A selective ER ‐phagy exerts procollagen quality control via a Calnexin‐ FAM 134B complex
2018; Springer Nature; Volume: 38; Issue: 2 Linguagem: Inglês
10.15252/embj.201899847
ISSN1460-2075
AutoresAlison Forrester, Chiara De Leonibus, Paolo Grumati, Elisa Fasana, M. Piemontese, Leopoldo Staiano, Ilaria Fregno, Andrea Raimondi, Alessandro Marazza, Giovanni Bruno, Maria Iavazzo, Daniela Intartaglia, Marta Seczyńska, Eelco van Anken, Iván Conte, Maria Antonietta De Matteis, Ivan Đikić, Maurizio Molinari, Carmine Settembre,
Tópico(s)Connexins and lens biology
ResumoArticle17 December 2018Open Access Source DataTransparent process A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex Alison Forrester Alison Forrester Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Chiara De Leonibus Chiara De Leonibus Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Paolo Grumati Paolo Grumati Institute of Biochemistry II, Goethe University Frankfurt – Medical Faculty, University Hospital, Frankfurt am Main, Germany Search for more papers by this author Elisa Fasana Elisa Fasana Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Marilina Piemontese Marilina Piemontese Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Leopoldo Staiano Leopoldo Staiano Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Ilaria Fregno Ilaria Fregno Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland Search for more papers by this author Andrea Raimondi Andrea Raimondi Experimental Imaging Center, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Alessandro Marazza Alessandro Marazza Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland Search for more papers by this author Gemma Bruno Gemma Bruno Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Maria Iavazzo Maria Iavazzo Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Daniela Intartaglia Daniela Intartaglia Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Marta Seczynska Marta Seczynska Institute of Biochemistry II, Goethe University Frankfurt – Medical Faculty, University Hospital, Frankfurt am Main, Germany Search for more papers by this author Eelco van Anken Eelco van Anken Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy Search for more papers by this author Ivan Conte Ivan Conte Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Maria Antonietta De Matteis Maria Antonietta De Matteis Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Department of Molecular Medicine and Medical Biotechnologies, University of Naples "Federico II", Naples, Italy Search for more papers by this author Ivan Dikic Corresponding Author Ivan Dikic [email protected] orcid.org/0000-0001-8156-9511 Institute of Biochemistry II, Goethe University Frankfurt – Medical Faculty, University Hospital, Frankfurt am Main, Germany Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Maurizio Molinari Corresponding Author Maurizio Molinari [email protected] orcid.org/0000-0002-7636-5829 Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Carmine Settembre Corresponding Author Carmine Settembre [email protected] orcid.org/0000-0002-5829-8589 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Department of Medical and Translational Science, University of Naples "Federico II", Naples, Italy Search for more papers by this author Alison Forrester Alison Forrester Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Chiara De Leonibus Chiara De Leonibus Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Paolo Grumati Paolo Grumati Institute of Biochemistry II, Goethe University Frankfurt – Medical Faculty, University Hospital, Frankfurt am Main, Germany Search for more papers by this author Elisa Fasana Elisa Fasana Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Search for more papers by this author Marilina Piemontese Marilina Piemontese Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Leopoldo Staiano Leopoldo Staiano Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Ilaria Fregno Ilaria Fregno Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland Search for more papers by this author Andrea Raimondi Andrea Raimondi Experimental Imaging Center, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Alessandro Marazza Alessandro Marazza Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland Search for more papers by this author Gemma Bruno Gemma Bruno Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Maria Iavazzo Maria Iavazzo Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Daniela Intartaglia Daniela Intartaglia Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Marta Seczynska Marta Seczynska Institute of Biochemistry II, Goethe University Frankfurt – Medical Faculty, University Hospital, Frankfurt am Main, Germany Search for more papers by this author Eelco van Anken Eelco van Anken Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy Search for more papers by this author Ivan Conte Ivan Conte Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Search for more papers by this author Maria Antonietta De Matteis Maria Antonietta De Matteis Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Department of Molecular Medicine and Medical Biotechnologies, University of Naples "Federico II", Naples, Italy Search for more papers by this author Ivan Dikic Corresponding Author Ivan Dikic [email protected] orcid.org/0000-0001-8156-9511 Institute of Biochemistry II, Goethe University Frankfurt – Medical Faculty, University Hospital, Frankfurt am Main, Germany Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Maurizio Molinari Corresponding Author Maurizio Molinari [email protected] orcid.org/0000-0002-7636-5829 Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Carmine Settembre Corresponding Author Carmine Settembre [email protected] orcid.org/0000-0002-5829-8589 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Department of Medical and Translational Science, University of Naples "Federico II", Naples, Italy Search for more papers by this author Author Information Alison Forrester1,‡, Chiara De Leonibus1,‡, Paolo Grumati2,‡, Elisa Fasana3,‡, Marilina Piemontese1, Leopoldo Staiano1, Ilaria Fregno3,4, Andrea Raimondi5, Alessandro Marazza3,6, Gemma Bruno1, Maria Iavazzo1, Daniela Intartaglia1, Marta Seczynska2, Eelco Anken7, Ivan Conte1, Maria Antonietta De Matteis1,8, Ivan Dikic *,2,9, Maurizio Molinari *,3,10 and Carmine Settembre *,1,11 1Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy 2Institute of Biochemistry II, Goethe University Frankfurt – Medical Faculty, University Hospital, Frankfurt am Main, Germany 3Faculty of Biomedical Sciences, Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Bellinzona, Switzerland 4Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland 5Experimental Imaging Center, San Raffaele Scientific Institute, Milan, Italy 6Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland 7Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Ospedale San Raffaele, Milan, Italy 8Department of Molecular Medicine and Medical Biotechnologies, University of Naples "Federico II", Naples, Italy 9Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, Germany 10School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland 11Department of Medical and Translational Science, University of Naples "Federico II", Naples, Italy ‡These authors contributed equally to this work *Corresponding author. Tel: +49 69 6301 5964; E-mail: [email protected] *Corresponding author. Tel: +41 91 8200319/352; E-mail: [email protected] *Corresponding author. Tel: +39 081 1923 0601; E-mail: [email protected] The EMBO Journal (2019)38:e99847https://doi.org/10.15252/embj.201899847 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 Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR-Cas9 or knockout-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with the ER-phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane-associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome-resistant misfolded clients from the ER. Synopsis Unfolded procollagen in the endoplasmic reticulum (ER) is an ER-associated degradation-resistant substrate that has to be cleared by autophagy. The ER chaperone Calnexin and the ER-phagy receptor FAM134B recognize misfolded procollagen and mediate its LC3-dependent delivery to the lysosome for autophagic degradation. A candidate deletion screen shows that calnexin and FAM134B are required for ER quality control of endogenous procollagens Calnexin acts as co-receptor recognizing misfolded procollagen within the ER lumen FAM134B binds misfolded procollagen through Calnexin and links it to LC3 on autophagosomal membranes Introduction Macroautophagy (hereafter referred to as autophagy) is a homeostatic catabolic process devoted to the sequestration of cytoplasmic material in double-membrane vesicles (autophagic vesicles, AVs) that eventually fuse with lysosomes where cargo is degraded (Mizushima, 2011). Autophagy is essential to maintain tissue homeostasis and counteracts both the onset and progression of many disease conditions, such as ageing, neurodegeneration and cancer (Levine et al, 2015). Substrates can be selectively delivered to AVs through receptor-mediated processes. Autophagy receptors harbour a LC3 or GABARAP interaction motif (LIR or GIM, respectively) that facilitate binding of the cargo to LC3 or GABARAP proteins, which decorate autophagosomal membranes (Stolz et al, 2014; Rogov et al, 2017). Proteins and entire organelles or their portions can be targeted to autophagy via receptor-mediated processes. A notable example is represented by ER-phagy, a selective form of autophagy in which portions of the ER are sequestered within AVs and transported to the lysosomes for degradation (Fregno & Molinari, 2018; Grumati et al, 2018). To date, the yeast Atg39, Atg40 and the mammalian FAM134B, SEC62, RTN3 and CCPG1 proteins have been identified as ER-phagy receptors (i.e. as LC3-binding proteins that decorate specific ER subdomains for capture by AVs) (Khaminets et al, 2015; Mochida et al, 2015; Fumagalli et al, 2016; Grumati et al, 2017; Smith et al, 2018). ER-phagy mediates the turnover of ER membranes and promotes recovery after ER stress, bacterial and viral infections (Khaminets et al, 2015; Chiramel et al, 2016; Fumagalli et al, 2016; Grumati et al, 2017; Lennemann & Coyne, 2017; Moretti et al, 2017; Smith et al, 2018). ER homeostasis relies on ER quality control mechanisms to prevent the accumulation of inappropriately folded cargoes within its lumen. Misfolded proteins are dislocated from the ER to the cytosol to be degraded by the 26S proteasome, a process known as ER-associated degradation (ERAD)(Preston & Brodsky, 2017). However, not all misfolded ER proteins are eligible for ERAD and thus must be cleared from the ER through other processes. Autophagy-dependent and autophagy-independent lysosomal degradation of proteins from the ER has also been reported (Ishida et al, 2009; Hidvegi et al, 2010; Houck et al, 2014; Fregno et al, 2018). However, the mechanism by which misfolded ER luminal proteins are recognized by the cytosolic autophagic machinery and delivered to the lysosomes remains to be understood. Collagens are the most abundant proteins in animals, and type I and type II collagen (COL1 and COL2) are the major protein components of bone and cartilage, respectively (Bateman et al, 2009). They are synthesized as alpha I and alpha II chains and folded into triple helices of procollagen (PC) in the ER. Properly folded PCs associate with the heat shock protein 47 (HSP47) chaperone and then leave the ER through sub-regions called ER exit sites (ERES), within COPII-coated carriers, and move along the secretory pathway (Malhotra & Erlmann, 2015). Previous studies estimated that approximately 20% of newly synthesized type I PC (PC1) is degraded by lysosomes as a consequence of inefficient PC1 folding or secretion (Bienkowski et al, 1986; Ishida et al, 2009). In case of mutations in PC or HSP47, the fraction of PC degraded increases significantly (Ishida et al, 2009). Similarly, a fraction of type II PC (PC2) produced by chondrocytes of the growth plates is degraded by autophagy, and inactivation of this catabolic pathway results in PC2 accumulation in the ER and defective formation of the extracellular matrix (Cinque et al, 2015; Bartolomeo et al, 2017; Settembre et al, 2018). Overall these data clearly indicate that aberrant PC molecules represent ERAD-resistant substrates where autophagic clearance emerges as a crucial and physiologically relevant event in the maintenance of cellular and organ homeostasis. However, to date, the mechanism by which ER-localized PCs are selectively disposed of by autophagy is still unknown. In this study, we sought to uncover the mechanisms that select non-native PC in the ER lumen for lysosomal delivery and clearance. We found that the misfolded PC molecules (e.g. HSP47 negative) are cleared from the ER through FAM134B-mediated ER-phagy. Notably, FAM134B binds PC molecules in the ER through the interaction with the transmembrane ER chaperone Calnexin (CANX) that acts as a specific FAM134B ER-phagy co-receptor for misfolded PCs. The formation of this complex allows the selective delivery of PC molecules to the lysosomes. Results Autophagy promotes degradation of intracellular procollagens preventing their accumulation in the ER Using three different collagen producing cell lines, mouse embryonic fibroblasts (MEFs) and human osteoblasts (Saos2) stably expressing the autophagosome membrane marker LC3 fused with GFP (GFP-LC3) (Kabeya et al, 2000), and rat chondrosarcoma cells (RCS) immunolabelled for LC3, we observed co-localization of LC3-positive vesicles (hereafter referred as autophagic vesicles, AVs) with PC1 (MEFs and Saos2) and PC2 (RCS) (Fig 1A–D). Similarly, we observed the co-localization of PC1 spots with the GFP-tagged double-FYVE domain-containing protein 1 (DFCP1), which labels sites for autophagosome biogenesis (omegasomes) (Fig EV1A and B). In vivo, osteoblasts of the mandible in Medaka fish embryos (stage 40), showed the presence of AVs containing PC2 molecules (Fig EV1C–E). Figure 1. PCs are autophagy substrates and accumulate in lysosomes A, B. Airyscan confocal analysis of PC1 (568, red) co-localization with GFP-LC3 (green) in (A) MEF (B) Saos2. Scale bars = 10 μm. The insets show higher magnification (A = x4.68; B = x6.76) and single colour channels of the boxed area. C. Airyscan confocal analysis of PC2 (647, red) co-localization with LC3 (488, green) in RCS cells. Scale bars = 10 μm. The insets show higher magnification (x7.33) and single colour channels of the boxed area. D. Quantification of GFP (A, B) or LC3 (C) vesicles positive for PC1 or PC2, expressed as % of total LC3 (mean ± SEM), n = 18 cells (MEFs and Saos2); n = 12 (RCS) from three independent experiments. E–G. Scanning confocal microscopy analysis of MEFs, Saos2 and RCS cells treated with BafA1, immunolabelled for PC1 or PC2 and LAMP1. Nuclei were stained with Hoechst. (E, F) Scale bars = 10 μm, (G) Scale bars = 5 μm. The insets show higher magnification (E = x4.99; F = x6.49; G = x2.01) and single colour channels of the boxed area. H. Transmission EM analysis in Saos2 cells, treated with BafA1, showing in detail a lysosome which contains immunolabelled PC1 (with nanogold particles), as indicated by arrows. I. Scanning confocal microscopy analysis of Saos2 WT and CRISPR-Cas9 IDUA Saos2 at steady state, immunolabelled for PC1 and LAMP1. Nuclei were stained with Hoechst. Scale bar = 10 μm. The insets show higher magnification (left = x3.09; right = x3.12) and single colour channels of the boxed area. Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell (mean ± SEM). n = 31 WT cells, n = 33 CRISPR cells counted; three independent experiments. Student's unpaired, two-tailed t-test ***P < 0.0001. J. WT and CRISPR-IDUA Saos2 lysed and analysed by Western blot. Data are representative of three independent experiments. Source data are available online for this figure. Source Data for Figure 1 [embj201899847-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. PC is an autophagy substrate A, B. Scanning confocal analysis of immunofluorescence for PC1 (568, red) and GFP-DFCP1 (green) in (A) MEF and (B) Saos2 and quantification of AVs positive for GFP-DFCP1 containing PC1 expressed as % of total DFCP1 per cell (mean ± SEM). The insets show higher magnification (A = x5.9; B = x6.04) and single colour channels of the boxed area. Scale bar = 10 μm. (A) n = 19 and (B) n = 14 cells counted per condition; three independent experiments. C. Schematic representation of a stage 40 medaka fish. Dotted box represents area of mandible analysed in (D and E). D. Scanning confocal image of mandible from stage 40 medaka, immunostained with LC3 (488, green) PC2 (568, red) and nuclei stained with Hoechst (blue). Dotted box represents area of mandible containing osteoblasts that was further analysed in (E). Scale bar = 20 μm. E. Airyscan confocal image of mandible at higher magnification, scale bar = 3 μm. Boxes on the right show magnification (x4.02) of boxed area. Download figure Download PowerPoint When MEFs, Saos2 and RCS cells were treated with the lysosomal inhibitor bafilomycin A1 (BafA1), PC molecules accumulated in the lumen of swollen endo/lysosomes (LAMP1-positive organelles, hereafter referred as lysosomes) (Fig 1E–G). These data were validated by PC1 immuno-electron microscopy (IEM) (Fig 1H). Western blot analysis confirmed the accumulation of intracellular PCs, as well as of the autophagy markers LC3-II and SQSTM1/p62, in cells treated with BafA1 compared to untreated cells (Fig EV2A). BafA1 washout induced a rapid clearance of PC1 and PC2 from lysosomes of MEFs and RCS, respectively, in line with the notion that PCs are degraded in this compartment (Fig EV2B and C). Click here to expand this figure. Figure EV2. PC1 and PC2 are autophagy substrates that are degraded in the lysosome A. MEFs, Saos2 and RCS were untreated or treated with 100 nM BafA1 for 6 h in MEFs, 100 nM BafA1 for 9 h in Saos2, 200 nM BafA1 for 6 h in RCS, then lysed and analysed by Western blot. Bands were visualized with antibodies against PC1, PC2, LAMP1, SQSTM1/p62, LC3 and β-actin. Western blots are representative of three independent experiments. B, C. (B) MEFs or (C) RCS treated with vehicle, 100 nM BafA1 for 4 h, followed by 4-h washout. Cells immunolabelled with LAMP1 (488, green) and PC1 (568, red). Nuclei were stained with Hoechst (blue). Scale bars = 10 μm. Source data are available online for this figure. Download figure Download PowerPoint Lysosomal storage disorders (LSDs) are genetic diseases characterized by a defective lysosomal degradative capacity due to mutations in genes encoding for lysosomal proteins. As a result, lysosomal substrates progressively accumulate within the lumen of lysosomes causing lysosomal swelling and cell dysfunction. We sought to determine whether PC molecules accumulate in the lysosomes of LSD osteoblasts. Saos2 osteoblasts in which the alpha-L-iduronidase gene was deleted using CRISPR-Cas9 technology (CRISPR-IDUA) represent a disease model of mucopolysaccharidosis type I (MPS I), a lysosomal storage disorder with severe skeletal manifestations (Oestreich et al, 2015). Similar to cells treated with BafA1, CRISPR-IDUA showed swollen lysosomes, suggesting an accumulation of undigested substrates in the lysosomal lumen (Fig 1I). Most importantly, the level of PC1 in lysosomes, and in the whole cell lysate, was higher in CRISPR-IDUA Saos2 compared to control cells (Fig 1I and J). To verify at which trafficking stage PC became an autophagy substrate, we performed a temperature shift assay where PC accumulates in the ER during incubation at 40°C, and is released from the ER upon shift of the temperature to 32°C. U2OS cells expressing GFP-LC3, mCherry-PC2 and ER marker RDEL-HALO, were imaged upon shift to 32°C (time 0 s). We observed that PC2 spots formed at the ER and progressively accumulated GFP-LC3 (Fig 2A and Movie EV1). Similarly in U2OS cells expressing phosphatidylinositol 3-phosphate (PtdIns(3)P) -recognition domain construct GFP-2•FYVE, mCherry-PC2 and ER marker RDEL-HALO, the PC2 was visible at an area of GFP-2•FYVE-positive ER, and dissociated from the main tubular ER structure releasing a vesicle positive for ER, GFP-2•FYVE and PC2 (Fig 2B and Movie EV2). Co-localization between GFP-LC3, the ER chaperone CANX and PC1 was also observed by Airyscan super-resolution confocal microscopy (Fig 2C). Similarly, we observed co-localization of PC1 spots with GFP-DFCP1 and CANX in MEFs and Saos2 cells (Fig EV3A). We also performed correlative light electron microscopy (CLEM) and electron tomography of GFP-LC3 expressing Saos2 cells, showing that PC1 and CANX are found together in a small vesicle contained within a larger LC3-positive vesicle (Fig 2D and E). Taken together, these data suggest that PC molecules are sequestered within LC3-positive vesicles when they are still within the ER. Figure 2. Autophagy sequesters PC molecules in the ER A, B. U2OS expressing (A) GFP-LC3 or (B) GFP-2-FYVE (green), mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification (A = x3.93; B = x3.42) from the boxed region are shown on the right. Scale bar = 10 μm. C. Airyscan analysis of Saos2 cells expressing GFP-LC3 (green) and immunolabelled for PC1 (405, blue) and CANX (647, red). The insets show higher magnification (x5.26) and single colour channels of the boxed area. Scale bar = 10 μm. D. Correlative light electron microscopy (CLEM) and electron tomography of Saos2 cells transfected with GFP-LC3 (green) and labelled for PC1 (568, red and nanogold particles) and CANX (647, blue). Cells were first imaged by confocal microscopy (top left panel), and then, the same region was retraced in EM (upper middle panel) and overlay is shown (upper right panel). Arrow indicates a LC3-positive vesicle containing CANX and PC1 molecules. E. Single tomography slice (left panel, taken from boxed are in D at a magnification of x2.84), overlay with immunofluorescence (IF) (central panel) and IF 3D rendering of AV (green) and the CANX positive vesicle containing gold particles of labelled collagen (blue and white, respectively) inside an AV (right panel). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Misfolded PC in the ER is targeted to lysosomes via autophagy DFCP1, a marker of autophagosome biogenesis co-localizes with PC1 and CANX. GFP-DFCP1 (green) expressing MEF and Saos2 immunolabelled for PC1 (568, red) and CANX (647, blue). Cells were imaged with scanning confocal microscopy. The insets show higher magnification (left = x5.57; right = x4.25) and single colour channels of the boxed area. Scale bars = 10 μm. Hsp47 is excluded from PC1 containing autophagosomes. GFP-LC3 (green) expressing MEFs immunolabelled for PC1 (568, red) and Hsp47 (647, blue). Cells were imaged by scanning confocal microscopy. The insets show higher magnification (x4.34) and single colour channels of the boxed area. Scale bars = 10 μm. Mutant PC2 is targeted to lysosomes at a higher rate than WT, and modulated via autophagy. RCS cells were transiently transfected with mCherry-PC2 WT, R789C or G1152D mutants (568, red) and treated for 6 h with 100 nM BafA1, and as indicated with SAR405 or Tat-BECLIN-1. Cells were fixed and immunolabelled for LAMP1 (488, green). Nuclei were stained with Hoechst (blue). The insets show higher magnification (top left to bottom right: x1.37, x1.2, x1.39, x1.34, x1.44, x1.07, x1.61, x1.7, x1.56). Scale bars = 10 μm. Bar graph shows quantification of LAMP1 vesicles positive for mCherry-PC2, expressed as % of total lysosomes per cell (mean ± SEM), minimum of n = 11 cells per genotype. Two-way ANOVA with Tukey's post hoc test performed and P-value adjusted for multiple comparisons. ns ≥ 0.05, **P < 0.005; ***P < 0.0001. Download figure Download PowerPoint The collagen-specific chaperone HSP47 was excluded from the AVs containing PC1 in MEFs, strongly suggesting that autophagy sequesters non-native PC1 molecules in the ER (Fig EV3B), in line with previous results (Ishida et al, 2009; Cinque et al, 2015). To further corroborate this notion, we studied two missense mutations in the COL2A1 protein (R789C and G1152D) that induce misfolding of the PC2 triple helix and accumulation within the ER of chondrocytes. The mutations cause a type II collagenopathy in humans, named spondyloepiphyseal dysplasia congenita (SEDC) (Murray et al, 1989). When expressed in chondrocytes, the R789C and G1152D mutants were targeted to the lysosomes at higher rates compared with WT COL2. Notably, pharmacological enhancement of autophagy with the autophagy inducing peptide Tat-BECLIN-1 (Shoji-Kawata et al, 2013) increased targeting of WT and of mutant PC2 molecules to lysosomes. Opposite results were observed by treating cells with the autophagy inhibitor SAR405 (Fig EV3C). Taken together, these data suggest that autophagy preferentially degrades non-native PC molecules and prevents their accumulation in the ER. FAM134B is required for autophagy of procollagen Distinct autophagy-related (ATG) proteins and receptors play an essential role in autophagosome formation and cargo recognition, respectively (Suzuki et al, 2017). To characterize the machinery that enables the delivery of PC molecules to lysosomes, we silenced genes belonging to different functional autophagy clusters in Saos2 cells treated with BafA1 and quantified the levels of PC1 within lysosomes. As expected, we found that the silencing of all genes tested involved in AV biogenesis significantly inhibited the delivery of PC1 to the lysosomes. Notably, among autophagy and ER-phagy receptors, we found that FAM134B silencing most effectively inhibited PC1 delivery to lysosomes (Fig 3A). Our siRNA data were further validated using MEFs knocked out for genes involved in AV biogenesis, namely Fip200 (Fip200−/−), Atg7 (Atg7−/−) or Atg16l−/− as well as in MEFs lacking Fam134b expression (CRISPR Fam134b) (Figs 3B and EV4A). The e
Referência(s)