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

Abstract

1-Deoxysphingolipids are non-canonical sphingolipids linked to several diseases, yet 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 applied organelle-specific bioorthogonal labeling to visualize the subcellular distribution of metabolically tagged sphingolipids. We observed that 1-deoxysphingolipids are retained in the endoplasmic reticulum (ER) and specifically in ER exit sites (ERESs), suggesting that they do not efficiently sort into vesicular carriers. Cell lines expressing disease-associated variants of serine palmitoyl-CoA transferase accumulated 1-deoxysphingolipids, which were accompanied by a reduction in ER membrane fluidity and enlargement of ERES. We found that the rates of membrane protein release from the ER were altered in response to 1-deoxysphingolipid metabolism in a manner dependent on the protein's affinity for ordered or disordered membranes. The dysregulation of sphingolipid metabolism can thus alter secretory membrane properties and affect protein trafficking.

Keywords: ceramides; endoplasmic reticulum; membrane fluidity; sphingolipids; trafficking.

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

A. SL and 1-deoxySL biosynthetic pathway depicting the intermediate lipid species and their chemical structures. B. The 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 or 0.1 μM alkyne-SA for 17 h, after which cells were fixed and the CuAAC reaction was carried with either PennGreen-azide or AZDye488 picolyl-azide. C. Imaging of RPE-1 cells fed with either alkyne-deoxySA or alkyne-SA and reacted with PennGreen-azide or AZDye488 picolyl-azide. Scale bar, 20 μm. Alkyne-deoxySA treatment led to a larger PennGreen-azide levels than for alkyne-SA. Each point represents an individual field of cells across two biological replicates. ****, P < 0.0001 by Mann-Whitney test. AZDye488 picolyl-azide labeling instead showed in localization: the alkyne-deoxySA showed a distinct ER localization, while alkyne-SA was observed at PM ruffles (arrows). D. Colocalization of 1-deoxySL-PennGreen products with Golgi (GalT-mApple) and ER (Sec61β-mApple) markers. Reprehensive images are shown to the left and quantification of the Pearson’s and Mander’s correlation coefficients to the right. PennGreen is correlated with both markers but overlaps 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 mCherry-Sec61β, with additional puncta 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. Cer produced in the ER can be exported to the Golgi via vesicular or non-vesicular trafficking, the latter is mediated by the CERT. B. CERT’s START domain makes polar contacts with the C1 hydroxyl of Cer (PDB: 2E3O). C. Measurements of CERT transport using FRET-based assay (top) and the structure of C12-NBD-DHCer/C12-NBD-deoxyDHCer (bottom). Donor liposomes (L_D) contained 93(mol)% di-oleoyl-phosphatidylcholine (DOPC), 5% of the NBD-labeled lipid, and 2% rhodamine-phosphatidylethanolamine (Rhod-PE). Acceptor liposomes (L_A) contained only DOPC. D. Lipid transport curves for C12-NBD-DHCer (top) or C12-NBD-deoxyDHCer (bottom) upon injection of CERT. 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. E. Initial transport rates calculated over the first transport phase. Linear fits are shown above, while calculated rates are plotted below. For rates, error bars show SEM (N = 4 independent experiments per condition). ****, P < 0.0001 by an unpaired Welch’s t-test. F. RPE-1 cells incubated with 0.1 μM 1-deoxySA and labeled with PennGreen-azide, as in Figure 1. 1-DeoxySL-PennGreen products are observed at puncta that co-localize with mCherry-Sec23. The Pearson’s colocalization coefficient (PCC) between 1-deoxySL-PennGreen and mCherry-Sec23 is shown for 17 cells. G. The centroid of 1-deoxySL-PennGreen puncta overlap with those of mCherry-Sec23 but show an average offset distance of 120 nm. An example of such overlap is shown with the corresponding two-channel intensity profile. Centroid-to-centroid distances for discrete 29 exit sites chosen from 5 individual cells are provided.

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

A. Immunoblotting of SPTLC-1 in RPE-1 cell line expressing additional copies of wild type (SPT^WT, orange) or mutant SPTLC1 (SPT^C133W, green). Upon incubation with the indicated concentration of doxycycline for 48 hours, WT and mutant SPTLC1 show similar expression levels. A separate blot for β-actin from the same samples is provided as a loading control. B. Growth of RPE-1, SPT^WT, and SPT^C133W is identical under no induction, while the latter shows a modest growth defect under 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 SLs and deoxySLs were compared between SPT^C133W cells cultured with or without doxycycline. D. Compared to induced SPT^WT cells, SPT^C133W cells show high accumulation of 1-deoxySLs (left) and only small changes to canonical Cers (middle) and complex SLs (right). E. Moderate accumulation of 1-deoxySLs in RPE-1 cells supplemented with 1 mM L-alanine (Ala). Normalized ion counts are comparable between panels C-F. F. In both SPT^C133W and Ala-fed RPE-1 cells, 1-deoxyCer and 1-deoxyDHCer species have an average N-acyl chain length (23 carbons) that is longer than canonical Cers (19 carbons). This VLCFA resembles the profile of Hexosyl- and LactosylCers (20 carbons), while SM species (17 carbons) show short N-acyl chain lengths. 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. Generating ER-localized membrane ordering profiles using segmentation of two-channel confocal fluorescence intensity of Laurdan. B. An example of GP_ER heatmap for a cell, showing distribution of the ordered and disordered channel intensities. Scale bar, 10 μm. C. Examples for 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. GP_ER for induced SPT^C133W cells is higher than that for SPT^WT cells. Similarly, Ala-fed RPE-1 cells show increased GP_ER. Points represent mean 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. 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. 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 RPE-1 measured by Laurdan GP when segmented with ER (mCherry-Sec61β), 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. 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; 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 mCherry-Sec23; the latter shows enlarged ERES (0.23 vs. 0.16 μm²) as measured by mCherry-Sec23 area. For the plot on the right, each individual point represents a mCherry-Sec23 puncta; n = 60 sites across 10 cells (SPT^WT), 40 across 11 cells (SPT^C133W). B. Ala-treated cells show an identical mCherry-Sec23 area (0.15 μm²; as untreated cells (RPE-1, n = 101 across 7 cells; Ala, n = 107 across 12 cells). C. The CERT inhibitor HPA-12 inhibits non-vesicular trafficking, leading to a loss of SM. 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 mCherry-Sec23 area of 0.16 μm² in n = 60 across 15 cells vs. 0.16 μm² in n = 59 across 15 cells for DMSO-treated control cells), while PDMP does (mean mCherry-Sec23 area of 0.20 μm² in n = 169 across 20 cells vs. 0.16 μm² in n = 151 across 16 cells for 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. RUSH experiments for monitoring release of different protein cargoes from the ER through the Golgi. B. Representative time course comparing ER release of TfR-mCherry upon biotin addition in SPT^WT and SPT^C133W cells. Dashed boxes indicate quantified Golgi regions; they are shown at the time point corresponding to initial Golgi accumulation observed. The plot on the right shows cargo 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 at 15 min. Time course data is shown in Figure S6C, 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. In this case, maximum Golgi-region intensity occurs at a similar time for both SPT^WT and SPT^C133W cells, but the former shows 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 mCherry-GPI at 10 min after biotin addition. Time course data is shown in Figure S6D. I. Ala-supplemented cells show identical mCherry-GPI ER release kinetics.