CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy
2020; Springer Nature; Volume: 39; Issue: 9 Linguagem: Inglês
10.15252/embj.2019102731
ISSN1460-2075
AutoresDavid Pla‐Martín, Désirée Schatton, Janica L. Wiederstein, Marie‐Charlotte Marx, Salim Khiati, Marcus Krüger, Elena I. Rugarli,
Tópico(s)Sirtuins and Resveratrol in Medicine
ResumoArticle9 March 2020Open Access Source DataTransparent process CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy David Pla-Martín David Pla-Martín orcid.org/0000-0002-5189-0723 Institute for Genetics, University of Cologne, Cologne, Germany Institute for Vegetative Physiology, University of Cologne, Cologne, Germany Search for more papers by this author Désirée Schatton Désirée Schatton Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Janica L Wiederstein Janica L Wiederstein Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Marie-Charlotte Marx Marie-Charlotte Marx Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Salim Khiati Salim Khiati MitoLab Team, Institut MitoVasc, UMR CNRS 6015, INSERM U1083, Université d'Angers, Angers, France Search for more papers by this author Marcus Krüger Marcus Krüger Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Elena I Rugarli Corresponding Author Elena I Rugarli [email protected] orcid.org/0000-0002-5782-1067 Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author David Pla-Martín David Pla-Martín orcid.org/0000-0002-5189-0723 Institute for Genetics, University of Cologne, Cologne, Germany Institute for Vegetative Physiology, University of Cologne, Cologne, Germany Search for more papers by this author Désirée Schatton Désirée Schatton Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Janica L Wiederstein Janica L Wiederstein Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Marie-Charlotte Marx Marie-Charlotte Marx Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Salim Khiati Salim Khiati MitoLab Team, Institut MitoVasc, UMR CNRS 6015, INSERM U1083, Université d'Angers, Angers, France Search for more papers by this author Marcus Krüger Marcus Krüger Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Elena I Rugarli Corresponding Author Elena I Rugarli [email protected] orcid.org/0000-0002-5782-1067 Institute for Genetics, University of Cologne, Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany Search for more papers by this author Author Information David Pla-Martín1,2,‡, Désirée Schatton1,3,‡, Janica L Wiederstein1,3, Marie-Charlotte Marx1,3, Salim Khiati4, Marcus Krüger1,3,5 and Elena I Rugarli *,1,3,5 1Institute for Genetics, University of Cologne, Cologne, Germany 2Institute for Vegetative Physiology, University of Cologne, Cologne, Germany 3Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany 4MitoLab Team, Institut MitoVasc, UMR CNRS 6015, INSERM U1083, Université d'Angers, Angers, France 5Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 221 47884244; E-mail: [email protected] The EMBO Journal (2020)39:e102731https://doi.org/10.15252/embj.2019102731 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 Mitochondria house anabolic and catabolic processes that must be balanced and adjusted to meet cellular demands. The RNA-binding protein CLUH (clustered mitochondria homolog) binds mRNAs of nuclear-encoded mitochondrial proteins and is highly expressed in the liver, where it regulates metabolic plasticity. Here, we show that in primary hepatocytes, CLUH coalesces in specific ribonucleoprotein particles that define the translational fate of target mRNAs, such as Pcx, Hadha, and Hmgcs2, to match nutrient availability. Moreover, CLUH granules play signaling roles, by recruiting mTOR kinase and the RNA-binding proteins G3BP1 and G3BP2. Upon starvation, CLUH regulates translation of Hmgcs2, involved in ketogenesis, inhibits mTORC1 activation and mitochondrial anabolic pathways, and promotes mitochondrial turnover, thus allowing efficient reprograming of metabolic function. In the absence of CLUH, a mitophagy block causes mitochondrial clustering that is rescued by rapamycin treatment or depletion of G3BP1 and G3BP2. Our data demonstrate that metabolic adaptation of liver mitochondria to nutrient availability depends on a compartmentalized CLUH-dependent post-transcriptional mechanism that controls both mTORC1 and G3BP signaling and ensures survival. Synopsis Mitochondria adapt their gene expression profile and metabolism in response to nutrient availability, to ensure proper energy production and cell survival. Here, ribonucleoprotein particles containing the RNA-binding protein CLUH are found to promote mitochondrial turnover and metabolic rewiring in murine hepatocytes, to ensure cell survival upon starvation. CLUH forms granules that control the translation of its target mRNAs. CLUH granules recruit mTOR kinase and other RNA-binding proteins, such as G3BP1 and G3BP2. CLUH inhibits bulk autophagy but promotes mitophagy. Rapamycin treatment or depletion of G3BPs rescues mitochondrial clustering occurring upon CLUH loss. Introduction Traditionally considered as the powerhouse of the cell, mitochondria contribute in several ways to cell and tissue metabolism, by producing biosynthetic intermediates, hosting catabolic reactions, and participating in signaling pathways (Chandel, 2014; Spinelli & Haigis, 2018). To adapt their metabolic function to cellular needs, mitochondria change shape, fuse or divide, interact with other organelles, and are replaced by balanced biogenesis and turnover (Eisner et al, 2018). When nutrients are abundant, the expression of a subset of mitochondrial proteins involved in oxidative phosphorylation (OXPHOS) and mitochondrial translation is promoted in a mTORC1-dependent manner to enable the production of ATP necessary for protein synthesis, which is an energetically costly process (Morita et al, 2013; Saxton & Sabatini, 2017). Turnover of actively respiring mitochondria via mitophagy ensures the maintenance of a healthy organellar population (Melser et al, 2013). Rewiring of mitochondrial metabolism is crucial to survive transitions from nutrient sufficiency to nutrient deprivation. During starvation, mitochondria are mainly catabolic organelles that use amino acids and lipids released by autophagy and convert them into ketone bodies and ATP to promote survival (Spinelli & Haigis, 2018). Inhibition of mTORC1 suppresses energy-consuming anabolic pathways and leads to mitochondrial hyperfusion as a mechanism to transiently protect mitochondria from autophagy and to suppress apoptotic cell death (Rambold et al, 2011; Morita et al, 2017). Prolonged starvation ultimately induces removal of mitochondria (Kristensen et al, 2008). Elucidating mechanisms that control the dynamic changes of mitochondrial metabolism and turnover to adapt them to energy needs is paramount to understand how organisms survive upon stress and starvation. An important feature of a successful mitochondrial adaptive response is to be fast, flexible, and reversible. Coordination of post-transcriptional events by ribonucleoproteins (RNPs) plays a fundamental role in living systems to respond in a quick and dynamic manner to environmental signals and stress (Keene, 2007; Gehring et al, 2017). RNA-binding proteins (RBPs) can control each step of the mRNA life cycle, determining stability or degradation, localization, and translation efficiency of mRNAs (Hentze et al, 2018). RBPs often assemble together with target transcripts in specific membrane-less subcellular compartments, such as stress granules (SGs), P-bodies, or other types of granules (Protter & Parker, 2016; Gomes & Shorter, 2019). These phase separations not only confer spatial regulation to the expression of groups of RNAs with a common function, but also integrate it with signaling pathways and allow sensing environmental changes (Kedersha et al, 2013; Yoo et al, 2019). Whether membrane-less organelles regulate mitochondrial function is currently unknown. CLUH (clustered mitochondria homolog) is an RBP that specifically binds several transcripts encoding mitochondrial proteins (Gao et al, 2014; Schatton et al, 2017). At least for a subset of these, CLUH promotes their stability and translation (Schatton et al, 2017). Mitochondrial proteins whose expression depends on CLUH belong to several pathways, including OXPHOS, tricarboxylic acid (TCA) cycle, amino acid degradation, fatty acid oxidation, and ketogenesis (Schatton et al, 2017). In the absence of CLUH, the mitochondrial proteome is severely depleted of polypeptides encoded by mRNAs under CLUH regulation (Gao et al, 2014; Schatton et al, 2017). Mitochondria appear abnormal in ultrastructure and display a characteristic clustering next to the nucleus. This phenotype, which has given the name to the gene, is extremely conserved upon deletion of CLUH orthologues in evolutionary distant species (Fields et al, 1998, 2002; Logan et al, 2003; Cox & Spradling, 2009; Gao et al, 2014; Schatton et al, 2017). CLUH-deficient cells show metabolic abnormalities characterized by respiratory deficiency, a shift toward a glycolytic metabolism, and impairment of the TCA cycle and β-oxidation (Schatton et al, 2017; Wakim et al, 2017). In vivo, CLUH plays a key role to allow survival during the fetal to neonatal transition, which is characterized by acute starvation and a shift to OXPHOS metabolism (Schatton et al, 2017). In the adult liver, CLUH is required to reach maximal respiratory capacity under nutrient sufficiency, but also to produce ketone bodies upon starvation (Schatton et al, 2017). Despite its crucial role for mitochondrial function in the liver, it is unclear whether CLUH is a general regulator of mitochondrial gene expression or whether it has a specific role during the metabolic switches in response to physiological nutrient fluctuations. Here, we show that in primary hepatocytes, CLUH assembles with its bound mRNAs in specific RNP particles that function not only as compartments that coordinate the translation of target mRNAs, but also as signaling hubs that control the dynamics of mTORC1 activation and modulate the function of other RBPs, such as Ras-GTPase-activating protein SH3 domain-binding proteins 1 and 2 (G3BPs). Through this mechanism, CLUH promotes turnover of mitochondria and metabolic rewiring. These data demonstrate a role of CLUH-dependent RNA granules in hepatocytes to coordinate mitochondrial catabolism and nutrient-sensing signaling pathways, thus ensuring survival upon starvation. Results CLUH and its target mRNAs form G3BP1-positive RNA granules We previously showed that CLUH plays a physiological role in the adult mouse liver upon starvation to allow amino acid catabolism, to produce ketone bodies, and to maintain glucose levels (Schatton et al, 2017). Intriguingly, CLUH subcellular localization in the mouse liver and in primary hepatocytes changed depending on the nutrient condition. CLUH displayed a cytosolic punctate localization in the liver of fed mice, but formed bigger aggregates in the tissue of mice subjected to food deprivation (Fig EV1A and B). Similarly, when hepatocytes were cultured in basal glucose-rich medium, CLUH decorated small cytosolic puncta and a few bigger foci. In contrast, incubation of hepatocytes for 2 h in HBSS, a low-glucose medium devoid of serum and amino acids, increased CLUH redistribution to bigger structures, often located in the perinuclear region (Fig EV1A and C). Click here to expand this figure. Figure EV1. CLUH forms granules upon starvation A. Confocal images of liver cryosections and primary hepatocytes of Li-CluhWT and Li-CluhKO mice stained with anti-CLUH antibody. Scale bar, 10 μm. B. Confocal images of liver cryosections of fed and starved Li-CluhWT and Li-CluhKO mice stained with anti-CLUH antibody. Right panels show 6.5× magnified boxed areas. Scale bar, 10 μm. C. Confocal images of primary hepatocytes cultured in indicated media and stained with anti-CLUH antibody. Right panels show 4.5× magnified boxed areas. Scale bar, 10 μm. D, E. Confocal images of primary hepatocytes cultured in indicated media and stained with (D) anti-G3BP1 or (E) anti-DCP1A and anti-CLUH antibodies. Right panel shows 5× enlargement of indicated area. Scale bar, 10 μm. Download figure Download PowerPoint These results raised the possibility of CLUH assembly together with client mRNAs in RNP particles, which play a regulatory role in response to nutrient availability. To detect whether known CLUH target mRNAs localize to these granules, we combined immunofluorescence with in situ hybridization in primary hepatocytes. We selected two target transcripts highly expressed in the liver, the expression of which is reduced in the absence of CLUH (Schatton et al, 2017): Pcx (encoding pyruvate carboxylase involved in the carboxylation of pyruvate to oxaloacetate) and Hadha (encoding hydroxyacyl-CoA dehydrogenase that catalyzes the last three steps of β-oxidation of long fatty acids). As a negative control, we analyzed the distribution of Actb mRNA. Under basal conditions, very little colocalization was observed between CLUH and each mRNA species (Fig 1A–F). However, we noticed that Pcx and Hadha mRNA molecules colocalized with CLUH only within the few granules present (Fig 1A and B). After HBSS starvation, the pattern of Pcx and Hadha mRNA molecules became visibly more aggregated, and the colocalization with CLUH significantly increased (Fig 1A, B, D and E). In contrast, colocalization of CLUH with Actb mRNA was not enhanced by starvation and remained at background levels (Fig 1C and F). Figure 1. CLUH forms specific RNA granules with its targets A–C. Confocal images of primary hepatocytes grown under indicated conditions and stained with anti-CLUH antibody and Hadha (A), Pcx (B), and Actb (C) mRNA in situ hybridization. Right panels show 5× magnified boxed areas. Scale bar, 10 μm. D–F. Manders' colocalization coefficient between Hadha (D), Pcx (E), and Actb (F) mRNA molecules and CLUH signal (n ≥ 50 cells isolated from 3 to 6 mice). G, H. Confocal images of primary hepatocytes grown in HBSS and stained with anti-CLUH and anti-TIA-1 (G) or anti-G3BP1 (H) antibodies. Right panels show 5× magnified boxed areas. Scale bar, 10 μm. Data information: In (D–F), data are presented as boxplots showing the median, the first quartile, and the third quartile. Error bars show minimum and maximum values. ***P ≤ 0.001 (Student's t-test). Download figure Download PowerPoint To further investigate the nature of the CLUH particles, we examined whether they contained markers of well-characterized RNP granules, such as SGs and P-bodies. SGs form under conditions of stress and contain mRNAs stalled in translation initiation together with specific RBPs (Panas et al, 2016; Protter & Parker, 2016), while P-bodies are constitutively present and contain translationally repressed mRNAs (Hubstenberger et al, 2017). Upon HBSS treatment, CLUH colocalized with TIA-1 and G3BP1, two RBPs that are present in SGs (Fig 1G and H). However, classical G3BP1-positive SGs induced by arsenite treatment did not contain CLUH (Fig EV1D). Furthermore, CLUH granules were not positive for DCP1A, a marker of P-bodies, although they were closely located (Fig EV1E). In conclusion, CLUH forms granules with its target mRNAs in primary hepatocytes. These granules are positive for other RBPs and are more prominent upon starvation, but they are not induced by a classical SG triggering stimulus. CLUH-dependent granules temporally regulate translation of target mRNAs G3BP1 and TIA-1 are markers for SGs where mRNAs are stalled after recruitment of the translation initiation complex (Panas et al, 2016; Protter & Parker, 2016). Therefore, our first hypothesis was that the assembly of CLUH and its target mRNAs in these granules reflected translational arrest upon nutrient stress. To investigate the translational status of the CLUH-positive granules, we made use of the ribopuromycylation assay (David et al, 2012), which reveals the subcellular localization of protein translation, by detecting the incorporation of puromycin into translating polypeptides with a specific antibody (Appendix Fig S1A). Hepatocytes cultured in basal medium showed a diffuse puromycin signal in the cytosol, indicating pervasive translation (Fig 2A and B). As expected, this signal was suppressed by pre-incubation with both homoharringtonine (HHT) and arsenite. HHT causes ribosome stalling at the initiator codon, but leaves unaffected downstream ribosomes already engaged in elongation, while arsenite is a potent inducer of SGs (Fig 2A and B). Upon HBSS treatment, the puromycin signal showed a more granular pattern (Fig 2A). While the total fluorescent intensity of puromycin signal was dramatically reduced by incubation with HHT (Fig 2B), the number of puromycin granules was reduced but not completely abolished by HHT, possibly indicating translation stalled at the level of elongation (Fig 2C). We found that colocalization of puromycin signal with CLUH was higher in HBSS than in basal medium and was significantly reduced by HHT treatment (Fig 2D and E, and Appendix Fig S1B). Furthermore, the percentage of puromycin-positive granules also showing CLUH signal was reduced when samples were pre-treated with HHT (Fig 2F). These data suggest the existence of two types of puromycin-positive compartments in HBSS, one harboring active translation (which disappears upon HHT treatment) and another where translation might be stalled. Both types of granules contain CLUH. Figure 2. CLUH granules contain stalled and active translation sites Confocal images of primary hepatocytes grown under indicated conditions and treatments after ribopuromycylation assay stained with anti-puromycin antibody. Scale bar, 10 μm. Quantification of fluorescence intensity per cell of experiment shown in (A). AU, arbitrary units (n = 90–110 cells per treatment isolated from 4 mice). Quantification of the number of puromycin granules under indicated conditions (n ≥ 50 cells isolated from 4 mice). Confocal images of primary hepatocytes stained with anti-puromycin and anti-CLUH antibodies. The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Fig S1B for each individual channel. Scale bar, 4 μm. Cells analyzed were isolated from 4 different mice with similar results. Arrows point to colocalizing particles. Manders' colocalization coefficient between puromycin and CLUH from experiment shown in (D) (n ≥ 50 cells isolated from 4 mice). Quantification of puromycin granules containing CLUH signal (n ≥ 80 cells isolated from 4 mice). Data information: In (B, C, E, F), data are presented as boxplots showing the median, the first quartile, and the third quartile. Error bars show minimum and maximum values. (C, F) ***P ≤ 0.001; **P ≤ 0.01 (Student's t-test). (E) ***P ≤ 0.001 (one-way ANOVA, Tukey's multiple comparison test). Download figure Download PowerPoint We combined in situ hybridization and the ribopuromycylation assay to correlate the translational state of the granules with specific CLUH mRNA targets. Remarkably, CLUH granules containing Hadha or Pcx mRNAs incorporated puromycin when cells were cultured in glucose, but were mostly puromycin-negative after HBSS incubation (Fig 3A, B, D and E, Appendix Fig S2A and B). We hypothesized that the starvation-induced CLUH granules are dynamic and regulate translation of mRNAs depending on cellular requirements. We therefore probed the Hmgcs2 transcript, which encodes the mitochondrial enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2, implicated in the first reaction of ketogenesis. Defective production of β-hydroxybutyrate is the main metabolic defect of mice lacking CLUH specifically in the adult liver after starvation (Schatton et al, 2017). Hmgcs2 mRNA molecules were detected both in basal and in HBSS conditions. However, the colocalization of CLUH and puromycin was prominent only in HBSS medium (Fig 3C and F; Appendix Fig S2C). To test whether these granules reflect stalled translation in HBSS, we performed control experiments with HHT. Pre-incubation with HHT completely abrogated the detection of Hadha, Pcx, or Hmgcs2 mRNA molecules together with CLUH and puromycin (Appendix Fig S3A–C), demonstrating that CLUH granules are compartments where these mRNAs are translated. Figure 3. CLUH granules are translationally active or dormant depending on the mRNA A–C. Confocal images of primary hepatocytes after ribopuromycylation experiment combined with mRNA in situ hybridization for (A) Hadha, (B) Pcx, and (C) Hmgcs2. Scale bar, 4 μm. D–F. Fluorescence profile of 100-pixel line from the merged channel shown in (A–C). The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Fig S2 for each individual channel. Cells analyzed were isolated from 6 different mice with similar results. Download figure Download PowerPoint CLUH granules form in the absence of G3BPs and are distinct from SGs To test whether CLUH overexpression triggers granule formation in the absence of any stress, we transfected an untagged version in HeLa cells and examined overexpressing versus non-overexpressing cells in the same dish, using a specific antibody (Figs 4A and B, and EV2A and B). CLUH overexpression induced the formation of peripheral granules, positive for G3BP1 and the homolog protein G3BP2, in approximately 40% of transfected cells (Figs 4B and C, and EV2C and D). However, CLUH granules were not abrogated by cycloheximide (CHX) treatment, in contrast to arsenite-induced G3BP1-granules, indicating that they are not SGs (Fig EV2E–H). We downregulated G3BP1, G3BP2, or both RBPs and examined whether overexpressed CLUH retained the ability to induce granules (Fig 4A–C). The formation of CLUH granules was affected neither by G3BP1 downregulation nor by concomitant downregulation of both G3BPs (Fig 4B and C). Significantly less CLUH granules were seen in cells depleted for G3BP2, although the levels of overexpressed CLUH were decreased (Fig 4A). Thus, CLUH overexpression is sufficient for granule formation, independently from G3BP1 and G3BP2. Figure 4. CLUH granules form in the absence of G3BPs and are distinct from SGs Representative Western blot of HeLa cells downregulated for G3BPs and overexpressing untagged CLUH. Asterisks indicate signal of previous incubation with anti-G3BP1 antibody. Pan-actin was used as loading control. Confocal images of anti-CLUH staining in HeLa cells downregulated for G3BPs and overexpressing untagged CLUH. Insets show 2.6× enlarged area. Scale bar, 10 μm. Quantification of cells with CLUH granules from experiments shown in (B) and Fig EV2B (n = 3 independent experiments, > 50 cells per experiment per condition). Live imaging of WT and CLUH KO HeLa cells transfected with G3BP1-GFP plasmid and incubated in HBSS medium with or without CHX. Cells were recorded for a maximum of 2 h. Insets show 2.5× enlarged areas. Scale bar, 10 μm. Total number of cells analyzed by live imaging for the indicated experiments shown in (D). "Positive" relates to a cell which formed G3BP1 granules at the end of the recording. Confocal images of anti-G3BP1 staining in primary hepatocytes derived from Li-CluhWT and Li-CluhKO mice grown in indicated media. Arrows point to G3BP1-granules. Scale bar, 10 μm. Quantification of percentage of cells with G3BP1-positive granules of experiment shown in (F). Cells were analyzed in a blind fashion (n = 3 mice per genotype; number of cells in total: WT basal, 449; KO basal, 303; WT HBSS, 146; KO HBSS, 161). Western blot of primary WT and KO hepatocytes stained with indicated antibodies. Asterisks indicate unspecific signal. Pan-actin was used as loading control. Quantification of Western blot shown in (H) (n = 3 per genotype per condition). mRNA levels of G3bp1 in primary hepatocytes grown under indicated conditions (n = 4 per genotype per condition). Data information: In (C, G, I, J), data are presented as histograms showing the mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 (one-way ANOVA, Tukey's multiple comparisons test). Source data are available online for this figure. Source Data for Figure 4 [embj2019102731-sup-0006-SDataFig4.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Overexpressed CLUH forms CHX resistant granules in HeLa cells A. Confocal images of WT and CLUH KO HeLa cells stained with anti-CLUH antibody. Scale bar, 10 μm. B. Confocal images of HeLa cells downregulated for G3BPs and overexpressing untagged CLUH (marked with asterisks). These images were overexposed to detect cells with CLUH expression at endogenous level CLUH. Asterisks indicate overexpressing cells. Scale bar, 10 μm. C, D. Confocal images of HeLa cells overexpressing untagged CLUH (C) or FLAG-tagged CLUH (D) stained with indicated antibodies. Scale bar, 10 μm. E. Confocal images of HeLa cells overexpressing untagged CLUH treated with or without CHX and stained with the indicated antibodies. Scale bar, 10 μm. F. Quantification of percentage of cells with CLUH granules of experiment shown in (E) (n = 3 independent experiments, > 50 cells per condition per replicate). G. Confocal images of HeLa cells treated with arsenite with or without CHX and stained with anti-G3BP1 antibody. Scale bar, 10 μm. H. Quantification of percentage of cells with G3BP1 granules of experiment shown in (G) (n = 3 independent experiments, > 50 cells per condition per replicate). I. Live imaging of WT and CLUH KO HeLa cells transfected with G3BP1-GFP plasmid and treated with arsenite with and without CHX. Cells were recorded for a maximum of 30 min. Scale bar, 10 μm. J. Total number of cells analyzed by live imaging for the indicated experiments. "Positive" indicates a cell which forms G3BP1 granules at the end of the recording. Data information: In (F, H), data are presented as histograms showing the mean ± SEM. (H) ***P ≤ 0.001 (Student's t-test). Download figure Download PowerPoint We then asked whether G3BP1 granules can still form in HeLa cells lacking CLUH (Wakim et al, 2017). To this purpose, we expressed G3BP1-GFP and used live imaging to follow the formation of granules upon HBSS incubation. We selected cells which did not show granules at the beginning of the imaging, to avoid analysis of SGs that are known to form upon G3BP1 overexpression (Protter & Parker, 2016). Strikingly, approximately 80% of WT cells formed G3BP1-positive granules in HBSS, while this rarely occurred in KO cells (Fig 4D and E). These granules were still formed upon CHX treatment, suggesting that most of them are not SGs (Fig 4D and E). In contrast, arsenite treatment induced classical CHX-sensitive SGs in both WT and KO cells (Fig EV2I and J). Therefore, CLUH is required for the efficient formation of starvation-induced G3BP1 granules in HeLa cells. Lastly, we obtained hepatocytes from liver-specific Cluh knock-out (KO) mice (Li-CluhKO) (Schatton et al, 2017) and used G3BP1 staining to detect granules. The number of cells showing G3BP1-positive granules was significantly lower in CLUH-deficient hepatocytes compared to control cells in both basal and HBSS conditions (Fig 4F and G). Intriguingly, the protein levels of G3BP1 were also lower in KO hepatocytes under basal conditions, but they increased similarly in HBSS in cells of both genotypes (Fig 4H and I). In contrast, G3BP1 mRNA levels we
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