Spatial control of nucleoporin condensation by fragile X‐related proteins
2020; Springer Nature; Volume: 39; Issue: 20 Linguagem: Inglês
10.15252/embj.2020104467
ISSN1460-2075
AutoresArantxa Agote‐Arán, Stéphane Schmucker, Kateřina Jeřábková, Inès Jmel Boyer, Alessandro Berto, Laura Pacini, Paolo Ronchi, Charlotte Kleiss, Laurent Guérard, Yannick Schwab, Hervé Moine, Jean‐Louis Mandel, Sébastien Jacquemont, Claudia Bagni, Izabela Sumara,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle24 July 2020Open Access Source DataTransparent process Spatial control of nucleoporin condensation by fragile X-related proteins Arantxa Agote-Aran Arantxa Agote-Aran Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Stephane Schmucker Stephane Schmucker Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Katerina Jerabkova Katerina Jerabkova Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Inès Jmel Boyer Inès Jmel Boyer Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Alessandro Berto Alessandro Berto Institut Jacques Monod, CNRS UMR7592-Université Paris Diderot, Sorbonne Paris Cité, Paris, France Ecole Doctorale SDSV, Université Paris Sud, Orsay, France Search for more papers by this author Laura Pacini Laura Pacini Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy Search for more papers by this author Paolo Ronchi Paolo Ronchi European Molecular Biology Laboratory, Electron Microscopy Core Facility, Heidelberg, Germany Search for more papers by this author Charlotte Kleiss Charlotte Kleiss Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Laurent Guerard Laurent Guerard Imaging Core Facility, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Yannick Schwab Yannick Schwab European Molecular Biology Laboratory, Electron Microscopy Core Facility, Heidelberg, Germany European Molecular Biology Laboratory, European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Heidelberg, Germany Search for more papers by this author Hervé Moine Hervé Moine Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Jean-Louis Mandel Jean-Louis Mandel Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Sebastien Jacquemont Sebastien Jacquemont Service de Génétique Médicale, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland CHU Sainte-Justine Research Centre, University of Montreal, Montreal, QC, Canada Search for more papers by this author Claudia Bagni Claudia Bagni Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy Department of Fundamental Neuroscience, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Izabela Sumara Corresponding Author Izabela Sumara [email protected] Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Arantxa Agote-Aran Arantxa Agote-Aran Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Stephane Schmucker Stephane Schmucker Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Katerina Jerabkova Katerina Jerabkova Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Inès Jmel Boyer Inès Jmel Boyer Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Alessandro Berto Alessandro Berto Institut Jacques Monod, CNRS UMR7592-Université Paris Diderot, Sorbonne Paris Cité, Paris, France Ecole Doctorale SDSV, Université Paris Sud, Orsay, France Search for more papers by this author Laura Pacini Laura Pacini Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy Search for more papers by this author Paolo Ronchi Paolo Ronchi European Molecular Biology Laboratory, Electron Microscopy Core Facility, Heidelberg, Germany Search for more papers by this author Charlotte Kleiss Charlotte Kleiss Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Laurent Guerard Laurent Guerard Imaging Core Facility, Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Yannick Schwab Yannick Schwab European Molecular Biology Laboratory, Electron Microscopy Core Facility, Heidelberg, Germany European Molecular Biology Laboratory, European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Heidelberg, Germany Search for more papers by this author Hervé Moine Hervé Moine Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Jean-Louis Mandel Jean-Louis Mandel Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Sebastien Jacquemont Sebastien Jacquemont Service de Génétique Médicale, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland CHU Sainte-Justine Research Centre, University of Montreal, Montreal, QC, Canada Search for more papers by this author Claudia Bagni Claudia Bagni Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy Department of Fundamental Neuroscience, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Izabela Sumara Corresponding Author Izabela Sumara [email protected] Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France Université de Strasbourg, Strasbourg, France Search for more papers by this author Author Information Arantxa Agote-Aran1,2,3,4,‡, Stephane Schmucker1,2,3,4,‡, Katerina Jerabkova1,2,3,4, Inès Jmel Boyer1,2,3,4, Alessandro Berto5,6,14, Laura Pacini7,15, Paolo Ronchi8, Charlotte Kleiss1,2,3,4, Laurent Guerard9, Yannick Schwab8,10, Hervé Moine1,2,3,4, Jean-Louis Mandel1,2,3,4, Sebastien Jacquemont11,12, Claudia Bagni7,13 and Izabela Sumara *,1,2,3,4 1Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France 2Centre National de la Recherche Scientifique UMR 7104, Strasbourg, France 3Institut National de la Santé et de la Recherche Médicale U964, Strasbourg, France 4Université de Strasbourg, Strasbourg, France 5Institut Jacques Monod, CNRS UMR7592-Université Paris Diderot, Sorbonne Paris Cité, Paris, France 6Ecole Doctorale SDSV, Université Paris Sud, Orsay, France 7Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy 8European Molecular Biology Laboratory, Electron Microscopy Core Facility, Heidelberg, Germany 9Imaging Core Facility, Biozentrum, University of Basel, Basel, Switzerland 10European Molecular Biology Laboratory, European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Heidelberg, Germany 11Service de Génétique Médicale, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland 12CHU Sainte-Justine Research Centre, University of Montreal, Montreal, QC, Canada 13Department of Fundamental Neuroscience, University of Lausanne, Lausanne, Switzerland 14Present address: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland 15Present address: UniCamillus –Saint Camillus International University of Health and Medical Sciences, Rome, Italy ‡These authors contributed equally to this work *Corresponding author. Tel: +33 3 88 65 35 21; Fax: +33 3 88 65 32 01; E-mail: [email protected] The EMBO Journal (2020)39:e104467https://doi.org/10.15252/embj.2020104467 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 Nucleoporins (Nups) build highly organized nuclear pore complexes (NPCs) at the nuclear envelope (NE). Several Nups assemble into a sieve-like hydrogel within the central channel of the NPCs. In the cytoplasm, the soluble Nups exist, but how their assembly is restricted to the NE is currently unknown. Here, we show that fragile X-related protein 1 (FXR1) can interact with several Nups and facilitate their localization to the NE during interphase through a microtubule-dependent mechanism. Downregulation of FXR1 or closely related orthologs FXR2 and fragile X mental retardation protein (FMRP) leads to the accumulation of cytoplasmic Nup condensates. Likewise, models of fragile X syndrome (FXS), characterized by a loss of FMRP, accumulate Nup granules. The Nup granule-containing cells show defects in protein export, nuclear morphology and cell cycle progression. Our results reveal an unexpected role for the FXR protein family in the spatial regulation of nucleoporin condensation. Synopsis Fragile X-related proteins and dynein inhibit ectopic phase separation of nucleoporins in the cytoplasm and facilitate their localization to the nuclear envelope during G1 phase of the cell cycle. Fragile X-related (FXR) proteins and dynein regulate localization of cytoplasmic nucleoporins. FXR-Dynein pathway downregulation induces aberrant cytoplasmic condensation of nucleoporins. Cellular models of Fragile X syndrome accumulate aberrant cytoplasmic nucleoporin condensates. FXR-Dynein pathway regulates nuclear morphology and cell cycle progression. Introduction Formation of supramolecular assemblies and membrane-less organelles such as the nucleolus, Cajal bodies, nuclear speckles, stress granules (SG), P-bodies, germ granules and PML bodies are important for cellular homeostasis (Boeynaems et al, 2018). Among the factors controlling their formation and turnover is the presence of intrinsically disordered regions (IDRs) in protein components, their ability to form multivalent protein–protein, and protein–RNA interactions (Feng et al, 2019) and proteins' local concentration. Indeed, many RNA-binding proteins (RBPs) have the ability to demix into liquid states (liquid droplets), which can be subsequently transformed into pathological amyloids (Lin et al, 2015; Harrison & Shorter, 2017; Shorter, 2019) that have been linked to many neurological disorders (Shin & Brangwynne, 2017). One example of a large protein assembly consisting of IDR-containing proteins is the nuclear pore complex (NPC), which plays an essential role in cellular homeostasis (Knockenhauer & Schwartz, 2016; Sakuma & D'Angelo, 2017). NPCs are large, multisubunit protein complexes (Beck & Hurt, 2017; Hampoelz et al, 2019a) spanning the nuclear envelope (NE) that constitute the transport channels controlling the exchange of proteins and mRNA between the nucleus and the cytoplasm. They are built from roughly 30 different nucleoporins (Nups) each present in multiple copies in the NPCs. The ring-like NPC scaffold is embedded in the NE and shows highly organized eight-fold symmetry (Knockenhauer & Schwartz, 2016). In contrast, the central channel of the NPC is formed from Nups containing disordered elements characterized by the presence of phenylalanine–glycine (FG) repeats, the so-called FG-Nups. The FG-Nups have the ability to phase separate into sieve-like hydrogels that constitute a selective and permeable barrier for diffusing molecules and transported cargos through the NPCs (Schmidt & Görlich, 2016). This ability of the FG-Nups to form permeable hydrogels can also be reconstituted in vitro and is highly conserved through the evolution (Frey et al, 2006; Frey & Görlich, 2007; Schmidt & Görlich, 2015). The cohesive abilities of FG-Nups allow not only for the formation of the permeability barrier but also for building the links with the structural scaffold elements of the NPC (Onischenko et al, 2017). The non-FG-Nups can also form condensates in cells as they are sequestered in the SGs (Zhang et al, 2018) and in various pathological aggregates in the nucleus and in the cytoplasm (Li & Lagier-Tourenne, 2018; Hutten & Dormann, 2020). A fraction of cytoplasmic nucleoporins was also identified in the promyelocytic leukaemia protein (PML)-positive structures, the so-called CyPNs (cytoplasmic accumulations of PML and nucleoporins), which could move on microtubules to dock at the NE (Jul-Larsen et al, 2009), although the cellular roles of the CyPNs remain to be understood. This indicates that Nups have an intrinsic capacity to aberrantly assemble, suggesting protective mechanisms may exist to prevent it in the cell. Indeed, in Drosophila embryos a large excess of soluble Nups has been reported (Onischenko et al, 2004), and in cells, Nups are synthesized as soluble proteins in the cytoplasm (Davis & Blobel, 1987). How the balance of soluble Nups is controlled, and what factors regulate the localized assembly of Nups is currently unknown. The fragile X-related (FXR) proteins (FXR1, FXR2 and fragile X mental retardation protein [FMRP]) are a family of RNA-binding proteins displaying a high degree of sequence and structural similarity and playing important roles in mRNA metabolism (Li & Zhao, 2014). Silencing of the FMR1 gene that encodes the FMRP protein (Santoro et al, 2012) leads to fragile X syndrome (FXS), the most common form of inherited intellectual human disability worldwide, for which no efficient therapy exists to date (Mullard, 2015). For this reason, the role of FXR proteins has been mostly investigated in brain, in the context of neurodevelopmental disorders (Bagni & Zukin, 2019), and genome-wide association studies suggest the involvement of this family in a wide spectrum of mental illnesses (Guo et al, 2015; Khlghatyan et al, 2018). More recent studies have linked FXR proteins to cancer progression, and in particular, FMRP and FXR1 were found overexpressed in different types of cancer (Lucá et al, 2013; Jin et al, 2016; Zalfa et al, 2017; Cao et al, 2019). Although many overlapping functions have been proposed for this protein family, different tissue, cellular and intracellular distributions of the FXR proteins suggest that they might have, in addition to their canonical role as RNA-binding proteins, independent functions (Darnell et al, 2009). Interestingly, the protein region containing the RGG box (arginine- and glycine-rich region) of FMRP has a low-complexity sequence composition and is unfolded and flexible (Ramos, 2003), implicating its role in the membrane-less assemblies. Here, we identify a novel role for the FXR protein family and dynein in the spatial regulation of nucleoporin condensation. Results FXR1 protein localizes to the NE and interacts with Nups FXR1 co-localizes with various cytoplasmic protein–RNA assemblies, but it is also present in the nuclear compartment in human cells (Tamanini et al, 1999; Oldenburg et al, 2014). In search for possible additional cellular functions of FXR1 independent of its role in RNA binding, we performed immunoprecipitations (IPs) of stably expressed GFP-FXR1 protein and analysed the interacting partners by mass spectrometry. Of the interacting proteins, including the known FXR1 partners, FXR2 and FMRP, four nucleoporins (Nups), Nup210, Nup188, Nup133 and Nup85, were detected specifically in GFP-FXR1 IPs (Dataset EV1). We confirmed the GFP-FXR1 interaction with endogenous Nup133 and Nup85, which are components of the evolutionary conserved Nup107-160 NPC sub-complex also called the Y-complex (Fig 1A) (Knockenhauer & Schwartz, 2016; Beck & Hurt, 2017). IP of stably expressed GFP-Nup85 also demonstrated an interaction with endogenous FXR1 in HeLa cells (Fig 1B), and both Nup85 and Nup133 co-immunoprecipitated with endogenous FXR1 in HEK293T cells (Fig 1C). Figure 1. FXR1 protein localizes to the NE and interacts with NUPs Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (shown a mean value, *P < 0.05, **P < 0.01; N = 3). Lysates of HeLa cells stably expressing GFP alone or 3xGFP-Nup85 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (SE, short exposure, LE, long exposure; shown a mean value, *P < 0.05; N = 3). Immunoprecipitation from HEK293T cell lysates using FXR1 antibody or IgG analysed by Western blot. The arrow points to the heavy chain of IgG (IgG HC; shown a mean value, *P < 0.05; N = 3). HeLa cells were treated with indicated siRNAs, synchronized by double thymidine block, and released for 12 h and analysed by immunofluorescence microscopy for the lamin B receptor (LBR) to label the NE, and FXR1. HeLa cells stably expressing GFP-FXR1 were analysed by immunofluorescence microscopy for GFP and mAb414, which labels FG-Nups. The magnified framed regions are shown in the corresponding numbered panels. The arrowheads indicate NE and cytoplasmic localization of GFP-FXR1. HeLa cells stably expressing GFP-Nup107 were synchronized by double thymidine block and released for 12 h, permeabilized with Triton/SDS or digitonin for antibodies to access the nuclear and cytoplasmic or cytoplasmic side of the nucleus, respectively, and analysed by immunofluorescence microscopy. Data information: Scale bars are 5 μm. Statistical significance was assessed by unpaired two-tailed Student's t-test. Source data are available online for this figure. Source data for Figure 1 [embj2020104467-sup-0009-SDataFig1.pdf] Download figure Download PowerPoint Both endogenous FXR1 and GFP-FXR1 localized to the nuclear envelope (Fig 1D and E) and also occasionally to small cytoplasmic foci labelled by the monoclonal antibody mAb414, which recognizes a panel of phenylalanine–glycine (FG) repeat-containing Nups (FG-Nups; Fig 1E). FXR1 localization to the NE and the cytoplasmic foci was abolished by treatment with FXR1 siRNA (Fig 1D), demonstrating antibody specificity. Treatment with digitonin, which in contrast to permeabilization protocol with the Triton and SDS can selectively permeabilize the plasma membrane while leaving the NE intact revealed that FXR1 localized to the outer nuclear membrane (ONM; Fig 1F). We conclude that FXR1 interacts with Nups and can localize to both the ONM and to cytoplasmic foci containing Nups. FXR1 inhibits aberrant assembly of cytoplasmic Nups To assess the biological function of the FXR1-Nup interactions, we treated cultured human cells with FXR1-specific siRNA oligonucleotides. Downregulation of FXR1 in HeLa cells led to an accumulation of FG-Nups in the cytoplasm in the form of irregular aggregate-like assemblies of various sizes (Figs 2A and B, and EV1, Appendix Fig S1A, B, E, F and H) and in U2OS cells (Fig 2G, Appendix Fig S1J). These Nup assemblies were observed using two different siRNAs targeting FXR1 and could be rescued by stable ectopic expression of a form of GFP-FXR1 that is resistant to one of the siRNAs used (Figs 2B and EV1). Figure 2. FXR1 inhibits aberrant assembly of cytoplasmic Nups A. HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and release for 12 h and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. B. HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 h and then analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules was quantified, and 1,000 cells were analysed for each graph (mean ± SD, **P < 0.01; ***P < 0.001, N = 3). The corresponding representative pictures are shown in Fig EV1, and the corresponding Western blot analysis is shown in Fig 3B. C–F. HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 h and analysed by immunofluorescence microscopy. Nups present in different NPC sub-complexes are depicted in the colour code corresponding to the NPC scheme shown on the right. Additional or complementary representative images and channels of cells depicted in (C) are shown in Appendix Figs S1 and S2B–D. Nuclear intensity of FG-Nups labelled by mAb414 (D), RanBP2 (E) and GFP-Nup107 (F) was quantified. A total of 1,800 cells were analysed for each graph (mean ± SD, *P < 0.05; **P < 0.01; N = 3). G, H. Asynchronously proliferating U2OS cells were treated with the indicated siRNAs and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules (G) was quantified, and nuclear intensity of FG-Nups labelled by mAb414 (H) was quantified. A total of 1,600 cells were analysed in (G), and 2,100 cells were analysed in (H) (mean ± SD, *P < 0.05; ***P < 0.001; N = 3). Data information: Scale bars are 5 μm. Statistical significance was assessed by unpaired two-tailed Student's t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. FXR1 specifically controls cytoplasmic Nups and nuclear shapeHeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block and released for 24 h and analysed by immunofluorescence microscopy for mAb414 (related to Figs 2B and 3B and C). Scale bars are 5 μm. Download figure Download PowerPoint Downregulation of FXR1 led to the cytoplasmic retention and co-localization in granules of at least 10 Nups spanning several functional and structural NPC groups, including FG-Nups (Nup98, Nup214; and RanBP2); transmembrane Nups (Nup210 and POM121); Y-complex Nups (Nup133 and Nup85, stably expressed GFP-Nup133, stably expressed GFP-Nup85 and stably expressed GFP-Nup107) as well as Nup88 and NPC-associated the RanGTPase activating protein (RanGAP1; Fig 2C; Appendix Figs S1A–H and S2C and D). Absent from the Nup granules were the nuclear ring Nup ELYS and the inner nuclear basket component Nup153, although their levels at the NE were both slightly reduced (Fig 2C; Appendix Figs S1I and S2B and C). FXR1 downregulation also moderately reduced the NE localization of FG-Nups, RanBP2 and stably expressed GFP-Nup107 in HeLa cells (Fig 2D–F) and of FG-Nups in U2OS cells (Fig 2H, Appendix Fig S1J). Collectively, these data show that loss of FXR1 induces inappropriate assembly of Nups in the cytoplasm. The FXR1 regulates nuclear morphology during G1 cell cycle phase We noticed that the Nup granule-containing cells often displayed strong nuclear atypia (Figs 1D, 2A and C, and EV1 and 2; Appendix Figs S1 and S2). Interestingly, downregulation of FXR1 did not affect the recruitment of the nuclear lamina components lamin B receptor (LBR; Fig 1D), lamin A (Fig EV2A and B), lamin B1 (Fig EV2C and D) or emerin (Fig EV2E and F) to the NE in interphase or telophase cells, while Lap2β recruitment was moderately increased upon FXR1 downregulation (Fig EV2E and G). However, these lamina and INM components displayed irregular distribution along with the misshaped nuclear rim and intranuclear foci (Fig EV2A, C and E). Moreover, the size of nucleus was moderately increased upon downregulation of FXR1 (Fig EV2H). Defects in nuclear architecture including irregular and blebbed nuclei (Fig 3A) could be largely rescued by stable ectopic expression of the siRNA-resistant form of GFP-FXR1 (Figs 3B and C, and EV1). Click here to expand this figure. Figure EV2. FXR1 does not drive recruitment of lamina-associated proteins A–H. HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and released for 9 (telophase) and 12 (interphase) h and analysed by immunofluorescence microscopy. The nuclear intensity of Lamin A (B), Lamin B1 (D), Emerin (F) and Lap2β (G) was quantified, and 2,000 cells were analysed (mean ± SD, *P < 0.05; ns, non-significant; N = 3). The nuclear area was quantified (H), and 3,300 cells were analysed (mean ± SD, **P < 0.01; N = 5). Data information: Scale bars are 5 μm. Statistical significance was assessed by one-sample two-tailed Student's t-test. Download figure Download PowerPoint Figure 3. The FXR1 regulates nuclear morphology during G1 cell cycle phase A. HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 h and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. Arrowheads point to nuclear blebs observed in FXR1-deficient cells. B, C. HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 h and analysed by Western blot (B) and immunofluorescence microscopy (C). The percentage of cells with irregular nuclei was quantified, and 1,000 cells were analysed (mean ± SD, **P < 0.01, ***P < 0.001; N = 3). The corresponding representative pictures are shown in Fig EV1. D–J. HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 h and analysed by immunofluorescence microscopy. Time from prophase till anaphase (D), from prophase till metaphase (E), from metaphase till anaphase (F) and from anaphase till chromatin decondensation (G) was quantified. The selected frames of the movies are depicted, and time is shown in minutes (H). Arrowheads point to nuclear blebs appearing during nuclear expansion of FXR1-deficient cells. Percentage of daughter cells with irregular nuclei was quantified in (I), and time from anaphase till nuclear blebs was quantified in (J). Sixty-six cells were analysed (mean ± SD, ***P < 0.001; N = 3). Data information: Scale bars are 5 μm. Statistical significance was assessed by unpaired two-tailed Student's t-test. Source data are available online for this figure. Source data for Figure 3 [embj2020104467-sup-0010-SDataFig3.pdf]
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