OCRL controls trafficking through early endosomes via PtdIns4,5P 2 -dependent regulation of endosomal actin
2011; Springer Nature; Volume: 30; Issue: 24 Linguagem: Inglês
10.1038/emboj.2011.354
ISSN1460-2075
AutoresMariella Vicinanza, Antonella Di Campli, Elena Polishchuk, Michele Santoro, Giuseppe Di Tullio, Anna Godi, Elena Levtchenko, Maria Giovanna De Leo, Roman Polishchuk, Lisette Sandoval, María‐Paz Marzolo, Maria Antonietta De Matteis,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle4 October 2011free access OCRL controls trafficking through early endosomes via PtdIns4,5P2-dependent regulation of endosomal actin Mariella Vicinanza Mariella Vicinanza Telethon Institute of Genetics and Medicine, Naples, Italy Search for more papers by this author Antonella Di Campli Antonella Di Campli Institute of Protein Biochemistry, CNR, Naples, Italy Search for more papers by this author Elena Polishchuk Elena Polishchuk Institute of Protein Biochemistry, CNR, Naples, Italy Search for more papers by this author Michele Santoro Michele Santoro Telethon Institute of Genetics and Medicine, Naples, Italy Search for more papers by this author Giuseppe Di Tullio Giuseppe Di Tullio Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy Search for more papers by this author Anna Godi Anna Godi Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, ItalyPresent address: Virus Reference Department, Centre for Infections Health Protection Agency, London, UK Search for more papers by this author Elena Levtchenko Elena Levtchenko Department of Paediatrics, University Hospitals Leuven, Leuven, Belgium Search for more papers by this author Maria Giovanna De Leo Maria Giovanna De Leo Telethon Institute of Genetics and Medicine, Naples, Italy Search for more papers by this author Roman Polishchuk Roman Polishchuk Telethon Institute of Genetics and Medicine, Naples, Italy Search for more papers by this author Lisette Sandoval Lisette Sandoval Laboratorio de Tráfico Intracelular y Señalización, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Santiago, Chile Millenium Nucleus in Regenerative Biology (MINREB), Pontificia Universidad Católica de Chile, Santiago, Chile Search for more papers by this author Maria-Paz Marzolo Maria-Paz Marzolo Laboratorio de Tráfico Intracelular y Señalización, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Santiago, Chile Millenium Nucleus in Regenerative Biology (MINREB), Pontificia Universidad Católica de Chile, Santiago, Chile Search for more papers by this author Maria Antonietta De Matteis Corresponding Author Maria Antonietta De Matteis Telethon Institute of Genetics and Medicine, Naples, Italy Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy Search for more papers by this author Mariella Vicinanza Mariella Vicinanza Telethon Institute of Genetics and Medicine, Naples, Italy Search for more papers by this author Antonella Di Campli Antonella Di Campli Institute of Protein Biochemistry, CNR, Naples, Italy Search for more papers by this author Elena Polishchuk Elena Polishchuk Institute of Protein Biochemistry, CNR, Naples, Italy Search for more papers by this author Michele Santoro Michele Santoro Telethon Institute of Genetics and Medicine, Naples, Italy Search for more papers by this author Giuseppe Di Tullio Giuseppe Di Tullio Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy Search for more papers by this author Anna Godi Anna Godi Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, ItalyPresent address: Virus Reference Department, Centre for Infections Health Protection Agency, London, UK Search for more papers by this author Elena Levtchenko Elena Levtchenko Department of Paediatrics, University Hospitals Leuven, Leuven, Belgium Search for more papers by this author Maria Giovanna De Leo Maria Giovanna De Leo Telethon Institute of Genetics and Medicine, Naples, Italy Search for more papers by this author Roman Polishchuk Roman Polishchuk Telethon Institute of Genetics and Medicine, Naples, Italy Search for more papers by this author Lisette Sandoval Lisette Sandoval Laboratorio de Tráfico Intracelular y Señalización, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Santiago, Chile Millenium Nucleus in Regenerative Biology (MINREB), Pontificia Universidad Católica de Chile, Santiago, Chile Search for more papers by this author Maria-Paz Marzolo Maria-Paz Marzolo Laboratorio de Tráfico Intracelular y Señalización, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Santiago, Chile Millenium Nucleus in Regenerative Biology (MINREB), Pontificia Universidad Católica de Chile, Santiago, Chile Search for more papers by this author Maria Antonietta De Matteis Corresponding Author Maria Antonietta De Matteis Telethon Institute of Genetics and Medicine, Naples, Italy Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy Search for more papers by this author Author Information Mariella Vicinanza1,‡, Antonella Di Campli2,‡, Elena Polishchuk2, Michele Santoro1, Giuseppe Di Tullio3, Anna Godi3, Elena Levtchenko4, Maria Giovanna De Leo1, Roman Polishchuk1, Lisette Sandoval5,6, Maria-Paz Marzolo5,6 and Maria Antonietta De Matteis 1,3 1Telethon Institute of Genetics and Medicine, Naples, Italy 2Institute of Protein Biochemistry, CNR, Naples, Italy 3Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy 4Department of Paediatrics, University Hospitals Leuven, Leuven, Belgium 5Laboratorio de Tráfico Intracelular y Señalización, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Santiago, Chile 6Millenium Nucleus in Regenerative Biology (MINREB), Pontificia Universidad Católica de Chile, Santiago, Chile ‡These authors contributed equally to this work *Corresponding author. Telethon Institute of Genetics and Medicine (TIGEM), Telethon, Via Pietro Castellino, 111, Naples 80131, Italy. Tel.: +39 081 613 2220; Fax: +39 081 560 9877; E-mail: [email protected] The EMBO Journal (2011)30:4970-4985https://doi.org/10.1038/emboj.2011.354 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 Mutations in the phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) 5-phosphatase OCRL cause Lowe syndrome, which is characterised by congenital cataracts, central hypotonia, and renal proximal tubular dysfunction. Previous studies have shown that OCRL interacts with components of the endosomal machinery; however, its role in endocytosis, and thus the pathogenic mechanisms of Lowe syndrome, have remained elusive. Here, we show that via its 5-phosphatase activity, OCRL controls early endosome (EE) function. OCRL depletion impairs the recycling of multiple classes of receptors, including megalin (which mediates protein reabsorption in the kidney) that are retained in engorged EEs. These trafficking defects are caused by ectopic accumulation of PtdIns4,5P2 in EEs, which in turn induces an N-WASP-dependent increase in endosomal F-actin. Our data provide a molecular explanation for renal proximal tubular dysfunction in Lowe syndrome and highlight that tight control of PtdIns4,5P2 and F-actin at the EEs is essential for exporting cargoes that transit this compartment. Introduction Mutations in OCRL, which encodes a phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) 5-phosphatase, result in oculo-cerebro-renal syndrome of Lowe (Lowe syndrome). This is a severe disease that is characterised by congenital cataracts, central hypotonia, and renal proximal tubular dysfunction, with low molecular weight (LMW) proteinuria and acidosis (Lowe et al, 1952; Attree et al, 1992). OCRL is localised to the plasma membrane (PM), clathrin-coated vesicles (CCVs), multiple endosomal compartments, and the trans-Golgi network (TGN; Ungewickell et al, 2004; Choudhury et al, 2005; Faucherre et al, 2005; Hyvola et al, 2006). OCRL is a multidomain protein with a split N-terminal pleckstrin-homology (PH) domain (Mao et al, 2009), a central 5-phosphatase catalytic domain (Tsujishita et al, 2001; Schmid et al, 2004), an ASPM-SPD-2-Hydin (ASH) domain (Ponting, 2006; Erdmann et al, 2007; McCrea et al, 2008), a C-terminal inactive Rho-GTPase-activating protein (GAP) domain (Faucherre et al, 2003), and multiple clathrin-binding motifs (Choudhury et al, 2005, 2009) and Rab-binding regions (Hyvola et al, 2006). Through these domains and motifs, OCRL interacts with key components of the membrane-trafficking machineries, such as clathrin, the clathrin adaptor AP2, small GTPases (like Rab5, Rab6, Rab14, and Arf6), endocytic adaptors (like APPL1), and the recently identified Ses proteins (Ungewickell et al, 2004; Choudhury et al, 2005, 2009; Hyvola et al, 2006; Lichter-Konecki et al, 2006; Erdmann et al, 2007; Fukuda et al, 2008; McCrea et al, 2008; Swan et al, 2010; Noakes et al, 2011; Pirruccello et al, 2011). In spite of the detailed knowledge we have gained over the last few years regarding the subcellular localisation, molecular organisation, and interactors of OCRL, we still lack a real and coherent understanding of the cellular roles of OCRL and thus of pathogenetic mechanisms of Lowe syndrome. The role of OCRL in membrane trafficking, for instance, has remained debated and ill defined. In spite of reports showing that the depletion of OCRL impairs endosome-to-Golgi trafficking (Choudhury et al, 2005) in mammal cells, or that depletion of a distant homologue of OCRL induces the appearance of giant endocytic vacuoles in Drosophila (Ben El Kadhi et al, 2011), other reports conclude that OCRL does not directly modulate endocytosis or post-endocytic membrane trafficking in mammal cells (Coon et al, 2009; Cui et al, 2010). On the other side, OCRL has been shown to have a role in actin cytoskeleton regulation, cell migration, and more recently in cytokinesis (Suchy and Nussbaum, 2002; Coon et al, 2009; Ben El Kadhi et al, 2011; Dambournet et al, 2011); however, the significance of these roles of OCRL for the pathogenesis of Lowe syndrome remains to be understood. Here, with the aim of uncovering roles of OCRL that are relevant for the pathogenesis of Lowe syndrome, we analysed the impact of the loss of OCRL (both in cells knocked down (KD) for OCRL using small-interfering (si)RNAs and in renal proximal tubule cells (PTCs) from Lowe syndrome patients) on membrane trafficking pathways that govern protein reabsorption in PTCs, as this process is compromised in patients with Lowe syndrome. These pathways involve the multiligand receptor megalin, which mediates retrieval of the major fraction of the LMW proteins that are present in the ultrafiltrate. This is achieved by continuous cycling of megalin between the apical PM, where it binds the LMW proteins and other ligands in the ultrafiltrate, and the endosomal compartment, where it releases its bound ligands (Christensen and Birn, 2002; Saito et al, 2010). We show here that via its 5-phosphatase activity, OCRL is essential for early endosome (EE) function. Indeed, OCRL-KD cells and OCRL mutations in PTCs from patients with Lowe syndrome result in a ‘traffic jam’ at the level of the EEs, where different classes of endocytic and signalling receptors are retained, including megalin. We demonstrate that this trafficking defect involves ectopic accumulation of the OCRL substrate PtdIns4,5P2, and PtdIns4,5P2- and N-WASP-dependent increases in F-actin on EE membranes. Our data provide a molecular explanation for PTC dysfunction in Lowe syndrome, and they also highlight how tight temporal and spatial control of PtdIns4,5P2 and F-actin on EE membranes is essential for effective sorting and export of cargoes that pass through this compartment. Results OCRL is required for endocytic recycling of megalin We assessed the involvement of OCRL in endocytic trafficking pathways that control protein reabsorption in PTCs, and that involve the multiligand receptor megalin (Christensen and Birn, 2002; Saito et al, 2010). To this end, and due to the difficulties of obtaining satisfactory staining of endogenous megalin by immunofluorescence, we combined two approaches: a study of the distribution and trafficking of megalin in kidney cell lines (HK2 and MDCK cells) expressing a tagged form of megalin, and an analysis of the uptake and recycling of specific megalin ligands in PTCs from healthy subjects and from patients with Lowe syndrome. For the transfected megalin model, we exploited the megalin mini-receptor model (HA–Meg4), an accepted surrogate for full-length megalin (Li et al, 2001; Marzolo et al, 2003; Takeda et al, 2003; Yuseff et al, 2007) expressed in HK2 cells. At steady state, HA–Meg4 was distributed mainly to the PM, to both peripheral and central endosomal structures as labelled by APPL1, EEA1, and Mannose-6 Phosphate Receptor (MPR) (Figure 1A and C; Supplementary Figure S1B and C). Interestingly, about 30% of the megalin-positive structures also contained OCRL (Figure 1A). However, this percentage of colocalisation varied over time for the population of HA–Meg4 that was moving synchronously from the PM through the endosomal compartments. This population was followed using an anti-HA antibody that binds the lumenal HA epitope of HA–Meg4 (Figure 1B). When incubated at 4°C, the anti-HA antibody stained the PM, and then after 5 min at 37°C, the anti-HA antibody appeared in peripheral structures 30% of which contained OCRL. After a further 15 min at 37°C, the anti-HA antibody was in perinuclear structures 78% of which contained OCRL (Figure 1B). Figure 1.OCRL associates with megalin-containing endosomes. (A) HK2 cells expressing the HA–megalin (HA-Meg4) mini-receptor at steady state were stained for megalin (green) and OCRL (red), as indicated. OCRL and megalin partially colocalised in endosomal perinuclear structures (28% of megalin-containing structures were positive for OCRL). Inset: detail of boxed area. (B) HK2 cells expressing HA–Meg4 were initially kept for 2 h in serum-free medium, then incubated with a mouse anti-HA monoclonal antibody on ice for 30 min (to label the PM pool of HA–Meg4). They were then warmed to 37°C for 5 and 20 min (t5 and t20), given an acid wash, fixed, and incubated with an anti-OCRL polyclonal antibody and with secondary anti-mouse and anti-rabbit antisera. Insets: enlargement of the boxed area, with merged images of HA–Meg4 (green) and OCRL (red). After 5 min at 37°C, about 30% of the megalin-positive puncta contained OCRL. After 20 min, 78% of the perinuclear structures that contained megalin also contained OCRL. (C) HK2 cells were treated with non-targeting siRNA (CTR) or OCRL siRNAs (OCRL KD) for 96 h, transfected with HA–Meg4 for the last 18 h, and double labelled with antibodies against HA (upper panels) and against EEA1 or APPL1 (lower panels) as indicated. Insets: merged images of the boxed area, with megalin (green) and endocytic markers (red). Scale bar, 10 μm. Download figure Download PowerPoint The distribution of HA–Meg4 was markedly affected by OCRL KD, as HA–Meg4 was less visible at the PM and accumulated in EEA1- and MPR-positive endosomes (Figure 1C; Supplementary Figure S1). These changes in HA–Meg4 distribution induced by OCRL KD prompted us to investigate its impact on the trafficking of megalin and of its ligands. We found that the levels of surface-exposed HA–Meg4 were markedly reduced in the OCRL-KD HK2 cells, compared with control HK2 cells, in spite of comparable total levels of HA–Meg4 (Figure 2A and B, see also Supplementary Figure S1D for immuno-electron microscopy). Furthermore, the uptake of the anti-HA antibody in HA–Meg4 HK2 cells was significantly lower with OCRL KD, compared with control cells (Figure 2A and B). This reduced uptake was mainly due to impaired exposure of HA–Meg4 at the cell surface in OCRL-KD cells, rather than to a defect in the internalisation process per se, as the fraction of internalised/bound anti-HA antibody was not different with OCRL KD, compared with control HK2 cells (Figure 2B). Furthermore, from experiments designed to directly follow re-exposure of the internalised HA–Meg4 at the cell surface, we concluded that the impaired exposure of HA–Meg4 at the cell surface was due to a defect in the recycling of HA–Meg4 to the PM (Figure 2C). Figure 2.OCRL is required for megalin recycling to the PM. (A) HK2 cells were treated with non-targeting siRNA (CTR) or OCRL siRNAs (OCRL KD) for 96 h and transiently transfected with HA–Meg4 for the last 18 h. The PM exposure of HA–Meg4 and its internalisation were measured through binding at 4°C (cell surface HA–Meg4) and internalisation at 37°C for 5 min (internalised anti-HA Ab) of an anti-HA monoclonal antibody. The total amount of HA–Meg4 expressed was measured using an anti-HA polyclonal antibody (total HA–Meg4) in permeabilised HA–Meg4 cells. (B) Quantitative analysis of cell surface HA-Meg4 measured in cells exposed to the anti-HA monoclonal antibody for 1 h at 4°C (left graph), internalisation of HA–Meg4 measured at 5 and 20 min as uptake of anti-HA monoclonal antibody by HA–Meg4 HK2 cells treated as described in (A) (middle graph). Right graph: ratios of internalised/bound anti-HA monoclonal antibody at 5 and 20 min. All of the fluorescence intensities of the anti-HA monoclonal antibody (either at the cell surface or internalised) are normalised for total HA–Meg4 content (measured as described in (A), and expressed as the ratio between the mean fluorescence intensity of the anti-HA monoclonal antibody and that of the anti-HA polyclonal antibody). (C) HA–Meg4 recycling: HA–Meg4 HK2 cells were loaded with the anti-HA monoclonal antibody at 37°C for 30 min (LOAD) and then chased in fresh medium for 20 and 40 min at 37°C, and acid washed (CHASE). Data are mean values±s.d. (n=100 cells; three independent experiments). *P<0.01 and **P<0.001. (D, E) Uptake of GST–RAP in control PTCs (CTR; D, E), Lowe PTCs (D, E), and OCRL-KD PTCs, HK2, and MDCK cells (E) (by siRNA treatment), as indicated. RAP internalisation was quantified in (E) as the amount of cell-associated fluorescence, and expressed as % CTR. Scale bar, 10 μm. Data are mean values±s.d. (n=100 cells; three independent experiments). Download figure Download PowerPoint Of note, the defects in megalin trafficking were apparent only upon almost complete depletion of OCRL (below 5% of control values, reached by treating HK2 cells for 96 h with the OCRL siRNA; Supplementary Figure S1A). Less complete depletion of OCRL (20% of control values) might explain why Cui et al (2010) failed to see an effect of OCRL siRNA treatment on uptake/degradation of lactoferrin, which they took as an index of endogenous megalin trafficking in HK2 cells. Furthermore, we observed the same defects in HA–Meg4 exposure at the cell surface in MDCK cells stably expressing HA–Meg4 either with transient (Supplementary Figure S2A–C) or with stable (Supplementary Figure S2D–F) KD for OCRL. We also followed megalin trafficking in PTCs from healthy subjects and Lowe syndrome patients (obtained and characterised as described in Supplementary data and Supplementary Figure S2G–I); and in particular, we analysed the uptake of one of its ligands: the receptor-associated protein (RAP) that escorts neosynthesised megalin in its ER-to-Golgi transport (Bu et al, 1995; Willnow et al, 1996). RAP binds the lumenal domain of megalin with high affinity, and because of this, RAP has become a widely used tool to study megalin dynamics. When RAP is administered exogenously to cells as a recombinant protein, it is taken up with an efficiency that strictly depends on the efficiency of the trafficking and recycling of megalin (Czekay et al, 1997; Yuseff et al, 2007). While PTCs from healthy subjects readily internalised exogenously administered RAP, the Lowe PTCs took up RAP much less efficiently (Figure 2D and E). Importantly, a similar difference in efficiency was seen with OCRL KD by RNA interference in PTCs from healthy subjects and in HA–Meg4 HK2 and MDCK cells (Figure 2E). Altogether, the above data indicate that OCRL is required for the correct trafficking of megalin, and they provide a possible explanation for the impairment of LMW protein reabsorption by PTCs in Lowe syndrome. We then determined whether the requirement for OCRL was restricted to megalin trafficking or was extended to the trafficking of other endocytic cargoes/receptors. OCRL is required for endosomal trafficking of different classes of receptors We assessed the impact of OCRL KD on the trafficking of different classes of receptors that follow distinct itineraries: (i) the transferrin receptor (TfR), which follows two recycling routes to the PM, one fast and direct from the EEs, and the other slower and through the recycling endosomes (REs) (Ullrich et al, 1996; Maxfield and McGraw, 2004); (ii) the cation-independent MPR, which normally recycles between the TGN, EEs, late endosomes (LEs), and the PM (Ghosh et al, 2003; Pfeffer, 2009); and (iii) the epidermal growth factor receptor (EGFR), which after EGF-induced internalisation, is sorted towards degradation compartments (LEs/lysosomes) (Scita and Di Fiore, 2010). Compared with control cells, the OCRL-KD cells showed lower PM binding of Tf at 4°C (Figure 3A and B) (indicative of lower exposure of the TfR at the cell surface), along with impaired uptake of Tf (Figure 3A and B). As reported above for megalin ligands, also in the case of Tf, the ratio of internalised/bound Tf and the rate of internalisation of surface-bound Tf were not significantly different in the OCRL-KD cells, as compared with control cells (Figure 3B). This indicates that there was no defect in the internalisation process per se. Indeed, slower recycling rates of internalised Tf (Figure 3C) were measured in OCRL-KD cells, as compared with mock cells. The immunofluorescence and immuno-electron microscopy analyses indicated that the TfR accumulated in anomalously enlarged endosomal structures at the cell periphery and was less concentrated at the PM, as compared with control cells (Figure 3D and E). Figure 3.OCRL KD impairs recycling of the TfR. (A) Control (CTR) or OCRL-KD HeLa cells were exposed to Alexa-Fluor-488 (A488)-Tf for 1 h at 4°C and then warmed to 37°C in complete medium for 5 min. Scale bar, 10 μm. (B) Quantification of cell-associated A488-Tf, evaluated as mean fluorescence intensities at indicated times, and expressed as indicated. (C) For Tf recycling, the cells were loaded with Alexa-Fluor-488-Tf for 1 h at 37°C (LOAD) and chased in complete medium for 40 and 60 min (CHASE). The fluorescence intensities remaining in the cells after 40 and 60 min of chase were quantified and expressed as percentages of the loaded Tf. Data are mean values±s.d. (n=150 cells; three independent experiments). (D) Steady-state distributions of the TfR and MPR in CTR and OCRL-KD HeLa cells. Insets: enlargement of the boxed area, with merged images of TfR (green) and MPR (red). Scale bar, 10 μm. (E) Steady-state localisation of the TfR in CTR and OCRL siRNA-treated HeLa cells was visualised by pre-embedding immuno-gold labelling with an anti-TfR antibody. E, endosomes; G, Golgi complex; PM, plasma membrane. Scale bar, 100 nm. Download figure Download PowerPoint The trafficking of the MPR was also significantly altered in the OCRL-KD cells. This was indicated by impaired MPR-dependent uptake of the recombinant lysosomal enzyme α-glycosidase (which was reduced by 50%) and by reduced binding at 4°C and subsequent internalisation at 37°C of an antibody directed against a luminal epitope of MPR in the OCRL-KD cells, as compared with control cells (Figure 4A and B). Furthermore, the recycling of the MPR from the endosomes to the Golgi complex was impaired in the OCRL-KD cells and in Lowe PTCs. This was seen as an increase in the MPR associated with peripheral endosomal structures that also contained the TfR, and a decrease in the central Golgi pool of the MPR in PTCs from Lowe patients, and OCRL-KD PTCs, as compared with control PTCs (Figure 4C and D) and in HeLa cells (Supplementary Figure S3A). The impaired endosome-to-Golgi trafficking of the MPR prompted us to investigate the transport of MPR-dependent lysosomal enzymes, and we found that OCRL-KD cells release greater amounts of lysosomal enzymes in their precursor forms into the extracellular medium, consistent with their mis-routing to the PM as secondary to MPR mistrafficking (Supplementary Figure S3B and C). Figure 4.OCRL KD impairs recycling of the MPR to the PM and the Golgi complex. (A) Uptake of human α-glycosidase (GAA) was determined in control (CTR) and OCRL-KD HeLa cells incubated with Alexa-Fluor-546 (A546)-recombinant human GAA (A546-GAA) for 2 h at 37°C. White lines, approximate cell contours. The amount of internalised A546-hGAA (quantified by fluorescence intensity) decreased by 50% (±5%) of the CTR in the OCRL-KD cells (n=80 cells; three independent experiments). (B) PM exposure of the MPR and its internalisation were measured through binding at 4°C (1 h 4°C) and internalisation for 15 min at 37°C (+15 min 37°C) of an anti-MPR antibody directed against the luminal epitope of MPR (anti-MPR Ab) in control (CTR) and OCRL-KD HeLa cells. After fixation and treatment with the Alexa-568 secondary antibody, the cell-associated fluorescence was quantified and expressed as %CTR. (C, D) Steady-state distributions of the MPR and TfR in PTCs from healthy subjects (CTR) and Lowe syndrome patients (Lowe). In (C), the insets show enlargements of the boxed areas, with merged images of the TfR (green) and the MPR (red). (D) Images from control PTCs (CTR), Lowe PTCs, and PTCs from healthy subjects knock down for OCRL via siRNA treatment (OCRL KD) labelled for the MPR, as acquired and quantified by Scan^R automated microscopy (see Supplementary data). The percentages of cells showing the different MPR staining patterns were classified as indicated; these are mean values±s.d. (n=1000 cells; three independent experiments, each performed in quadruplicate). *P<0.01 and **P<0.001. Scale bar, 10 μm. (E) Colocalisation of the MPR with Rab proteins in control (CTR) and OCRL-KD HeLa cells transiently expressing GFP-tagged Rab4 and Rab5. Insets: enlargements of boxed areas, with merged images of Rab4 or Rab5 (green) and the MPR (red). Scale bar, 10 μm. Download figure Download PowerPoint Furthermore, we tested the impact of OCRL KD on EGF-induced trafficking of the EGFR. After 30 and 60 min of EGF stimulation, in control cells the EGFR was efficiently degraded (by up to 70%), while at the same time the EGFR levels remained high in the OCRL-KD cells (Supplementary Figure S4B and C), and the EGFR appeared to have accumulated in TfR-positive structures (Supplementary Figure S4A). Importantly, this delayed degradation of the EGFR was accompanied by more persistent signalling, as indicated by the prolonged phosphorylation state of ERK (Supplementary Figure S4B and C). The finding that OCRL depletion affects endosomal trafficking in HeLa cells might appear surprising considering the tissue specificity of the clinical manifestations of Lowe syndrome. Although the reasons for this tissue specificity remain to be defined, it appears likely that they involve compensatory mechanisms in non-affected tissues, such as high levels of the INPP5B 5-phosphatase. These compensatory mechanisms might be defective in the HeLa cell line. Indeed, Cui et al (2010) reported a lack of detectable levels of INPP5B mRNA in HeLa cells, and we also found very low levels of the INPP5B protein in this cell line (data not shown). Furthermore, it is likely that in addition to the cells that generate overt clinical signs, other cells might also suffer from trafficking defects in Lowe syndrome patients, as suggested by the platelet dysfunction (Lasne et al, 2010) and the higher blood levels of lysosomal enzymes reported in Lowe patients (Ungewickell and Majerus, 1999) and as supported by our data that OCRL-depleted cells release more lysosomal enzymes compared with control cells (Supplementary Figure S3B and C). OCRL controls receptor trafficking at EEs Altogether, the above data indicate that OCRL has a pivotal role in the trafficking and recycling of different classes of receptors. This prompted us to identify which of the different endosomal stations were these receptors pass through depends on OCRL for its correct functioning. To this end, we compared the distribution of TfR and MPR, two receptors with distinct endosomal recycling pathways: EE–LE for MPR (Ghosh et al, 2003; Pfeffer, 2009) and EE–RE for TfR (Ullrich et al, 1996; Maxfield and McGraw, 2004). In control cells, TfR and MPR show only 34% colocalisation while in OCRL-KD cells (Figure 3D) and in Lowe PTCs (Figure 4C) they show 72 and 63% colocalisation, respectively. This raises the question whether this increased colocalisation is due to the mis-routing of the TfR to LEs or to a retention/misrouting of the MPR into EEs/REs, respectively, and thus, whether the compartment affected by this OCRL KD is part of the EEs, REs, or LEs. To address this question, we took advantage of the well-defined Rab map (Stenmark, 2009) that allows identification of the nature of different endosomal compartments according to the types of their associated Rabs. We defined the EE nature of the ‘mixed’ (i.e., containing both TfR and MPR) endosomal compartment induced by the loss of OCRL by analysing a panel of Rabs (as Rabs 4, 5, 7, 9, and 11). Indeed, the peripheral structures containing the TfR and MPR in the OCRL-KD cells were marked by the EE Rabs, Rab4 and Rab5 (showing colocalisation of 87 and 58%, respectively; Figure 4E) and much less by the othe
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