1-Deoxysphingolipids dysregulate membrane properties and cargo trafficking in the early secretory pathway

Abstract

1-Deoxysphingolipids are non-canonical sphingolipids linked to several diseases, but their cellular effects are poorly understood. Here, we utilize lipid chemical biology approaches to investigate the role of 1-deoxysphingolipid metabolism on the properties and functions of secretory membranes. We first applied organelle-specific bioorthogonal labeling to visualize the subcellular distribution of metabolically tagged 1-deoxysphingolipids in RPE-1 cells, observing that they are retained in the endoplasmic reticulum (ER). We found that 1-deoxysphingolipids can be transported by the non-vesicular transporter CERT in vitro but are retained at ER exit sites (ERES) in cells, suggesting that they do not efficiently sort into vesicular carriers. Cells expressing disease-associated variants of serine palmitoyl-CoA transferase (SPT) accumulated long-chain 1-deoxysphingolipids, which reduced ER membrane fluidity and enlarged ERES. We observed that the rates of membrane protein release from the ER were altered in response to mutant SPT expression, in a manner that was dependent on the cargo affinity for ordered or disordered membranes. We propose that dysregulation of sphingolipid metabolism alters secretory membrane properties, which can then modulate protein trafficking.

Figure 1.. Organelle-specific labeling of 1-deoxySLs identifies their accumulation in early secretory membranes.

A. Schematic of SL and 1-deoxySL biosynthetic pathway depicting the intermediate lipid species and their chemical structures. B. Schematic of the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) approach utilized in this study to identify the localization of 1-deoxySL in subcellular compartments. RPE-1 cells were fed either with either 0.1 μM alkyne-deoxySA, a precursor for 1-deoxySL biosynthesis with an alkyne handle, or 0.1 μM alkyne-SA, and incubated for 17 hours to facilitate the metabolic incorporation, after which cells were fixed and CuAAC reaction was carried with either PennGreen-azide, for specific localization of the lipid in early secretory membranes or Cy3-azide, for labeling of the alkyne lipid in all cell membranes. To compare with the distribution of canonical SL products, alkyne-SA labeling was performed in parallel. C. Imaging of RPE-1 cells fed with either alkyne-deoxySA or alkyne-SA and reacted with PennGreen-azide or Cy3-azide. Scale bar, 20 μm. Alkyne-deoxySA treatment led to a larger PennGreen-azide corrected total cell fluorescence (CTCF) after washing of unreacted dye, indicating an accumulation of 1-deoxySL in the early secretory pathway. Each point represents an individual field of cells acquired and processed identically across two biological replicates. ****, P < 0.0001 by Mann-Whitney test. In contrast, the reaction with Cy3-azide did not show increased staining for the alkyne-deoxySA. D. Colocalization analysis of 1-deoxySL-PennGreen products with Golgi (GalT-mApple) and ER (Sec61β-mApple) markers. Represent images are shown to the left and quantification of the Pearson’s and Mander’s correlation coefficients to the right. PennGreen fluorescence intensity is strongly correlated with both markers, but overalaps more with the ER. Each point represents an individual cell. ****, P < 0.0001 by Mann-Whitney test. Scale bar, 20 μm. E. Airyscan images showing that co-localization of 1-deoxySL-PennGreen products with the ER network labeled by Sec61β-mCherry, with additional punctae that are not labeled by Sec61β-mCherry dispersed through the network indicated by the arrows in the merged image. Scale bar, 2.5 μm.

Figure 2.. 1-DeoxySLs are transported by CERT but accumulate at sites of vesicular trafficking.

A. Schematic of known Cer trafficking mechanisms in cells. Cer produced in the ER can be exported to the Golgi via vesicular or non-vesicular trafficking, the latter is mediated by the LTP CERT. B. The CERT START domain makes polar contacts (Q467, E446, and a coordinated water) with the C1 hydroxyl of Cer (arrow, PDB: 2E3O), which would not be made with 1-deoxy cargoes. C. Measurements of CERT transport activity for Cer and 1-deoxyCer cargoes using FRET-based liposome assay (top) and C12-NBD-DHCer/C12-NBD-deoxyDHCer (bottom). Protein preparation and probe synthesis are described in the Materials and Methods. Donor liposomes (L_D) contained 93 mol% di-oleoyl-phosphatidylcholine (DOPC), 5 mol% of the NBD-labeled lipid, and 2 mol% rhodamine-phosphatidylethanolamine (Rhod-PE). Acceptor liposomes (L_A) contained only DOPC. D. Lipid transport curves for liposomes containing 10 μM C12-NBD-DHCer (top) and C12-NBD-deoxyDHCer (bottom) upon injection of 200 nM CERT to a cuvette containing donor and acceptor liposomes. Absolute lipid transport rates are generated by normalizing FRET donor (NBD) emission with liposome standards. CERT transports C12-NBD-deoxyDHCer faster than C12-NBD-DHCer, and transport of both is slowed in the presence of 1 μM of the competitive inhibition HPA-12. Initial transport rates are reported on the right. Error bars show SEM (N = 4 independent experiments per condition). ****, P < 0.0001 by an unpaired Welch’s t-test. E. RPE-1 cells incubated with 0.1 μM 1-deoxySA for 17 hours before fixation, CuAAC labeling with PennGreen-azide, and washing, as in Figure 1. The resulting 1-deoxySL-PennGreen products are observed at punctae that co-localize with the ERES marker SEC23-mCherry. Pearson’s colocalization coefficient (PCC) between 1-deoxySL-PennGreen and SEC23-mCherry is shown for 17 cells. F. The centroid of 1-deoxySL-PennGreen punctae overlap with those of SEC23-mCherry, which label ERES, but show an average offset distance of 120 nm. A single example of such overlap is shown with the corresponding two-channel profile intensity along the dashed line. Measurements of centroid-to-centroid distances for 29 exit sites are also shown.

Figure 3.. Endogenous overproduction of 1-deoxySLs in RPE-1.

A. Immunoblotting of SPTLC-1 and β-actin in RPE-1 cell line expressing additional copies of wild type (SPT^WT, orange) or mutant SPTLC1 (SPT^C133W, green) under a titratable Tet promoter. Upon incubation with the indicated concentration of doxycycline for 48 hours, cell lysates were obtained and analysed. WT and mutant SPTLC1 show similar expression levels. B. Growth of RPE-1, SPT^WT, and SPT^C133W is identical under no induction, while the latter shows a modest growth defect under 1 μg/mL doxycycline induction. Error bars indicate SEM for N = 3 independent wells measured with an IncuCyte system. C. Induction of SPT^C133W causes high accumulation of 1-deoxy products (left) and depletions of canonical Cers (middle) and complex SLs (right). Levels of canonical SL and deoxySL were compared between SPT^C133W cells cultured with or without 1 μg/mL doxycycline for 48h. D. Compared to induced SPT^WT cells, SPT^C133W cells show high accumulation of 1-deoxySLs (left) and only minor changes to canonical Cers (middle) and complex SLs (right). E. Moderate accumulation of 1-deoxySLs in RPE-1 cells supplemented with 1 mM L-alanina (Ala) for 48 hours. F. In both SPT^C133W and Ala-fed RPE-1 cells, 1-deoxyCer and 1-deoxyDHCer species have an average N-acyl chain length (mean of 23 carbons) that is longer than canonical Cers (19 carbons). This VLCFA resembles the profile of Hexosyl- and Lactosyl Cers (20 carbons), while SM species (17 carbons) show short N-acyl chain lengths. For all lipidomic analyses, error bars indicate SEM (extracts from N = 3 independent culture dishes). *, P < 0.05; ** P < 0.01; ****, P < 0.0001 by 2-way ANOVA.

Figure 4.. 1-DeoxySL metabolism alters membrane fluidity in the secretory pathway.

A. Methodology for generating ER-localized membrane ordering profiles using segmentation of two-channel confocal fluorescence intensity of the solvatochromic dye Laurdan. An organelle marker, Sec61β, is used to mask the ER signal. B. An example of GP_ER heatmap for an RPE-1 cell, showing distribution of the ordered and disordered channel intensities. Scale bar = 10 μm. C. Examples of 1-deoxySL-accumulating cells. The profile on the right shows a region of ER in an induced SPT^C133W cell with elevated ordered channel signal, reflecting a less fluid membrane. D. The mean GP_ER for induced SPT^C133W cells is higher than that for SPT^WT cells. Similarly, 1 mM Ala-fed RPE-1 cells show increased GP_ER compared to cells lacking Ala supplementation. Points represent total GP_ER values computed across individual cells (n = 20 (SPT^C133W), 24 (SPT^WT), 19 (RPE-1), 17 (Ala)). *, P < 0.05 by Mann-Whitney test. E. Variability in GP_ER within single cells is reduced in both SPT^C133W and Ala-fed cells, corresponding to more homogenous regions of high GP_ER. The SD of GP_ER across all pixels within a single cell was computed for the same cells analyzed in D. **, P < 0.01 by Mann-Whitney test. F. Accumulation of canonical Cer in the ER can also reduce membrane fluidity. Cells treated with the CERT inhibitor HPA-12 show increased GP_ER (plot on left) and regions of high order tubules (micrographs on right). ****, P < 0.001 by Mann-Whitney test. G. Membrane fluidity decreases along the secretory pathway in unstressed cells, demonstrated by a monotonic increase in Laurdan GP when segmented with ER (Sec61β-mCherry), Golgi (SiT-mApple) and PM (Cell Mask Deep Red) markers. H. In SPT^C133W cells, the increase in ordering reflected between GP_ER and GP_Golgi is lost. It remains in SPT^WT. I. Ala-fed cells retain an ER to Golgi GP difference, despite increases in GP_ER. Points show mean and SEM of individual cell organelle GP in separate experiments for GP_ER and GP_Golgi and fields of cells for GP_PM; n, SPT^WT = 24 (ER), 18 (Golgi), 8 (PM); n, SPT^C133W = 20 (ER), 15 (Golgi), 8 (PM); n, RPE-1 = 15 (ER), 12 (Golgi), 4 (PM); n, Ala = 17 (ER), 10 (Golgi), 8 (PM). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by 2-way ANOVA.

Figure 5.. Alterations to SL metabolism alter ER exit site morphology.

A. Individual SPT^WT and SPT^C133W cells transfected with the COPII protein SEC23-mCherry; the latter shows enlarged ERES (0.23 vs. 0.16 μm²) as measured by SEC23-mCherry area. For the plot on the right, each individual point represents an individual SEC23-mCherry puncta across multiple dishes; n = 60 (SPT^WT), 40 (SPT^C133W). B. Ala treatment does not enlarge the ERES of RPE-1 cells, with treated and untreated cells featuring an identical SEC23-mCherry area of 0.15 μm² (RPE-1, n = 101; Ala, n = 107). C. Testing the roles of vesicular and non-vesicular Cer routes from the ER to Golgi. The CERT inhibitor HPA-12 inhibits non-vesicular trafficking, leading to a loss of SM levels compared to the DMSO control. The GCS inhibitor PDMP affects the vesicular trafficking route and leads to a loss of glycosylated SLs. D. HPA-12 treatment does not alter ERES size (mean SEC23-mCherry area of 0.16 μm² in n = 59 ERES vs. 0.16 μm² in n = 59 ERES of DMSO-treated control cells), while PDMP does (mean SEC23-mCherry area of 0.20 μm² in n = 169 ERES vs. 0.16 μm² in n = 151 ERES of DMSO-treated control cells). *, P < 0.05; ****, P < 0.001 by Mann-Whitney test. Scale bars = 11 μm (whole cell) or 1.5 μm (inset).

Figure 6.. Accumulation of 1-deoxySLs modulates protein cargo release from the ER.

A. Schematic of RUSH experiments for monitoring protein cargo release from the ER through the Golgi. On the left are different cargoes analyzed in this study: disordered-membrane cargoes include TfR, E-cadherin, and TNF-α, while apical membrane cargoes include GPI-anchored proteins and Gp135. B. Representative live cell time course comparing ER release of TfR-mCherry upon biotin addition in SPT^WT and SPT^C133W cells. The dashed box indicates the Golgi region for which fluorescence is quantified; it is shown at the time point corresponding to initial Golgi accumulation observed, which occurs earlier for SPT^C133W. The plot on the right shows the fluorescence intensity within Golgi regions as a fraction of the whole cell. Arrows indicate times for maximum cargo concentration in the Golgi region. Number of cells (n) for each condition is provided. Scale bars = 10 μm. C. Similar data for the basolateral membrane protein E-cadherin-mApple. D. Fixed cell RUSH experiments with TfR show an early release population co-localizing with the Golgi marker in SPT^C133W cells 15 minutes after biotin addition. Time course data is shown in Figure S7C, alongside that of another disordered membrane cargo (TNF-α-mCherry). E. Ala-supplemented cells also show an early release of TfR-mCherry. F. Representative live cell time course for ER release of GPI-anchored mCherry. GPI-anchored proteins localize to apical membranes. In this case, maximum Golgi-region intensity occurs at a similar time for both SPT^WT and SPT^C133W cells, but the former show increased levels at early time points. Scale bars = 10 μm. G. Similar data for the apical transmembrane protein Gp135, which shows slower ER exit kinetics in SPT^C133W cells. H. Fixed cell RUSH data shows a population of SPT^C133W cells with unreleased GPI-mCherry at 10 minutes after biotin addition. Time course data is shown in Figure S7D. I. Ala-supplemented cells show identical GPI-mCherry ER release kinetics.