Sterol transfer, PI 4P consumption, and control of membrane lipid order by endogenous OSBP
2017; Springer Nature; Volume: 36; Issue: 21 Linguagem: Inglês
10.15252/embj.201796687
ISSN1460-2075
AutoresBruno Mesmin, Joëlle Bigay, Joël Polidori, Denisa Jamecna, Sandra Lacas‐Gervais, Bruno Antonny,
Tópico(s)Electrochemical Analysis and Applications
ResumoArticle4 October 2017free access Transparent process Sterol transfer, PI4P consumption, and control of membrane lipid order by endogenous OSBP Bruno Mesmin orcid.org/0000-0002-5437-3246 Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Search for more papers by this author Joëlle Bigay Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Search for more papers by this author Joël Polidori Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Search for more papers by this author Denisa Jamecna Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Search for more papers by this author Sandra Lacas-Gervais Université Côte d'Azur, Centre Commun de Microscopie Appliquée, Nice, France Search for more papers by this author Bruno Antonny Corresponding Author [email protected] orcid.org/0000-0002-9166-8668 Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Search for more papers by this author Bruno Mesmin orcid.org/0000-0002-5437-3246 Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Search for more papers by this author Joëlle Bigay Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Search for more papers by this author Joël Polidori Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Search for more papers by this author Denisa Jamecna Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Search for more papers by this author Sandra Lacas-Gervais Université Côte d'Azur, Centre Commun de Microscopie Appliquée, Nice, France Search for more papers by this author Bruno Antonny Corresponding Author [email protected] orcid.org/0000-0002-9166-8668 Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France Search for more papers by this author Author Information Bruno Mesmin1, Joëlle Bigay1, Joël Polidori1, Denisa Jamecna1, Sandra Lacas-Gervais2 and Bruno Antonny *,1 1Université Côte d'Azur, Inserm, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France 2Université Côte d'Azur, Centre Commun de Microscopie Appliquée, Nice, France *Corresponding author. Tel: +33493957775; E-mail: [email protected] EMBO J (2017)36:3156-3174https://doi.org/10.15252/embj.201796687 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 The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol-binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi network (TGN). Using the inhibitor OSW-1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4-kinase PI4KIIIβ triggers large periodic traveling waves of PI4P across the TGN. These waves are cadenced by long-range PI4P production by PI4KIIα and PI4P consumption by OSBP. Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4-kinases to control the lipid composition of subcellular membranes. Synopsis Cholesterol transporter OSBP uses a large amount of phosphatidylinositol 4-phosphate to control cholesterol distribution and consequently the gradient of membrane lipid order along the secretory pathway. Acute OSBP inhibition reduces cholesterol transfer from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN). Reduced cholesterol transfer to the TGN weakens the lipid gradient between pre- and post-Golgi membranes. The cholesterol transfer activity of OSBP consumes most of the phosphatidylinositol 4-phosphate (PI4P) present at the TGN. The OSBP-dependent PI4P consumption across the TGN shows a steady or oscillatory behaviour depending on activity of PI4P producing kinases PI4KIIIβ and PI4KIIα, respectively. Introduction Cholesterol, a major lipid of mammalian cell membranes, comes from different sources (diet and synthesis), travels along many routes, and visits most intracellular compartments. These dynamic processes require a combination of sterol sensors, transporters, and effectors (Ikonen, 2008). In the case of cholesterol transporters, a few proteins have been associated with defined routes. These include NPC proteins for cholesterol export from lysosomes (Sleat et al, 2004; Cheruku et al, 2006), and Lam and STARD4 proteins for the retrograde transfer of cholesterol between the plasma membrane (PM) and the endoplasmic reticulum (ER; Mesmin et al, 2011; Gatta et al, 2015). However, what drives the export of cholesterol after its synthesis in the ER has remained enigmatic. This export is paramount for the establishment of a cholesterol gradient along organelles of the secretory pathway. Cholesterol levels must be low at the ER to maintain its biosynthetic properties and high at the PM to make this membrane an impermeable barrier. Oxysterol-binding protein (OSBP) was identified as a binding protein for oxysterols, by-products of cholesterol that downregulate the expression of genes involved in cholesterol synthesis (Ridgway et al, 1992). However, subsequent studies indicated that OSBP is dispensable for this signaling pathway (Nishimura et al, 2005). Instead, examination of the domain architecture of OSBP suggested that it could act as a cholesterol transfer protein at membrane contact sites (MCS) between ER and the trans-Golgi network (TGN; Levine, 2004). OSBP contains three well-defined regions: A PH (pleckstrin homology) domain, a FFAT (two phenylalanines in an acidic tract) motif, and an ORD (OSBP-related domain). The PH domain binds selectively to the TGN through a dual interaction with the phosphoinositide phosphatidylinositol-4-phosphate (PI4P) and the protein Arf1-GTP (Levine & Munro, 2002). The FFAT motif binds to the ER receptor VAP-A, a transmembrane protein that controls ER localization of a myriad of cytosolic proteins (Loewen et al, 2003). Last, the ORD binds not only oxysterols but also cholesterol (Im et al, 2005). Because these domains are separated by regions that can act as flexible spacers, the overall architecture of OSBP suggests a ferry-bridge mechanism in which OSBP connects the ER to the TGN through its FFAT motif and PH domain, respectively, and transfers cholesterol between the two apposed membranes through its C-terminal ORD domain (Levine, 2004). The ferry-bridge model of OSBP has been successfully tested using purified components (Mesmin et al, 2013). OSBP can connect artificial membranes through its FFAT motif and PH domain, respectively, and this tethering conditions the fast (≈2 s) transfer of sterol by the ORD domain between apposed membranes. In addition, various cellular and biochemical observations using mutated forms of OSBP concur to make the ferry bridge a plausible model for its functioning at MCS (Mesmin et al, 2013). Lastly, this model might apply to several cytosolic proteins that share with OSBP a similar domain organization. Two prominent examples are CERT and FAPP2, which transfer ceramide and glucosylceramide, respectively (Hanada et al, 2003; D'Angelo et al, 2007). In its simplest formulation, the ferry-bridge model is tantamount to a passive conduit through which specific lipids rapidly cross the ER/TGN interface. In the case of OSBP and its yeast homologue Osh4, however, various observations suggest that the mechanism is active because the phosphoinositide PI4P brings energy into it (de Saint-Jean et al, 2011; Mesmin et al, 2013; Moser von Filseck et al, 2015b). This lipid is synthesized by various PI4-kinases, which reside in organelles of the late secretory pathway, including the TGN. In contrast, PI4P is hydrolyzed by the ER-resident enzyme Sac1. This segregation creates a sharp PI4P gradient from the TGN where PI4P levels are high to the ER where PI4P levels are low. Reconstitution experiments indicate that this gradient makes the functioning of OSBP and Osh4 akin to lipid pumps (de Saint-Jean et al, 2011; Mesmin et al, 2013; Moser von Filseck et al, 2015b). After sterol delivery to a donor liposome that imitates the TGN, the ORD of OSBP extracts PI4P from there and transfers PI4P back to ER-like liposomes, where PI4P is further hydrolyzed by Sac1 (Mesmin et al, 2013). Thus, PI4P might act as a fuel to direct the forward transfer of sterol, a function that departs from the classical role of phosphoinositides as membrane signposts. Does the OSBP cycle fairly describe the cellular function of OSBP? This question remains largely unanswered. First, evidence exists that OSBP in mammalian cells and Osh4 in yeast act in signaling pathways (Wang et al, 2005; Mousley et al, 2012; Bao et al, 2015). This does not exclude a role in lipid transport but complicates the analysis. Second, functional studies of OSBP in model organisms are scarce. In mammals, deletion of OSBP is lethal at early stages of embryogenesis (Brown and Goldstein, personal communication). In Drosophila, deletion of OSBP leads to a defect in spermatogenesis by preventing the formation of sterol-rich speckles in the cytoplasm during the process of spermatid individualization (Ma et al, 2010). However, Drosophila is auxotroph for cholesterol, hence not well adapted to cover all functions of OSBP. In yeast, the role of Osh4 in the control of sterol flows remains highly debated (Raychaudhuri et al, 2006; Georgiev et al, 2011; Beh et al, 2012). The most compelling evidence in favor of the OSBP cycle comes from the observation that some RNA viruses build a cholesterol-rich replication organelle from the ER by hijacking OSBP and PI4-kinases (Roulin et al, 2014; Strating et al, 2015). Addressing the relevance of the OSBP cycle in the cell requires overcoming two issues: (i) rapidly switching off endogenous OSBP, and (ii) following its two lipid ligands with a good spatiotemporal resolution. Here, we take advantage of a recently described drug, OSW-1, which specifically inactivates endogenous OSBP (Burgett et al, 2011), and of fluorescent probes to follow sterol, PI4P, and lipid order (Hao et al, 2002; Levine & Munro, 2002; Balla et al, 2005; Niko et al, 2016). We show that endogenous OSBP contributes massively to the ER to TGN transfer of sterols and, for this, uses about half of PI4P present in the cell. The presence of a PI4-kinase in the vicinity of OSBP is key to support the cycle. If not, the system starts to oscillate because of the mismatch between the membranes that are adapted to host OSBP and the membranes that are permissive to the production of PI4P. Results ORPphilin OSW-1 is a potent inhibitor of the OSBP cycle ORPphilins are natural drugs that inhibit the growth of a unique subset of cancer cell lines, suggesting a similar target (Burgett et al, 2011). Affinity chromatography identified OSBP and its close homologue ORP4 as receptors for ORPphilin OSW-1 (Burgett et al, 2011). Furthermore, numerous observations suggest that OSBP and ORP4 are the main and probably sole cellular targets of ORPphilins. This includes a strong correlation between the cytotoxicity of ORPphilins and their affinities for OSBP/ORP4 (Burgett et al, 2011). Thus, ORPphilins provide unique pharmacological tools to evaluate the function of OSBP and/or ORP4 in a cellular context. A prerequisite, however, is to determine the impact of ORPphilins on the molecular activities of these lipid-transfer proteins. We assessed the effect of OSW-1 on three biochemical activities of OSBP. These are cholesterol transfer, PI4P transfer, and membrane tethering. In all cases, the assay consists in analyzing mixtures containing purified OSBP and two populations of synthetic liposomes, LE and LG, which mimicked the ER and the TGN, respectively (Mesmin et al, 2013). LE contained a fraction of DGS-NTA(Ni) lipids to which the cytosolic domain of VAP-A was attached through an histidine tag, as previously described (Mesmin et al, 2013). We first followed the transfer of dehydroergosterol (DHE), a naturally fluorescent cholesterol analog, from LE to LG in the presence of increasing concentration of OSW-1 (Fig 1A). OSW-1 blocked OSBP-catalyzed transfer of DHE with an apparent Ki of 50 nM, as compared to 200 nM in the case of 25-hydroxycholesterol (25-OH; Fig 1B). Sterol transfer by the ORD domain alone was similarly inhibited by OSW-1 (Fig 1B, right panel) suggesting that the lipid-binding pocket of OSBP is the target of OSW-1. This observation corroborates previous findings showing that ORPphilins compete with 25-OH for binding to OSBP (Burgett et al, 2011). Note that at the concentration of OSBP used in the experiments shown in Fig 1 (100 nM), we can hardly measure affinity lower than 50 nM. Figure 1. Characterization of OSW-1 as a potent OSBP inhibitor Experimental strategy for lipid exchange assays. Inhibitory effect of OSW-1 and 25-OH on DHE transfer mediated by full-length OSBP (0.1 μM; left panel) or by its ORD domain alone (0.1 μM; right panel). Liposomes mimicking the ER (LE) and the Golgi (LG; 63 μM lipids each) contained 18% DHE and 2.5% Dansyl-PE, respectively. VAP-A-His was used at 1 μM. Data show the percent of maximal transfer activity obtained without drug. Effect of OSW-1 and 25-OH on OSBP-mediated PI4P transfer. LG contained 2 mol% PI4P and LE contained 2 mol% cholesterol (300 μM total lipids each). VAP-A-His was used at 3 μM. OSW-1 gradually inhibited PI4P transfer whereas 25-OH slightly stimulated PI4P transfer. See also Appendix Fig S1A and B. Data are mean ± SEM (n = 3). Experimental strategy for liposome aggregation measurements. OSW-1 stabilizes liposome tethering in the presence of Sac1. Aggregation of LE and LG liposomes by OSBP as followed by DLS. The cuvette contained LE and LG liposomes (25 μM lipids each). LE had 2 mol% DGS-NTA(Ni) and was decorated with VAP-A-His (0.2 μM) and Sac1-His (10 nM) when indicated. LG contained 2 mol% PI4P otherwise indicated. When indicated, OSBP (0.2 μM) was added and promoted liposome aggregation. The presence of Sac1-His diminished the growth of liposome aggregates (left panel). OSW-1 antagonized the Sac1 effect in a dose-dependent manner (right panel). Size of liposome aggregates obtained from 10 autocorrelation curves after the reactions. Error bars represent SD. HeLa cells coexpressing TagBFP-βGalT1, GFP-VAP-A, and mCherry-OSBP. βGalT1 labels the TGN and VAP-A the ER network, whereas OSBP is mostly cytosolic (top). Upon OSW-1 treatment (20 nM, for 1 h at 37°C), OSBP and VAP-A concentrate to a perinuclear region (bottom). Scale bar: 20 μm. See also Movie EV1. Thin-section EM of cells expressing GFP-VAP-A and mCherry-OSBP and treated with OSW-1. Scale bar: 250 nm. Evolution of the Pearson's correlation coefficient between mCherry-OSBP and GFP-VAP-A over time. When indicated, OSW-1 (5 or 20 nM final concentration) was added to the medium. DMSO was added in control experiment (0 nM). Data represent mean ± SEM (error bars; n = 4). Download figure Download PowerPoint We next measured the effect of OSW-1 on the ability of OSBP to transfer PI4P (Fig 1C). OSW-1 decreased the PI4P transfer rate in a dose-dependent manner (Ki ≈ 100 nM) indicating that this step of the OSBP cycle is inhibited by OSW-1 as well. In marked contrast, 25-OH was ineffective at blocking PI4P transfer and rather slightly accelerated the reaction (Fig 1C and Appendix Fig S1A and B). Last, we assessed the effect of OSW-1 on the membrane-tethering activity of OSBP (Fig 1D–F). We mixed LE and LG liposomes and monitored their aggregation by dynamic light scattering (DLS). In the presence of VAP-A on LE and PI4P on LG, OSBP caused massive liposome aggregation as previously reported (Fig 1E, orange curve; Mesmin et al, 2013). When liposomes LE were further supplemented with Sac1, liposome aggregation was strongly reduced reaching levels similar to that observed when LG did not contain PI4P (compare red and black curves). By transferring PI4P from LG to LE where Sac1 can hydrolyze PI4P, OSBP promotes the disappearance of a key determinant for its own membrane attachment (Mesmin et al, 2013). Figure 1E and F shows that OSW-1 did not change the initial rate of liposome aggregation but increased the steady-state level (half-maximal effect = 35 nM), suggesting that OSW-1 does not modify the ability of OSBP to tether membranes per se but stabilizes membrane tethering by inhibiting PI4P transfer. As such, the drug maintains high levels of PI4P in liposomes LG, which is optimal for their robust aggregation with liposomes LE by OSBP. To test whether OSW-1 also stabilizes OSBP-mediated membrane tethering in a cellular context, we coexpressed mCherry-tagged OSBP, GFP-tagged VAP-A, and a TGN marker [residues 1–82 of β-1,4-galactosyltransferase-1 (βGalT1) tagged with TagBFP] in HeLa cells. Overexpressed OSBP is mainly cytosolic due to its PI4P exchange activity, which facilitates PI4P hydrolysis by Sac1 and therefore limits OSBP membrane attachment over time (Fig 1G; Mesmin et al, 2013). Cells were then treated with 20 nM OSW-1 and tracked by fluorescence microscopy during 1 h at 37°C. Movie EV1 shows that upon addition of OSW-1, OSBP rapidly shifted from the cytosol to βGalT1-positive perinuclear regions. This shift was accompanied by a dramatic recruitment of VAP-A in the same structures (Fig 1G). By thin-section EM, we observed extensive appositions of ER and Golgi membranes in cells expressing OSBP and VAP-A and treated with 20 nM OSW-1 (Fig 1H). Measurements of the Pearson's correlation coefficient between OSBP and VAP-A over time (Fig 1I) indicated that OSW-1 promotes the formation of ER-TGN MCS in a dose-dependent manner. Note that the cellular effects reported here with OSW-1 are reminiscent to what we observed with 25-OH (Mesmin et al, 2013) except that nanomolar concentrations of OSW-1 were enough to achieve what normally required micromolar amounts of 25-OH (Appendix Fig S1C–E). Altogether, the experiments shown in Fig 1 indicate that OSW-1 blocks OSBP in a conformation where the protein stably bridges TGN- and ER-like membranes but no longer transfers sterols and PI4P. As such, it surpasses 25-OH by three criteria: a better affinity, a higher specificity, and the ability to block not only cholesterol transfer but also PI4P transfer by OSBP. Therefore, OSW-1 appears as an ideal tool to target endogenous OSBP and to address the relevance of the sterol/PI4P cycle in a cellular context. Inhibition of endogenous OSBP affects intracellular sterol distribution Whereas OSBP is ubiquitously expressed, ORP4 is only present in a few tissues (testis, brain, and heart; Lehto et al, 2001; Udagawa et al, 2013). In the following, we applied OSW-1 to two cell lines: immortalized retinal pigmental epithelial cells (hTERT-RPE1) and HeLa cells. A previous comprehensive analysis of protein stoichiometry in HeLa cells indicates that OSBP is present in a 100-fold mole excess over ORP4 (Hein et al, 2015). Using specific antibodies against OSBP and ORP4, we confirmed these findings and observed that OSBP is also present in large excess over ORP4 in RPE1 cells (Appendix Fig S2). In these cell lines, OSW-1 should mostly exert its effect through OSBP inhibition. To determine the contribution of endogenous OSBP to sterol transfer, we performed UV-sensitive imaging of DHE-loaded RPE1 cells in the presence or in the absence of OSW-1. DHE mirrors [3H]-cholesterol in its cellular distribution and dynamics and can be tracked in living cells making it a fair reporter of cholesterol flows (Hao et al, 2002). Figure 2A shows RPE-1 cells pulse-labeled with DHE and further incubated for 2 h with or without 20 nM OSW-1 at 37°C. In control cells, DHE was typically enriched at the PM and in a perinuclear region positive for βGalT1. Note that the DHE signal appearing in this region might derive from both TGN and endocytic recycling compartment (ERC) since these two organelles are often located right next to each other in this area. In OSW-1-treated cells, DHE was redistributed (Fig 2A and Appendix Fig S3). First, the DHE signal increased by about 2.4-fold in lipid droplets, as judged by its co-localization with the neutral lipid stain LipidTOX. Second, we observed a 1.5-fold drop in the TGN/ERC labeling by DHE, indicating a significant decrease in the amount of cholesterol in this region that normally holds a major fraction of cellular cholesterol (Hao et al, 2002). We also noticed a subtle decline in DHE at the cell periphery and the apparition of a diffuse DHE signal, which eventually concentrated around the nucleus or in a large hazy perinuclear region, probably corresponding to the ER (Fig 2A and Appendix Fig S3). This unusual DHE distribution contrasted with the sharp TGN/ERC pattern found in control cells. Together, these observations point to a blockage of sterol export from the ER at the expense of sterol traffic toward late secretory compartments. Figure 2. Contribution of endogenous OSBP to intracellular sterol transport and lipid membrane order OSW-1 affects intracellular DHE distribution. Widefield images of RPE-1 cells transfected with TagBFP-βGalT1 for 18 h. Cells were pulse-chased with DHE and incubated with OSW-1 (20 nM) for 2 h at 37°C, or DMSO (1%) as a control. Cells were labeled with the lipid droplet marker LipidTOX Green (LT) 15 min prior imaging. Note the hazy distribution of DHE, as well as its increased co-localization with lipid droplets, in OSW-1-treated cells. Scale bar: 20 μm. Right panels show the amount of DHE present in the perinuclear region (top) or in lipid droplets (bottom) from DMSO (190 cells) and OSW-1-treated cells (240 cells), from two independent experiments. Each point represents the signal from a single cell with the mean fluorescence indicated by the black lines. Student's t-test P-values are shown. Ratio imaging of RPE-1 cells stained with 100 nM of PA. Scale bar: 20 μm. The pseudo-colors represent the long (550–700 nm) to short (470–550 nm) emission wavelength ratio upon excitation at 405 nm, according to the scale shown on the right. The bottom panel represents the pixel distribution of the ratio (or tonal) value from cells treated with OSW1 or control cells. Data represent means ± SEM (error bars; n = 3). Student's t-test P-values are shown over the indicated ranges to compare OSW1 and DMSO-treated cells. Download figure Download PowerPoint We performed a similar analysis of DHE distribution in cells that have been treated for 72 h with siRNA against OSBP. OSBP silencing [≈90%, see Fig 3 below and Dong et al (2016)] caused a decrease in DHE labeling of TGN/ERC of similar amplitude as that observed after OSW-1 addition, suggesting that OSBP is the main target of OSW-1 in these cells (compare Figs 2A and EV1A). However, this effect was not accompanied by a significant change of DHE in lipid droplets. Instead, we observed a large increase of DHE in late endosomal structures (Fig EV1B). Compared to the short (2 h) OSW-1 treatment, the long (72 h) exposure of cells to siRNA against OSBP is more likely to favor compensatory effects preventing toxic cholesterol accumulation at the ER. In this respect, we noted that cholesterol transfer proteins acting between the ER and late endosomal compartments have been very recently identified (Wilhelm et al, 2017; Zhao & Ridgway, 2017). Figure 3. Massive consumption of PI4P by OSW-1 target A. Epifluorescence images of RPE-1 cells co-transfected with TagBFP-βGalT1 and the PI4P probe GFP-P4MSidM for 18 h. When indicated, cells were incubated with 25-OH (20 μM) or OSW-1 (20 nM) during 45 min, washed, and fixed with PFA (3%) before imaging. Scale bar: 20 μm. In the graph showing the TGN/cytosol ratio of GFP-P4MSidM, each point represents a single cell measurement (135–150 cells per condition, three independent experiments). The dim cytosol area of OSW-1-treated cell is demarcated by a white dashed line. Horizontal lines on the graph indicate the mean values. Student's t-test P-values are shown. B. Time-lapse microscopy of RPE-1 cells co-transfected with TagBFP-βGalT1 and the PI4P probe GFP-P4MSidM for 18 h and treated with OSW-1 (20 nM) as indicated. Top: individual frames from a movie of GFP-P4MSidM (Movie EV2) featuring the TGN region. An inverted grayscale table was used, and fluorescence is shown in black. Scale bar: 5 μm. Bottom: time course of GFP-P4MSidM at the TGN (defined by the βGalT1 mask) or in the cytosol (gray curve). The arrow indicates drug addition into the cell medium (t = 0). Data are means ± SEM of four to eight independent experiments. C, D. Same as in (B) with RPE-1 cells stably expressing GFP-P4MSidM and either treated for 72 h with siRNA against OSBP (C) or overexpressing (24 h) mCherry-OSBP (D). Left: individual frames from time series. Scale bar: 20 μm. Central graphs: normalized intensity of GFP-P4MSidM at the TGN (colored curves) and in the cytosol (gray); mean of four independent experiments ± SEM. Student's t-test P-values are shown. Right graphs: the specific GFP-P4MSidM level at the TGN was obtained by subtracting the cytosol value from the TGN value. Panel (C) also shows a Western blot of the expression level of endogenous OSBP under the various conditions. In (D), the purple and black arrowheads indicate cells transfected or not with mCherry-OSBP, respectively. The dotted lines report the curves of panel (B) obtained on control cells. E. Quantification of PIP species by LC-MS/MS of RPE-1 cells subjected to OSW-1 (20 nM) treatment during 30 min at 37°C, or DMSO as a control. The relative peak areas are normalized to the highest one. Data represent means ± SEM (error bars; n = 3). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Effect of OSBP silencing on DHE subcellular distribution A, B. RPE1 cells transfected for 72 h with siRNA against OSBP or control siRNA (si-NT) were subsequently pulse-chased for 2 h at 37°C with DHE and analyzed as in Fig 2A for the amount of DHE in the TGN region as identified by the marker TagBFP-βGalT1 (A), in lipid droplets (LipidTOX; B), or in late endosomal compartments (DND-99; B). Scale bars: 20 μm. Each point represents the signal from a single cell structure in (A) and from a single field of cells in (B). All data are from three independent experiments and the means are shown. P-values from Student's t-test are quoted on the graphs. Download figure Download PowerPoint Altogether, the experiments shown in Figs 2 and EV1 indicate that endogenous OSBP makes a major contribution to the transfer of cholesterol from the ER to the TGN. Inhibition of endogenous OSBP affects lipid order of cell membranes Cholesterol influences the physicochemical properties of cellular membranes by modifying the lateral order of lipids. If OSW-1 prevents normal cholesterol trafficking from early to late membranes, this should impact on membrane lipid order. We used a recently developed pyrene-derivative dye (PA) that is highly sensitive to lipid order (Niko et al, 2016). PA is bright and photostable, and its emission shifts from 520 nm, when embedded in a high-order membrane environment, to 580 nm, when embedded in a low-order membrane environment (Niko et al, 2016). This shift is favored by lipid saturation as well as by increasing the amount of cholesterol in model membranes (Fig EV2A). RPE1 cells were incubated with 100 nM of the PA dye for 10 min at 37°C and subsequently imaged at two emission channels (470–550 and 550–700 nm). The long-to-short wavelength ratio was eventually converted into a pseudo-colored scale (Figs 2B and EV2B). In agreement with previous observations, control cells displayed a large diversity of lipid order among intracellular compartments (Niko et al, 2016). We could differentiate regions of low ratio values (colored blue) comprising the PM and lipid droplets, from regions of high ratio values (colored red), which included the nuclear membrane and possibly the rest of the ER. Intermediate ratio values (yellow/green colors) defined a well-outlined perinuclear region, which might include the ERC/TGN. Click here to expand this figure. Figure EV2. Effect of OSW-1 on lipid order Fluorescence properties of PA (1 μM) added to POPC liposomes (200 μM) containing increasing amount of cholesterol. Excitation: 430 nm. Left: emission spectra. Right: plot of the maximum emission wavelength as a function of cholesterol concentration. For comparison, the dashed vertical lines on the spectrum panel indicate the extreme emission wavelength of PA on highly ordered (sphingomyelin + cholesterol liposomes) and highly disordered membrane (C18:1-C18:1 PC) as previously reported (Niko et al, 2016). The solid vertical lines indicate the range of PA emission under our experimental conditions (POPC liposomes ± cholesterol). Gallery o
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