Article6 August 2013free access Source Data Mfn2 modulates the UPR and mitochondrial function via repression of PERK Juan Pablo Muñoz Juan Pablo Muñoz Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Saška Ivanova Saška Ivanova Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Jana Sánchez-Wandelmer Jana Sánchez-Wandelmer Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Paula Martínez-Cristóbal Paula Martínez-Cristóbal Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Eduard Noguera Eduard Noguera Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Ana Sancho Ana Sancho Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Angels Díaz-Ramos Angels Díaz-Ramos Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author María Isabel Hernández-Alvarez María Isabel Hernández-Alvarez Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author David Sebastián David Sebastián Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Caroline Mauvezin Caroline Mauvezin Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Manuel Palacín Manuel Palacín Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Antonio Zorzano Corresponding Author Antonio Zorzano Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Juan Pablo Muñoz Juan Pablo Muñoz Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Saška Ivanova Saška Ivanova Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Jana Sánchez-Wandelmer Jana Sánchez-Wandelmer Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Paula Martínez-Cristóbal Paula Martínez-Cristóbal Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Eduard Noguera Eduard Noguera Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Ana Sancho Ana Sancho Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Angels Díaz-Ramos Angels Díaz-Ramos Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author María Isabel Hernández-Alvarez María Isabel Hernández-Alvarez Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author David Sebastián David Sebastián Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Caroline Mauvezin Caroline Mauvezin Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Manuel Palacín Manuel Palacín Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Antonio Zorzano Corresponding Author Antonio Zorzano Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author Author Information Juan Pablo Muñoz1,2,3, Saška Ivanova1,2,3, Jana Sánchez-Wandelmer1,2,3, Paula Martínez-Cristóbal1,2,3, Eduard Noguera1,2,3, Ana Sancho1,2,3, Angels Díaz-Ramos1,2,3, María Isabel Hernández-Alvarez1,2,3, David Sebastián1,2,3, Caroline Mauvezin1,2,3, Manuel Palacín1,2 and Antonio Zorzano 1,2,3 1Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain 2Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain 3CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Barcelona, Spain *Corresponding author. Institute for Research in Biomedicine (IRB Barcelona), C/Baldiri Reixac 10, 08028 Barcelona, Spain. Tel.:+34 93 4037197; Fax:+34 93 4034717; E-mail: [email protected] The EMBO Journal (2013)32:2348-2361https://doi.org/10.1038/emboj.2013.168 Correction(s) for this article Mfn2 modulates the UPR and mitochondrial function via repression of PERK14 January 2014 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 Mitofusin 2 (Mfn2) is a key protein in mitochondrial fusion and it participates in the bridging of mitochondria to the endoplasmic reticulum (ER). Recent data indicate that Mfn2 ablation leads to ER stress. Here we report on the mechanisms by which Mfn2 modulates cellular responses to ER stress. Induction of ER stress in Mfn2-deficient cells caused massive ER expansion and excessive activation of all three Unfolded Protein Response (UPR) branches (PERK, XBP-1, and ATF6). In spite of an enhanced UPR, these cells showed reduced activation of apoptosis and autophagy during ER stress. Silencing of PERK increased the apoptosis of Mfn2-ablated cells in response to ER stress. XBP-1 loss-of-function ameliorated autophagic activity of these cells upon ER stress. Mfn2 physically interacts with PERK, and Mfn2-ablated cells showed sustained activation of this protein kinase under basal conditions. Unexpectedly, PERK silencing in these cells reduced ROS production, normalized mitochondrial calcium, and improved mitochondrial morphology. In summary, our data indicate that Mfn2 is an upstream modulator of PERK. Furthermore, Mfn2 loss-of-function reveals that PERK is a key regulator of mitochondrial morphology and function. Introduction Endoplasmic reticulum (ER) stress is triggered in response to a variety of stimuli that lead to a number of alterations of the ER, such as dysregulated redox homoeostasis, abnormal protein folding, and reduced calcium levels. Pathologies such as neurodegeneration, diabetes, cancer, and cardiovascular disease are characterized by the presence of ER stress (Hotamisligil, 2010). At least three proteins located in the ER membrane (PERK, IRE-1α, and ATF6) respond to ER stress stimuli by triggering the Unfolded Protein Response (UPR). These pathways activate several transcription factors involved in cell survival during the early stages of stress and in apoptotic cell death during acute or chronic stress. The PERK branch participates in the reduction of protein synthesis and activation of autophagy, apoptosis, and chaperone expression (Harding et al, 1999; Lu et al, 2004b). PERK-stimulated CHOP favors apoptosis (Harding et al, 2000; Rouschop et al, 2010), and, conversely, it has also been reported that CHOP protects neuronal cells after ischaemia reperfusion (Halterman et al, 2010). PERK activation decreases protein synthesis through eIF2α phosphorylation (Lu et al, 2004a), and, in addition, sustained PERK activation induces GADD34 expression, which relieves protein synthesis inhibition through eIF2α dephosphorylation (Novoa et al, 2001). The IRE-1α branch activates the expression of XBP-1 transcription factor, which is involved in the synthesis of chaperones and other ER proteins. The IRE-1α pathway is also required for autophagy activation (Ogata et al, 2006). Interestingly, XBP-1 deficiency promotes autophagy in neuronal cells (Hetz et al, 2009). This observation points to a complex role of the IRE-1α pathway in autophagic regulation. The transcription factor ATF6 is also stimulated during ER stress. In addition, calcium signals participate in this activation (Xu et al, 2004). This transcription factor induces XBP-1 and chaperones and participates in cell survival. Thus, reduced apoptosis occurs in response to ischaemia reperfusion in hearts overexpressing ATF6 (Doroudgar et al, 2009). Tumour cell survival also requires ATF6 through the activation of the mTOR pathway (Schewe and Aguirre-Ghiso, 2008; Doroudgar et al, 2009). ATF6 also mediates the synthesis of proteins and lipids required for ER biogenesis and participates in the synthesis of proteins required to activate ERAD, a mechanism for the degradation of ER proteins (Yamamoto et al, 2004; Maiuolo et al, 2011). Recent data demonstrate that PERK facilitates the stimulation of the ATF6 pathway (Teske et al, 2011). These studies indicate that UPR branches are exquisitely regulated and that several proteins are involved in the cross talk between branches. Mitofusin 2 (Mfn2) is a GTPase protein localized in the outer mitochondrial membrane and in mitochondrial-associated membranes. In addition to controlling ER morphology, this protein participates in mitochondrial fusion and regulates the transfer of calcium from the ER to mitochondria (Santel and Fuller, 2001; Legros et al, 2002; Bach et al, 2003; Ishihara et al, 2004; de Brito and Scorrano, 2008). Moreover, Mfn2 deficiency upregulates markers of the UPR in liver, skeletal muscle, and cultured cells (Ngoh et al, 2012; Sebastian et al, 2012), and normalization of ER stress by treatment with tauroursodeoxycholic acid ameliorates deficient insulin signalling in liver-specific Mfn2-knockout mice (Sebastian et al, 2012). Here we demonstrate that Mfn2 is a novel PERK regulator and that it is required for the normal activation of apoptosis and autophagy during the ER stress response. Moreover, Mfn2 deficiency causes mitochondrial dysfunction through sustained activation of PERK. Results Mfn2 knockout (KO) cells show massive ER expansion in response to ER stress On the basis of the observations linking Mfn2 to ER biology, we examined the response of Mfn2-deficient cells to agents that cause ER stress. To this end, mouse embryo fibroblasts (MEFs) were incubated with 1 μM thapsigargin (Tg) for 12 h in order to induce ER stress. Under these conditions, Mfn2 KO MEFs underwent dramatic ER expansion, characterized by multilamellar structures, compared to wild-type (WT) MEFs (Figure 1A and B). To provide further evidence of the effects of ER stress on ER morphology, cells were transfected with a plasmid encoding an RFP protein that is localized in the ER luminal compartment (ER-RFP), and thereafter treated with Tg and visualized by epifluorescence microscopy. Under basal conditions, Mfn2 ablation caused ER fragmentation, which was detectable with the luminal ER marker (Supplementary Figure S1A). This finding is in keeping with prior data (de Brito and Scorrano, 2008). Supplementary Figure S1A also shows increased ER luminal expansion during ER stress (induced by Tg, tunicamycin, or brefeldin A) in Mfn2 KO cells compared to WT cells. Movie 1 (Supplementary Data) shows the formation of the expanded ER in Mfn2 KO cells upon the induction of ER stress. Labelling of the ER membrane by transfection of Sec61β-GFP also showed a dramatic ER expansion in Mfn2 KO cells in response to Tg (Figure 1C). However, labelling of ER membranes with Sec61β-GFP did not show alterations in ER morphology in these cells under basal conditions (Figure 1C), which is in keeping with prior data in COS cells (Friedman et al, 2011). No alterations in ER morphology were detected in Mfn1 KO cells in response to Tg (Figure 1C). Reexpression of Mfn2 in Mfn2 KO cells rescued normal mitochondrial morphology and prevented ER expansion in response to ER stress (Supplementary Figure S1B–E). Figure 1.Mfn2 ablation promotes abnormal ER expansion during ER stress conditions. (A) WT or Mfn2 KO MEFs (Mfn2 KO cells) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology. Scale bar: 1 μm. (B) EM images of Tg-treated Mfn2 KO cells show accumulation of ER membrane stacking. Scale bar: 1 μm. (C) WT, Mfn2 KO or Mfn1 KO cells were transfected with the Sec61β-GFP plasmid and treated with 1 μM Tg for 24 h. Confocal microscopy images show ER vacuolization in Mfn2 KO cells treated with Tg. Scale bar: 10 μm. Insets show × 10 zoomed images. Scale bar: 5 μm. (D) WT and Mfn2 KO cells were treated with Tg 1 μM for 3 h and then incubated with brefeldin A-bodipy to stain ER and Golgi. Representative flow-cytometry histograms (upper panel). Mean fluorescence intensity was used to quantify ER expansion (n=5) (lower panel). Data are mean±s.e.m. *P<0.05 versus WT group. Download figure Download PowerPoint The enhanced ER expansion in Mfn2 KO cells upon Tg treatment was further documented by labelling ER–Golgi with brefeldin A-bodipy (Hetz et al, 2006) (Figure 1D). Deficiency of Mfn1 or Mfn2 was also induced in MEFs by lentiviral expression of shRNAs, which caused mitochondrial fragmentation (Supplementary Figure S2A and B). Labelling of the ER membrane by transfection of Sec61β-GFP also showed marked ER expansion in Mfn2-silenced MEF cells treated with Tg (Supplementary Figure S2C). Mfn1 deficiency did not cause ER expansion upon Tg treatment (Supplementary Figure S2C). Mfn2 KO cells show defective apoptosis in response to ER stress On the basis of the initial observations indicating that Mfn2 ablation causes abnormal ER expansion in response to ER stress, next we examined whether Mfn2 loss-of-function also alters the biological responses driven by this stress. In this regard, apoptosis was induced through activation of caspase 3 (Rao et al, 2002) in response to ER stress. Mfn2 loss-of-function significantly enhanced caspase activity in MEFs and in C2C12 cells under basal conditions (Supplementary Figure S3A) (see Supplementary Figures S2 and S4 for details on Mfn2 deficiency induced in these cells). WT MEFs showed caspase 3 activation and increased caspase activity in response to tunicamycin, brefeldin A, or Tg (Figure 2A–C). Under these conditions, Mfn2 ablation greatly reduced caspase 3 processing and caspase activity (Figure 2A–C), and reexpression of Mfn2 normalized the activation of this enzyme (Supplementary Figure S3B). Reduced apoptosis was also detected by cytometry assays (subG1 DNA fragmentation) in Mfn2 KO cells (Figure 2D). Mfn1 KO cells showed reduced caspase 3 processing and caspase activity upon Tg, tunicamycin, or brefeldin A treatment, although the values observed were higher than in Mfn2 KO cells (Supplementary Figure S3C and D). Silencing of Mfn2 in 3T3-L1 or in C2C12 cells also reduced caspase 3 processing in response to ER stress (Figure 2E and Supplementary Figure S3F). In contrast, agents that induce apoptosis independently of ER stress, such as staurosporine or TNFalpha/cycloheximide (CHX), activated caspase 3 processing to a greater extent in Mfn2 KO cells compared to WT cells (Supplementary Figure S3E). Figure 2.Mfn2 ablation prevents caspase activation during ER stress and promotes paraptosis-like cell death. (A) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h. Total and cleaved caspase 3 levels were detected by western blot. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group. (B, C) WT and Mfn2 KO cells were treated with 0.5 μg/ml tunicamycin (Tm), 100 ng/ml brefeldin A (Bref), or 1 μM Tg for 24 h. Total and cleaved caspase 3 levels were detected by western blot (B) and caspase activity (C) by measurement of DEVD-AFC substrate processing. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group. (D) Flow-cytometry analysis of the Sub G1 DNA fragmentation in methanol-fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h. Data are given as mean±s.e.m. (n=3). *P<0.05 versus WT group. (E) Scr (stably expressing scrambled shRNA) and Mfn2 knockdown (KD) (stably expressing shRNA directed against Mfn2) 3T3-L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h. Total and cleaved caspase 3 were detected by western blot. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group. (F) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h. Lactate dehydrogenase (LDH) release was analyzed by flow cytometry to assess necrotic cell death. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group. (G) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z-VAD-fmk and stained for annexin V/PI. Data are mean±s.e.m. (n=4). *P<0.05 versus WT+Tg group. (H) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h (3 h of pre-incubation with CHX). Florescence microscopy images show that CHX prevents cytoplasmic vacuolization. Scale bar: 10 μm. (I) WT (black circles) and Mfn2 KO cells (white circles) were incubated with 1 μM Tg for varying times, and ALIX protein was detected by western blot. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group.Source data for this figure is available on the online supplementary information page. Source Data for Figure 2 [embj2013168-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint Next, we analyzed whether Mfn2 KO cells die as a result of another form of cell death upon ER stress. Lactate dehydrogenase release (an index of cell necrosis) increased in control cells during ER stress, but this did not occur in Mfn2-ablated cells (Figure 2F). We explored whether Mfn2 KO cells undergo a paraptotic-like death, which is characterized by swelling of the mitochondria and of the ER, reduced ALIX expression (Sperandio et al, 2000; Yoon et al, 2010), and involves externalization of phosphatidylserine (PS) independently of caspases (Wang et al, 2004). In keeping with this, Mfn2 KO cells became annexin V-positive in response to Tg, and PS externalization was not inhibited with the pan-caspase inhibitor z-VAD (Figure 2G). In addition, in response to ER stress, Mfn2 KO cells underwent vacuolization, which was rescued by CHX (Figure 2H), and showed lower ALIX protein expression (Figure 2I). Propidium iodide (PI) permeability assays coupled to annexin V labelling in control and Mfn2 KO cells showed increased PI/annexin V labelling in response to Tg. This labelling was inhibited by addition of CHX in Mfn2 KO cells but not in WT cells (Supplementary Figure 3G). In summary, our data show that Mfn2 loss-of-function impairs the normal activation of apoptosis during ER stress, and instead paraptosis-like cell death is triggered. This observation suggests that Mfn2 plays a key role in the maintenance of a normal response to ER stress. Mfn2 KO cells show defective autophagy in response to ER stress ER stress also activates autophagy (Ogata et al, 2006), and the accumulation of multilamellar ER structures detected in Mfn2−/− cells (Figure 1) pointed to alterations in the clearance process. To analyze autophagy in cells subjected to ER stress, control and Mfn2 KO cells were treated with Tg for a range of times, and autophagy was assayed by immunodetection of LC3b-I and LC3b-II. ER stress caused an increased abundance of LC3b-II in control cells, which is coherent with the activation of autophagosome formation (Figure 3A). Under basal conditions, Mfn2 KO cells showed a slightly increased LC3b-II abundance (Figure 3A). ER stress-induced LC3b-II was inhibited in Mfn2 KO cells (Figure 3A), thereby suggesting a defect in autophagy progression. Incubation with the lysosomal inhibitor bafilomycin A1 increased LC3b-II levels in WT cells but not in Mfn2 KO ones (Figure 3B). Mfn2 reexpression normalized LC3b-II levels in Mfn2 KO cells treated with Tg (Supplementary Figure S5A). These observations indicate that autophagosomal formation is impaired during ER stress upon ablation of Mfn2 in MEFs. Figure 3.Mfn2 loss-of-function decreases autophagy in response to ER stress. (A, upper panel) WT (black circles) or Mfn2 KO cells (white circles) were treated with 1 μM Tg for a range of times. (A, lower) Densitometric quantification. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group; #P<0.05 versus WT non-Tg-treated group. (B, upper panel) WT or Mfn2 KO cells were treated with Tg for 0, 6 or 12 h, in the presence or absence of Bafilomycin (Baf, 100 nM). LC3b-I and LC3b-II expression was measured by western blot. (B, lower panel) Densitometric quantification of LC3b-II levels (relative to tubulin). Data are mean±s.e.m. (n=3). *P<0.05 versus WT+Baf+Tg group. (C) WT or Mfn2 KO cells stably expressing mCherry-GFP-LC3b were treated with 1 μM Tg, 100 ng/ml Brefeldin (Bref), or 0.5 μg/ml tunicamycin (Tm) for 24 h and examined by confocal microscopy. Scale bar: 10 μm. (D) Quantification of mCherry+/GFP- (mCherry-LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P<0.05 versus WT group. (E) Acidic compartments of WT or Mfn2 KO cells were stained with Lysotracker Green and analyzed by flow cytometry. Data are given as mean±s.e.m. (n=3). *P<0.05 versus WT group. (F) LAMP1 expression was immunodetected in WT and Mfn2 KO cells in basal conditions. (G) Expression of Beclin-1 or LC3b transcripts in WT or Mfn2 KO cells treated with or without 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3 [embj2013168-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint To obtain further insight into the autophagic steps that may be altered in Mfn2-deficient cells, we expressed mCherry-EGFP-LC3 protein in these cells. ER stress markedly enhanced the abundance of acidic autophagolysosomes—detected as red punctuates in mCherry-EGFP-LC3B-expressing cells—which is consistent with increased autophagic activity (Figure 3C and D). Under these conditions, Mfn2 ablation reduced the abundance of acidic autophagolysosomes during ER stress induced by Tg, brefeldin A, or tunicamycin (Figure 3C and D). mCherry-EGFP-LC3B remained as yellow punctuate structures in Mfn2 KO cells, thereby indicating its presence in autophagosomes (Figure 3C and D). In keeping with these data, reduced Lysotracker staining and LAMP1 expression were detected in these cells (Figure 3E and F). The expression of the autophagic genes LC3b or beclin-1 was also dysregulated in Mfn2 KO cells upon ER stress (Figure 3G). In contrast to the data obtained in Mfn2 KO cells, ablation of Mfn1 did not alter ER stress-induced autophagy (Supplementary Figure S5B–D), and Lysotracker staining was enhanced compared to WT cells (Supplementary Figure S5E). Autophagy was also analyzed in 3T3-L1 fibroblasts and in C2C12 myoblasts upon Mfn2 silencing (Supplementary Figure S4). Reduced autophagic activity, assessed by LC3b-II abundance or the degradation of long-lived proteins, was detected in Mfn2-deficient cells in response to ER stress (Supplementary Figure S5F–H). In parallel, a reduced degradation rate of long-lived proteins was detected in Mfn2-deficient 3T3-L1 cells in response to tunicamycin (Supplementary Figure S5H). In all, our data show that Mfn2 loss-of-function impairs the normal progression of autophagy during ER stress. This loss-of-function is characterized by reduced autophagosome formation, reduced lysosome abundance, and reduced autophagolysosome formation. Mfn2 deficiency dysregulates PERK, IRE-1, and ATF6 UPR branches in response to ER stress To understand how Mfn2 ablation leads to a disrupted biological response to ER stress, next we monitored the UPR response (Figure 4A). Mfn2 KO cells showed enhanced PERK phosphorylation as well as induced expression of downstream signalling proteins, ATF4, GADD34, and CHOP (Figure 4B and C). These observations indicate enhanced PERK branch signalling during ER stress in Mfn2 KO cells. Under these conditions, the activation of eIF2α was reduced in these cells during ER stress (Figure 4B and C). Tunicamycin also caused overactivation of the PERK pathway in Mfn2 KO cells, which was characterized by enhanced phosphorylation of eIF2α, and greater ATF4 expression (Figure 4D). Figure 4.Mfn2 modulates the UPR response. (A) Scheme on UPR branches. (B) Immunodetection of p-PERK,PERK, GADD34, p-eIF2α, eIF2α, ATF4, CHOP, and XBP-1s in WT and Mfn2 KO cells treated with 1 μM
