Human Fis1 regulates mitochondrial dynamics through inhibition of the fusion machinery

Article6 March 2019Open Access Source DataTransparent process Human Fis1 regulates mitochondrial dynamics through inhibition of the fusion machinery Rong Yu Rong Yu Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden Search for more papers by this author Shao-Bo Jin Shao-Bo Jin Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Urban Lendahl Urban Lendahl Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Monica Nistér Corresponding Author Monica Nistér [email protected] orcid.org/0000-0002-1261-3790 Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden Search for more papers by this author Jian Zhao Corresponding Author Jian Zhao [email protected] orcid.org/0000-0001-5659-730X Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden Search for more papers by this author Rong Yu Rong Yu Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden Search for more papers by this author Shao-Bo Jin Shao-Bo Jin Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Urban Lendahl Urban Lendahl Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Monica Nistér Corresponding Author Monica Nistér [email protected] orcid.org/0000-0002-1261-3790 Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden Search for more papers by this author Jian Zhao Corresponding Author Jian Zhao [email protected] orcid.org/0000-0001-5659-730X Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden Search for more papers by this author Author Information Rong Yu1, Shao-Bo Jin2, Urban Lendahl2, Monica Nistér *,1,‡ and Jian Zhao *,1,‡ 1Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden 2Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +46 8 51770309; E-mail: [email protected] *Corresponding author. Tel: +46 8 51770585; E-mail: [email protected] The EMBO Journal (2019)38:e99748https://doi.org/10.15252/embj.201899748 See also: M Liesa et al (April 2019) 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 Mitochondrial dynamics is important for life. At center stage for mitochondrial dynamics, the balance between mitochondrial fission and fusion is a set of dynamin-related GTPases that drive mitochondrial fission and fusion. Fission is executed by the GTPases Drp1 and Dyn2, whereas the GTPases Mfn1, Mfn2, and OPA1 promote fusion. Recruitment of Drp1 to mitochondria is a critical step in fission. In yeast, Fis1p recruits the Drp1 homolog Dnm1p to mitochondria through Mdv1p and Caf4p, but whether human Fis1 (hFis1) promotes fission through a similar mechanism as in yeast is not established. Here, we show that hFis1-mediated mitochondrial fragmentation occurs in the absence of Drp1 and Dyn2, suggesting that they are dispensable for hFis1 function. hFis1 instead binds to Mfn1, Mfn2, and OPA1 and inhibits their GTPase activity, thus blocking the fusion machinery. Consistent with this, disruption of the fusion machinery in Drp1−/− cells phenocopies the fragmentation phenotype induced by hFis1 overexpression. In sum, our data suggest a novel role for hFis1 as an inhibitor of the fusion machinery, revealing an important functional evolutionary divergence between yeast and mammalian Fis1 proteins. Synopsis Yeast Fis1 regulates mitochondrial dynamics by recruiting dynamin-related fission factors, but the role of mammalian Fis1 has remained elusive. Human Fis1 (hFis) promotes Drp1- and Dyn2-independent mitochondrial fragmentation by inhibiting mitofusins and OPA1 GTPase activity, revealing evolutionary divergence. Fission factors Drp1 and Dyn2 are dispensable for hFis1-mediated mitochondrial fragmentation. hFis1 binds pro-fusion mitofusins Mfn1 and Mfn2, and OPA1. hFis1 inhibits the GTPase activities of Mfn1, Mfn2, and OPA1, but not of Drp1 and Dyn2. Overexpression of hFis1 reduces mitochondrial fusion, whereas knockdown of hFis1 enhances mitochondrial fusion. Introduction Mitochondria are dynamic organelles that constantly alter their shape in response to changes in cellular physiological conditions or as a result of pathophysiology. The mitochondrial network is determined through a balance between mitochondrial fission and fusion events, collectively referred to as mitochondrial dynamics, which produces fragmented or fused mitochondria, respectively and is closely associated with mitochondrial function (Chan, 2012; Schrepfer & Scorrano, 2016). Mitochondrial fragmentation can be caused by stimulation of fission activity and/or inhibition of mitochondrial fusion. Conversely, inhibition of fission and/or stimulation of fusion leads to mitochondrial elongation. An aberrant balance between fission and fusion has been observed in a number of human diseases and impacts a broad range of cellular functions (Ong & Hausenloy, 2010; Romanello et al, 2010; Yoon et al, 2011; Rehman et al, 2012; Youle & van der Bliek, 2012; Schrepfer & Scorrano, 2016; Chen & Chan, 2017; Trotta & Chipuk, 2017). Mitochondria are evolutionarily derived from the endosymbiosis of alpha-proteobacteria, which likely was a unique event in the history of eukaryotic evolution. The mitochondrial division machinery probably arose considerably later, but there are indeed a number of highly conserved proteins regulating mitochondrial dynamics in yeast and mammals (Okamoto & Shaw, 2005; Zhao et al, 2013; Kraus & Ryan, 2017; Ramachandran, 2018), indicating that at least some of the fundamental mechanisms controlling mitochondrial dynamics have a long evolutionary history. Several of these proteins are related to dynamin, a GTPase which pinches off vesicles from the cell surface (Pagliuso et al, 2018). Thus, in mammals, mitochondrial fission is executed by the GTPases dynamin-related protein 1 (Drp1) and dynamin-2 (Dyn2) (Smirnova et al, 1998; Lee et al, 2016). Drp1 is primarily distributed in the cytosol and translocated to the mitochondrial surface during mitochondrial division, where it assembles into higher-order complexes at endoplasmic reticulum (ER)–mitochondrial contact sites to wrap around the mitochondria inducing mitochondrial fission via its GTPase activity. The final abscission step is carried out by Dyn2 (Otera et al, 2013; Friedman & Nunnari, 2014; Lee et al, 2016; Mishra & Chan, 2016; Prudent & McBride, 2016; Kraus & Ryan, 2017). Mitochondrial fusion is regulated by three other dynamin-related GTPases: Mitofusins (Mfn1 and Mfn2), which are located in the mitochondrial outer membrane (MOM) and optic atrophy 1 (OPA1), located in the mitochondrial inner membrane (MIM) (Willems et al, 2015; Schrepfer & Scorrano, 2016). Mfn1 and Mfn2 are essential for fusion of mitochondrial outer membranes, whereas the mitochondrial inner membrane-associated OPA1 is required for fusion of the inner membranes (Willems et al, 2015; Schrepfer & Scorrano, 2016; Pagliuso et al, 2018). The pro-fission GTPase Drp1 does not contain a membrane-localizing pleckstrin homology (PH) domain, transmembrane (TM), or any other membrane-anchoring domain and therefore needs to be actively recruited to the mitochondrial surface by MOM-anchored receptors in order to execute its function. In yeast, recruitment of Dnm1p (the yeast Drp1 homolog) to mitochondria is carried out by the evolutionarily conserved membrane-anchored protein Fis1p (the yeast Fis1 homolog) through interaction with Mdv1p and Caf4p, promoting mitochondrial fission (Hoppins et al, 2007). As Fis1 is evolutionarily conserved from yeast to humans, mammalian Fis1 is believed to have a similar role to its homolog Fis1p in yeast, i.e., to recruit Drp1 to mitochondria and promote mitochondrial fission. In keeping with a pro-fission function, overexpression of Fis1 causes extensive mitochondrial fragmentation and depletion of Fis1 causes mitochondrial elongation (James et al, 2003; Yoon et al, 2003; Stojanovski et al, 2004; Jofuku et al, 2005; Yu et al, 2005). Interestingly, while Mitofusins, OPA1, Drp1, Dyn2, and Fis1 are highly conserved between yeast and mammals, Mdv1p and Caf4p lack obvious mammalian homologs (Okamoto & Shaw, 2005; Hoppins et al, 2007; Bui & Shaw, 2013; Zhao et al, 2013). Instead, mammalian cells contain a set of proteins, Mff and MIEF1/2 (MiD51/49), which are anchored in the mitochondrial outer membrane and serve to mediate recruitment of Drp1 to the mitochondrial surface (Gandre-Babbe & van der Bliek, 2008; Otera et al, 2010; Palmer et al, 2011; Zhao et al, 2011; Koirala et al, 2013; Liu et al, 2013; Loson et al, 2013; Yu et al, 2017). Furthermore, increased or decreased levels of Fis1 do not seem to regulate the amount of Drp1 at mitochondria in mammalian cells (Suzuki et al, 2003; Lee et al, 2004), and according to several reports, Fis1 is largely dispensable for Drp1 recruitment to mitochondria and Drp1-mediated fission (Otera et al, 2010, 2013; Bui & Shaw, 2013; Koirala et al, 2013; Loson et al, 2013; Osellame et al, 2016). Given the absence of Mdv1p and Caf4p in mammals, the actual role of hFis1 in the mitochondrial fission process remains enigmatic (Okamoto & Shaw, 2005; Hoppins et al, 2007; Bui & Shaw, 2013; Otera et al, 2013; Zhao et al, 2013). Some reports argue that mammalian Fis1 interacts with Drp1 and can serve as a receptor for Drp1 (James et al, 2003; Yoon et al, 2003), whereas other studies reveal that mammalian Fis1 is largely dispensable for Drp1 recruitment (Suzuki et al, 2003; Lee et al, 2004). In this study, we address the functional role of human Fis1 (hFis1). We show that both Drp1 and Dyn2 are dispensable for hFis1-mediated mitochondrial fragmentation. hFis1 instead interacts robustly with Mfn1, Mfn2, and OPA1, and overexpression of hFis1 blocks their GTPase activity, leading to reduced mitochondrial fusion and shifting the balance of mitochondrial dynamics toward fission. In conclusion, our data suggest a novel function for hFis1 in regulating mitochondrial dynamics in mammals and reveal an evolutionary divergence of Fis1 function between yeast and mammals. Results Drp1 and Dyn2 are largely dispensable for hFis1-mediated mitochondrial fragmentation We first assessed whether Fis1-induced fragmentation was dependent on the presence of Drp1. Overexpression of human Fis1 (hFis1) triggered extensive mitochondrial fragmentation in wild-type (WT) 293T cells (i.e., in the presence of endogenous Drp1), resulting in small and punctuate mitochondria in most (92 ± 0.4%) cells compared to empty vector control (1.5 ± 2.3%) (Fig 1A, upper panel; summarized in Fig 1E), in line with several previous reports (James et al, 2003; Yoon et al, 2003; Stojanovski et al, 2004; Yu et al, 2005; Alirol et al, 2006). To explore the role of Drp1 in hFis1-mediated fragmentation, we next generated a DRP1-deficient 293T cell line (Drp1−/−) using CRISPR/Cas9-mediated gene editing (Appendix Fig S1), and ablation of Drp1 as expected halted mitochondrial fission, resulting in a super-fused tubular mitochondrial network (Fig 1A, lower left panel). Overexpression of hFis1, however, still efficiently induced mitochondrial fragmentation in the Drp1−/− 293T cells (85.9 ± 1.2%), although there was a slight decrease in the number of cells with fragmented mitochondria in comparison with WT 293T cells (Fig 1A, lower right panel; summarized in Fig 1E). These results indicate that Drp1 is largely dispensable for mitochondrial fragmentation induced by hFis1. Figure 1. Drp1 is not required for hFis1-induced mitochondrial fragmentation Confocal images of mitochondrial morphology in wild-type (WT) and Drp1−/− 293T cells transfected with empty vector (left panel) and Myc-hFis1 (right panel), stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Quantitative analyses of fragmented mitochondria size (mean area (μm2) per mitochondrion) after Myc-hFis1 overexpression in WT and Drp1−/− 293T cells using ImageJ software (Particle analysis) in three independent experiments. In each cell, only dispersed individual mitochondria were analyzed. The total number of mitochondria (mito) analyzed is indicated for each condition. Quantitative analysis of mean mitochondria number per cell in WT and Drp1−/− 293T cells overexpressing Myc-hFis1 using Image J software (Particle analysis) in three independent experiments. Schematic representation of hFis1 mutants used in this experiment. Percentages of cells with indicated mitochondrial morphologies in WT and Drp1−/− 293T cells transfected with empty vector (control), Myc-hFis1 and either Myc- or GFP-tagged mutants as indicated in three independent experiments. Data information: Data are expressed as means ± SEM and were statistically analyzed by Student's t-test. n represents the number of cells analyzed (B, C, and E). Download figure Download PowerPoint To further investigate whether hFis1-induced fragmentation could also occur in other types of human cells in the absence of endogenous Drp1, we generated a DRP1-deficient (Drp1−/−) HeLa cell line using CRISPR/Cas9-mediated gene editing (Appendix Fig S2). Similarly, this led to a super-fused tubular mitochondrial network (Appendix Fig S2D), and hFis1 overexpression still triggered mitochondrial fragmentation in Drp1−/− HeLa cells (38.8 ± 2.3%) (Fig EV1). Overall, this confirms that hFis1 can promote mitochondrial fragmentation in the absence of Drp1, but loss of Drp1 partially reduces hFis1-induced fragmentation. Click here to expand this figure. Figure EV1. Drp1 is largely dispensable for mitochondrial fragmentation induced by hFis1 in HeLa cells (related to Fig 1) Confocal images of mitochondrial morphology in wild-type and Drp1−/− HeLa cells transfected with empty vector (left panel) and Myc-hFis1 (right panel), stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Insets represent high magnification views of the boxed areas. Percentages (mean ± SEM) of cells with indicated mitochondrial morphologies in wild-type and Drp1−/− HeLa cells transfected with empty vector (control) or Myc-hFis1 in three independent experiments (n represents the number of cells analyzed). Download figure Download PowerPoint While hFis1-induced fragmentation occurred also in the absence of Drp1, there were some noticeable differences between overexpression of hFis1 in wild-type (control) and Drp1−/− (deficient) cells: The size of fragmented (punctate) mitochondria was larger with an average size ~0.48 ± 0.01 μm2 in Drp1−/− cells compared to an average size of ~0.28 ± 0.01 μm2 in WT 293T cells. At the same time, the number of mitochondria was lower in Drp1-deficient cells (Fig 1B and C), i.e., mitochondria were more fragmented in WT cells, whereas most mitochondria in Drp1−/− cells appeared as larger spheres. A similar phenotype was also observed in Drp1−/− HeLa cells expressing Myc-hFis1 (Fig EV1). These subtle differences in mitochondrial phenotype may be attributed to the continuously ongoing Drp1-mediated fission occurring in WT but being blocked in Drp1−/− cells. To further elaborate on the role of hFis1 in mitochondrial dynamics, we generated several hFis1 mutants (Fig 1D) and tested their effects on mitochondrial morphology in WT and Drp1−/− 293T cells. As previously reported (Yoon et al, 2003; Stojanovski et al, 2004), the deletion mutant Myc-hFis1ΔTM/C, lacking the TM domain and the C-terminal tail (from residues 123–152), was diffusely distributed in the cytosol and had no effect on mitochondrial morphology in WT and Drp1−/− 293T cells (Figs 1E and EV2A). In contrast, GFP-hFis1Δ1–121 (lacking the cytosolic domain of hFis1, i.e., consisting of GFP fused to the hFis1 TM domain and C-terminal tail including residues 122–152) was still localized to the mitochondrial surface (Stojanovski et al, 2004) and triggered mitochondrial aggregation in both WT (47 ± 3.1%) and Drp1−/− 293T cells (51.1 ± 4.1%) (Figs 1E and EV2A). These data indicate that both the cytosolic domain and the C-terminal region including the TM domain and C-terminal tail are required for hFis1-induced mitochondrial fragmentation regardless of whether Drp1 is present or not in cells. We further evaluated the effects of the ER (endoplasmic reticulum) mistargeting mutant Myc-hFis1K149/151A (i.e., full-length hFis1 carrying lysine to alanine mutations in the residues 149 and 151) (Stojanovski et al, 2004; Delille & Schrader, 2008) on mitochondrial morphology. When expressed in WT and Drp1−/− 293T cells, Myc-hFis1K149/151A significantly reduced the number of cells with fragmented mitochondria to 24.2 ± 0.8% in WT cells and to 7.9 ± 1.6% in Drp1−/− cells (Figs 1E and EV2A). To further explore the function of the TM domain and C-terminal tail of hFis1, we generated a hFis1/yTom5C mutant, in which the TM and C-terminal tail domains of hFis1 were replaced with the corresponding regions of the mitochondrial C-tail-anchored yeast protein Tom5, as previously reported (Jofuku et al, 2005). Expression of this construct largely abolished hFis1-induced mitochondrial fragmentation in both WT and Drp1−/− 293T cells (Figs 1E and EV2A), implying that hFis1 fission function was lost in spite of the fact that the mutant mainly localized on mitochondria (Fig EV2A). In summary, these data establish that intact N-terminal cytosolic as well as C-terminal TM and tail domains are all required for hFis1-mediated mitochondrial fragmentation to occur both in the presence and in the absence of Drp1. Click here to expand this figure. Figure EV2. Subcellular localization and mitochondrial phenotypes of hFis1 mutants; hFis1 overexpression does not induce apoptosis and autophagy in WT and Drp1−/− 293T cells (related to Fig 1) A. Representative confocal images of mitochondrial morphology in wild-type (left panel) and Drp1−/− 293T cells (right panel) transfected with different hFis1 mutants as indicated, stained with MitoTracker (red) followed by immunostaining with anti-Myc (green) or anti-GFP antibody (green). B, C. Overexpression of WT hFis1 and mutants does not affect apoptosis and autophagy as analyzed by immunoblotting with PARP and LC3B antibodies. WT 293T (B) and Drp1−/− 293T (C) cells were transiently transfected with 0.5 μg of empty vector, Myc-hFis1, Myc-hFis1ΔTM/C, GFP-hFis1Δ1–121, Myc-hFis1K149/151A, or Myc-hFis1/yTom5C plasmid. Cells were harvested after transfection for 20 h and analyzed by Western blotting with the indicated antibodies. Source data are available online for this figure. Download figure Download PowerPoint To extend the observations on the mitochondrial phenotype, we assessed the consequences of hFis1 overexpression on apoptosis and autophagy in WT 293T and Drp1−/− 293T cells. As shown in Fig EV2B and C, overexpression of WT hFis1 and mutants did not increase the amounts of cleaved PARP or LC3B-II, confirming that the observed hFis1-induced mitochondrial fragmentation is not an unspecific effect resulting from apoptosis or autophagy that is potentially associated with excessive expression of mitochondrial proteins. Dynamin-2 (Dyn2) has recently been reported to regulate the final abscission step of mitochondrial fission after Drp1 recruitment and polymerization (Lee et al, 2016). We therefore tested whether Dyn2 is required for hFis1-mediated mitochondrial fragmentation. In WT 293T cells, depletion of Dyn2 by small interfering RNA (siRNA) resulted in a moderately elongated mitochondrial network in 16.5 ± 2.7% of cells compared to control (3.4 ± 0.8%, P = 0.0006) (Fig 2A–C), in line with previous observations (Lee et al, 2016). Overexpression of hFis1 in Dyn2-deficient cells still caused extensive mitochondrial fragmentation in 78.9 ± 2.8% of cells, comparable to the effect of hFis1 overexpression in WT 293T cells (84.1 ± 3.0%) (Fig 2A–C), indicating that Dyn2 is also dispensable for hFis1-mediated fragmentation. Moreover, depletion of Dyn2 by siRNA in Drp1−/− 293T cells did not prevent mitochondrial fragmentation induced by hFis1 overexpression in most of cells (90.6 ± 3.5%) compared to controls (94.7 ± 1.7%) (Fig 2D–F), indicating that simultaneous depletion of Drp1 and Dyn2 does not prevent hFis1-induced fragmentation either. Taken together, these data show that hFis1 can induce mitochondrial fragmentation under Drp1/Dyn2-deficient conditions. Figure 2. Dyn2 is not required for hFis1-induced mitochondrial fragmentation Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA (control) and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels) as indicated. Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Western blot of Dyn2, Myc-hFis1, Drp1, and GAPDH in WT 293T cells collected from the experiments in (A). Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector or Myc-hFis1 in three independent experiments. Confocal images of mitochondrial morphology in Drp1−/− 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels). Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Representative examples of tubular, fragmented, and tubular cluster phenotypes are indicated. Western blot of Dyn2, Myc-hFis1, and GAPDH in Drp1−/− 293T cells collected from the experiments in (D). Percentages of cells with indicated mitochondrial morphologies in Drp1−/− 293T cells treated with scrambled siRNA or Dyn2 siRNA, followed by transfection with empty vector or Myc-hFis1 as indicated in three independent experiments. Data information: Data are expressed as means ± SEM and were statistically analyzed by Student's t-test. n represents the number of cells analyzed (C and F). Source data are available online for this figure. Source Data for Figure 2 [embj201899748-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Mfn1, Mfn2, and OPA1 are major hFis1-binding proteins The data described above indicate that mitochondrial fission can occur in a Drp1- and Dyn2-dispensable manner and that hFis1 plays a role as an alternative fission mediator. To assess the role of hFis1 further, we examined whether hFis1 instead influenced the mitochondrial pro-fusion machinery. Depletion of hFis1 by siRNA in Drp1−/− 293T cells altered mitochondrial morphology, resulting in a more super-fused tubular clustering phenotype of mitochondria localized asymmetrically to one side of the nucleus (Fig 3A). This phenotype was quite reminiscent of the phenotype seen in Drp1−/− cells overexpressing either of the pro-fusion GTPases Mfn1, Mfn2, or OPA1 (Fig 3B and C). Figure 3. Mfn1, Mfn2, and OPA1 but not Drp1 and Dyn2 are major hFis1-binding partners A. Confocal images of mitochondrial morphology in Drp1−/− 293T cells transfected with scrambled siRNA (control) or with hFis1-siRNA and then stained with MitoTracker (red). B. Confocal images of mitochondrial morphology in Drp1−/− 293T cells transfected with either Mfn1-Myc, Mfn2-Myc, or OPA1-Myc, stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). C. Percentages (mean ± SEM) of cells with indicated mitochondrial morphologies in Drp1−/− 293T cells transfected with either scrambled siRNA (Ctr), hFis1 siRNA, Mfn1-Myc, Mfn2-Myc, or OPA1-Myc in three independent experiments for each condition (n represents the number of cells analyzed). D. hFis1 interacts with Mfn1, Mfn2, and OPA1 as well as Drp1, but not with Dyn2 at endogenous levels following chemical crosslinking. Wild-type (WT) and Drp1−/− 293T cells were in vivo crosslinked with 1% formaldehyde (FA), and cell lysates were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies. E, F. hFis1 binds to Mfn1, Mfn2, and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from WT 293T (E) and HeLa (F) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies. G, H. Interaction of hFis1 with Mfn1/2 and with OPA1 are independent events. WT 293T cells were treated with control, OPA1 (G), or Mfn1 plus Mfn2 (H) siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies. I. Interactions between Mfn1/2 and OPA1 occur independent of hFis1. WT 293T cells were treated with control or hFis1 siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG (negative control) or mouse anti-OPA1 antibody as indicated, followed by immunoblotting with indicated antibodies. J. Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression. 293T cells were transfected with empty vector or Myc-hFis1, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG or mouse anti-Mfn1 antibody, followed by immunoblotting with indicated antibodies. Source data are available online for this figure. Source Data for Figure 3 [embj201899748-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint These results may imply a functional link between hFis1 and the pro-fusion machinery. We first addressed this by examining the interaction of hFis1 with the pro-fission and pro-fusion GTPases. A weak interaction between endogenous hFis1 and Drp1 was observed following in vivo chemical crosslinking (Hajek et al, 2007; Zhao et al, 2011) before co-immunoprecipitation (co-IP) (Fig 3D), in agreement with previous studies (Yoon et al, 2003; Yu et al, 2005). No interactions were detected between hFis1 and Dyn2, Miro1 (a MOM-anchored protein) (Fransson et al, 2003) or MTGM (a MIM-anchored protein, also known as Romo1) (Chung et al, 2006; Zhao et al, 2009), irrespective of whether Drp1 was present or not (Fig 3D). In contrast, hFis1 robustly interacted with Mfn1, Mfn2, and OPA1 at endogenous levels under conditions of chemical crosslinking (Fig 3D). To further assess whether the interaction of hFis1 with Mfn1, Mfn2, and OPA1 was transient or stable, we also performed co-IP in the absence of chemical crosslinking. hFis1 still efficiently bound to Mfn1, Mfn2, and OPA1 at endogenous levels to form stable protein complexes, whereas no interactions were detected between hFis1 and either Drp1, Dyn2, Miro1, or MTGM (Fig 3E). A similar interaction pattern at endogenous levels was obtained by co-IP in HeLa cells without chemical crosslinking (Fig 3F). Together, these data indicate that hFis1 robustly interacts with Mfns and OPA1, while the interaction between hFis1 and Drp1 is weak and transient, and is detectable only after chemical crosslinking, in agreement with a previous report (Yoon et al, 2003). Furthermore, knockdown of either OPA1 (Fig 3G) or Mfn1/2 (Fig 3H) using siRNAs in WT 293T cells did not affect the binding of hFis1 to respectively Mfn1/Mfn2 or OPA1, indicating that the binding of hFis1 to Mfn1/2 and to OPA1 were independent events. Likewise, knockdown of hFis1 by siRNA did not affect the interaction of OPA1 with Mfn1 and Mfn2 (Fig 3I), and overexpression of Myc-hFis1 did not affect the endogenous interaction between Mfn1 and Mfn2 (Fig 3J). In conclusion, these data suggest that Mfn1/2 and OPA1, but not the pro-fission proteins Drp1 and Dyn2, are major hFis1-binding GTPase partners. We next set out to establish which region in hFis1 is responsible for the interactions with Mfns and OPA1. hFis1 is anchored to the MOM through a single C-terminal TM domain with the bulk of the protein including a tetratricopeptide repeat (TPR) domain (consisting of TPR1 and TPR2) facing the cytosol (Fig 4A) (James et al, 2003; Yoon et al, 2003; Suzuki et al, 2005). To define the hFis1 region responsible for the interactions with Mfns and OPA1, several N- or C-terminally truncated, Myc-tagged hFis1 mutants were expressed in hFis1−/− 293T cells generated by CRISPR/Cas9-mediated gene editing (Appendix Fig S3). The interaction of these hFis1 mutants with Mfn1, Mfn2, and OPA1 was evaluated by co-IP, and the results are summarized in Fig 4B, left panel and C. Like full-length hFis1, hFis1Δ1−31, and hFis1Δ1−60 interacted with the three pro-fusion GTPases, indicating that the first N-terminal 60