, this Ca2+ transfer must be finely regulated since too little or too much Ca2+ transfer is noxious (Scorrano et al, 2003; Csordás et al, 2006; Cárdenas et al, 2016). Observations of reduced mitochondrial Ca2+ and increased ER Ca2+ levels in Mfn2 depleted cells (Chen et al, 2012; Seidlmayer et al, 2019) support the view that Mfn2 regulates mitochondrial metabolism by tethering the ER and mitochondria together and thereby regulating mitochondrial Ca2+. However, this has not been studied thoroughly. By expressing Mfn2 targeted at the ER or mitochondria in Mfn2 KO or Mfn1/2 DKO cells, here, we show that the presence of Mfn2 in the ER is needed in order to tether ER and mitochondria together and enhance mitochondrial energetics. The reestablishment of ER-mitochondria contact via the expression of ER-located Mfn2 or the artificial tether ChiMERA (Kornmann et al, 2009) is sufficient to correct the bioenergetic defects in Mfn2 KO fibroblasts. The rescue of neuritic arbor abnormalities in Mfn2 KO neurons by ER-located Mfn2 or ChiMERA provides a proof of concept that targeting the ER-mitochondria contacts may be a suitable therapeutic option in pathologic conditions in which Mfn2 is involved. Results Defects in mitochondrial bioenergetics in Mfn2 KO cells do not depend on mitochondrial morphology Mfn2 is a potent regulator of mitochondrial bioenergetics, but the mechanism by which Mfn2 maintains mitochondria bioenergetics is not well understood. Elongated mitochondria are associated with increased mitochondrial bioenergetics (Liesa & Shirihai, 2013). However, although Mfn1 KO fibroblasts (Appendix Fig S1A) showed more globular mitochondria than Mfn2 KO fibroblasts (Fig 1A–D), they did not show bioenergetic alterations (Fig 1E–I and Appendix Fig S1B). Meanwhile, Mfn2 KO cells showed reduced mitochondrial membrane potential (MMP) (Fig 1E) and lower basal and maximal oxygen consumption rate (OCR) (Fig 1F) when analyzed using a fluorescent O2 sensor probe, which resulted in reduced respiratory control ratio (RCR) and a reduced spare respiratory capacity (SRC) (Fig 1G and H). Analysis of the OCR using Seahorse XF24 showed a similar pattern (Appendix Fig S1B). Figure 1. Defects in mitochondrial bioenergetics in Mfn2 KO cells do not depend on mitochondrial morphology A–D. (A) Representative images of WT, Mfn1 KO, and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria-targeted RFP (mt-RFP). Scale bar: 5 µm. (B) Percentage of cells displaying globular mitochondria (n = 310–668 cells analyzed in 3–6 independent experiments). Data are presented as mean ± SEM. (C) Analysis of aspect ratio (AR) calculated as major axis/minor axis and (D) circularity calculated as 4*π*Area/Perimeter2 (n = 300 mitochondria in three independent experiments). Data are presented as mean ± SEM. E. Mitochondrial membrane potential (MMP) was determined by measuring TMRM fluorescence (n = 90 cells analyzed in three independent experiments). Data are presented as mean ± SEM. F–H. (F) Oxygen consumption rate (OCR) normalized to the amount of protein in WT, Mfn1 KO, and Mfn2 KO fibroblasts. Proton leak was measured after application of oligomycin (1 µM), maximal after CCCP application (10 µM), and oligomycin (n = 3 independent experiments). Data are presented as mean ± SEM. (G) Respiratory control ratio (RCR) was calculated as maximal oxygen consumption rate (OCR)/H+ leak OCR (n = 3 independent experiments). Data are presented as mean ± SEM. (H) Spare respiratory capacity was calculated as maximal OCR-basal OCR (n = 3 independent experiments). Data are presented as mean ± SEM. I. WT MEFs and Mfn1 or Mfn2 KO MEFs were incubated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n = 3 independent experiments). Data are presented as mean ± SEM. Data information: *P < 0.05, one-way ANOVA followed by Tukey's post hoc test. Download figure Download PowerPoint Cells adjust metabolic pathways to maintain relatively constant total ATP levels, and therefore, there were not differences in the ATP levels between the different cell lines (Fig EV1A). However, in agreement with the reduced respiratory capacity in Mfn2 KO cells, inhibition of glycolysis with 2-deoxy-D-glucose (2-DG) caused a drop in ATP levels in Mfn2 KO cells with respect to WT and Mfn1 KO cells (Fig 1I). Click here to expand this figure. Figure EV1. Bioenergetics defects in Mfn2 KO cells are independent of mitochondrial morphology A. ATP levels in the indicated cell lines when metabolic flexibility is allowed (n = 3 independent experiments). Data are presented as mean ± SEM. B. Representative Western blots of the indicated proteins in Mfn2 KO fibroblasts transfected with plasmids expressing Mfn1, DN-Drp1, or a control plasmid (CT) (n = 3 independent experiments). C–F. Expression of Mfn1 restores mitochondrial morphology in Mfn2 KO MEFs. WT or Mfn2 KO MEFs were co-transfected with plasmids encoding mt-RFP and Mfn1 or control plasmids. (C) Representative images. Scale bar = 20 µm. (D) Percentage of cells displaying globular mitochondria (n = 54–414 cells analyzed in three independent experiments), (E) aspect ratio, and (F) circularity (n = 260–300 mitochondria analyzed in three independent experiments). Data are presented as mean ± SEM. G–J. Expression of DN-Drp1 restores mitochondrial morphology in Mfn2 KO MEFs. WT or Mfn2 KO MEFs were co-transfected with plasmids encoding mt-RFP and DN-Drp1 or control plasmids. (G) Representative images. Scale bar = 20 µm. (H) Percentage of cells displaying globular mitochondria (n = 135–470 cells analyzed in three independent experiments), (I) aspect ratio and (J) circularity (n = 300 mitochondria were analyzed in three independent experiments). Data are presented as mean ± SEM. K, L. Restoration of mitochondrial morphology by expression of (K) Mfn1 or (L) DN-Drp1 in Mfn2 KO MEFs has no effect on mitochondrial membrane potential (n = 90 cells analyzed in three independent experiments). Data are presented as mean ± SEM. M, N. Restoration of mitochondrial morphology by expression of (M) Mfn1 or (N) DN-Drp1 in Mfn2 KO MEFs has no effect on basal oxygen consumption rate (n = 3 independent experiments). Data are presented as mean ± SEM. Data information: *P < 0.05, one-way ANOVA followed by Tukey's post hoc test. Download figure Download PowerPoint Mitochondrial morphology can affect uptake of cationic dyes used to measure MMP (Kowaltowski et al, 2002). However, restoration of mitochondrial morphology in Mfn2 KO cells by expressing exogenous Mfn1 or a dominant-negative mutant of the pro-fission protein Drp1 (DN-Drp1) (Fig EV1-EV5) did not modify the MMP or the basal oxygen consumption in Mfn2 KO cells (Fig EV1-EV5). Click here to expand this figure. Figure EV2. ER and mitochondrial Ca2+ levels are deregulated in Mfn2 KO cells A. Mander's coefficient of WT and Mfn1 KO cells co-transfected with plasmids encoding mitochondria-targeted RFP and ER-targeted GFP (n = 15 cells analyzed in three independent experiments). Data are presented as mean ± SEM. B. Restoration of mitochondrial morphology does not restores ER-mitochondria colocalization. Mander's coefficient of WT and Mfn2 KO cells co-transfected with plasmids encoding mitochondria-targeted RFP and ER-targeted GFP and the indicated plasmids (n = 15 cells analyzed in three independent experiments). Data are presented as mean ± SEM. C, D. ERMITO-Luc detects changes in ER-mitochondria contacts induced by manipulation of mTOR pathway. RLuc activity in cells transfected with ERMITO-Luc and treated with (C) the mTOR inhibitor PP242 (25 µM) (n = 4 independent experiments) or (D) the mTOR activator MHY 1485 (10 µM) (n = 6 independent experiments). Data are presented as mean ± SEM. E. There are not differences in the RLuc activity in the WT and Mfn2 KO cells transfected with plasmid expressing full-length Renilla Luciferase driven by the CMV promoter (n = 3 independent experiments). Data are presented as mean ± SEM. F. RLuc activity in WT and Mfn1 KO cells transfected with ERMITO-Luc (n = 3 independent experiments). Data are presented as mean ± SEM. G, H. Diminished Ca2+ levels in Mfn2 KO mitochondria. (G) Mitochondrial Ca2+ was released from mitochondria by applying CCCP (10 µM), and the rise in cytoplasmic Ca2+ was analyzed with Fluo-4 (n = 30 cells analyzed from three independent experiments). (H) Analysis of mitochondria Ca2+ levels in WT, and Mfn2 KO cells were transfected with the mitochondria-targeted Ca2+ sensor mt-Cepia (n = 30 cells from three independent experiments). Data are presented as mean ± SEM. I. Increased ER Ca2+ levels in Mfn2 KO cells analyzed by measuring cytoplasmic Ca2+ rise with Fluo-4 after thapsigargin (1 µM) treatment (n = 30 cells from three independent experiments). Data are presented as mean ± SEM. J. Caffeine induces higher ER Ca2+ release to the cytoplasm in Mfn2 KO cells. ER Ca2+ release was stimulated with caffeine (20 mM) as indicated, and cytoplasmic Ca2+ levels were analyzed with Fluo-4 (n = 30 cells from three independent experiments). Data are presented as mean ± SEM. K. The source of Ca2+ after caffeine treatment is from de ER. Inhibition of the RyR with dantrolene (10 µM) prevents caffeine-induced increase in mitochondrial Ca2+ levels (n = 30 cells from three independent experiments). Data are presented as mean ± SEM. Data information: *P < 0.05, one-way ANOVA followed by Tukey's post hoc test. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Restoration of bioenergetics defects in Mfn2 KO cells require proper Ca2+ transfer from the ER to mitochondria A, B. ChiMERA is weakly expressed in MEFs. (A) Representative Western blots of the indicated proteins in Mfn2 KO fibroblasts transfected with RFP plus ChiMERA, GFP, or control (CT) plasmids as indicated (n = 3 independent experiments). Data are presented as mean ± SEM. (B) Representative confocal images of Mfn2 KO transfected cells with ChIMERA or GFP. Green fluorescent signal cannot be observed in ChIMERA expressing cells, although GFP molecule can be detected by three-step immunofluorescence using antibodies anti-GFP. Scale bar = 10 µm. C. MCU mRNA and protein levels were analyzed by qPCR and Western blot, respectively, of MEFs transfected with siRNA control (siCT) or a pool of 4 siRNAs targeting MCU (siMCU) (n = 3 independent experiments). D. MCU KD (siMCU) abolish mitochondrial Ca2+ uptake when the IP3R is stimulated with ATP (100 µM). WT and Mfn2 KO cells were transfected with siRNA control (siCT) or targeting MCU (siMCU). Mitochondrial Ca2+ uptake was analyzed. (n = 28–30 cells from three independent experiments). Data are presented as mean ± SEM. E, F. Inhibition of IP3R with (E) 2APB (50 µM) or (F) xestospongin C (XeC) (1.5 µM) prevents mitochondrial Ca2+ uptake caused by ATP treatment (n = 30 cells from three independent experiments). Data are presented as mean ± SEM. G–I. Treatment with 2APB or XeC prevents ChiMERA-mediated rescue of (G) MMP (n = 90 cells analyzed in three independent experiments), (H) ATP levels (n = 3 independent experiments), and (I) basal oxygen consumption rate (n = 3 independent experiments) in Mfn2 KO cells. Data are presented as mean ± SEM. J, K. 2APB or XeC treatment does not affect cell viability. WT and Mfn2 KO MEFs were treated with XeC (1.5 µM) or 2APB (50 µM) for 16 h, and then cell viability (J) was analyzed by fixing cells, DAPI staining and counting non-pyknotic nuclei as percentage of the total number of nuclei (n = 3 independent experiments). (K) Total number of nuclei per field was counted (n = 3 independent experiments). Data are presented as mean ± SEM. L. Mitochondrial mass is not affected by 2APB or XeC treatment. Representative Western blot of the indicated proteins of WT and Mfn2 KO MEFs treated for 16 h with XeC (1.5 µM), 2APB (50 µM) or vehicle (CT) (n = 3 independent experiments). M. Mfn2 KO show diminished expression levels of MCU but this is not rescued by ChiMERA expression. WT and Mfn2 KO cells were transfected with RFP and ChiMERA or control plasmid. After 24 h, the transfected cells were sorted and plated for another 24 h, when protein extracts were obtained. Representative Western blot of the indicated proteins (n = 3 independent experiments). N. Western blot with the indicated antibodies to characterize cultures of tamoxifen-inducible Mfn2 KO astrocyte cultures (n = 3 independent experiments). O–Q. Bioenergetic parameters were analyzed in WT and Mfn2 KO astrocytes transfected with ChiMERA or control plasmid. (O) MMP (n = 30 cells from three independent cultures). (P) Analysis of ATP levels after 6 h treatment with 2-DG (10 mM) (n = 3 independent experiments). (Q) Oxygen consumption rates. Proton leak was measured after application of oligomycin (1 µM), maximal after CCCP application (10 µM), and oligomycin (n = 5 independent experiments). Data are presented as mean ± SEM. Data information: *P < 0.05, one-way ANOVA followed by Tukey's post hoc test except (C) that was used two-tailed Student's t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. ER-Mfn2 localizes in the ER A, B. (A) Representative immunofluorescence imaging of Mfn1/2 DKO MEFs transfected with ER-Mfn2 and ER-RFP or mt-RFP and (B) colocalization of ER-Mfn2 with mitochondria or ER analyzed by Mander's coefficient (n = 10 cells analyzed in three independent experiments). Scale bar = 5 µm. Data are presented as mean ± SEM. *P < 0.05, two-tailed Student's t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Mitochondria only targeted Mfn2 restores mitochondrial morphology to the same extent as Mfn2 in Mfn1/2 DKO cells A. Representative immunofluorescence imaging of Mfn1/2 DKO MEFs transfected with mt-Mfn2 and ER-RFP or mt-RFP. Scale bar: 5 µm. B. Colocalization of mt-Mfn2 with mitochondria or ER analyzed by Mander's coefficient (n = 10 cells analyzed in three independent experiments). Data are presented as mean ± SEM. *P < 0.05, two-tailed Student's t-test. C–F. mt-Mfn2 expression restores mitochondrial morphology to the same extent as Mfn2 expression. Mfn1/2 DKO cells were transfected with control (CT), ChiMERA, or the indicated Mfn2 expression vectors and (C) percentage of cells displaying globular mitochondria (n = 310–668 cells analyzed in 3–6 independent experiments), (D) aspect ratio, and (E) circularity were calculated (n = 300 mitochondria analyzed from three independent experiments). Data are presented as mean ± SEM. (F) Representative images. White scale bar = 10 µm. Yellow scale bar = 5 µm. Data information: *P < 0.05, one-way ANOVA followed by Tukey's post hoc test. Download figure Download PowerPoint These results indicate that the potential for Mfn2 to maintain mitochondrial bioenergetics is independent of its capacity to modify mitochondrial morphology. Mfn2 KO cells show alterations in ER and mitochondrial Ca2+ that may mediate impaired bioenergetic function Mfn2 modulates the ER stress response (Ngoh et al, 2012; Muñoz et al, 2013) and ER stress regulates mitochondrial bioenergetics (Bravo et al, 2011; Balsa et al, 2019; Carreras-Sureda et al, 2019). We analyzed the three branches of the unfolded protein response (UPR) and found increased activation of the ATF6 and Xbp1 pathways but no changes in the PERK-eIF2a pathway (Appendix Fig S2A–C). Use of the chemical chaperone 4-phenylbutyric acid (4PBA) blocked activation of the UPR in Mfn2 KO cells (Appendix Fig S2B and C), but 4PBA had no effect on bioenergetics defects in Mfn2 KO cells (Appendix Fig S2D–F). Mfn2 has also been reported to modulate mitophagy (Chen & Dorn, 2013; Gong et al, 2015) which could be responsible for reduced mitochondrial bioenergetics in Mfn2 KO cells. However, we observed no changes in the colocalization of LC3 with mitochondria in Mfn2 KO cells or in protein levels of VDAC and Hsp60, two well-known markers of mitochondrial mass (Appendix Fig S2G and H). Mfn1 and Mfn2 are ubiquitinated by Parkin and degraded upon induction of mitophagy (Gegg et al, 2010; Tanaka et al, 2010), but protein levels of Mfn1 remained unaltered in Mfn2 KO cells (Appendix Fig S2H). Thus, alterations in ER stress or mitophagy do not seem to be the mechanisms responsible for bioenergetics defects in Mfn2 KO cells. Ca2+ transfer from the ER to mitochondria is essential to maintain mitochondrial energetics (Cárdenas et al, 2010). Ca2+ released from the ER is taken up by mitochondria through the MCU in regions of close contact between the two organelles (Rizzuto et al, 1993; Rizzuto et al, 1998; Baughman et al, 2011; De Stefani et al, 2011). A small fraction of Mfn2 (5–10%) localizes in the ER where it interacts with mitochondrial Mfns and tethers the organelles together (de Brito & Scorrano, 2008). However, whether Mfn2
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