Traceless synthesis of ceramides in living cells reveals saturation-dependent apoptotic effects

Results and Discussion

To determine the feasibility of this approach, we synthesized a small collection of palmitic acid salicylaldehyde esters with different ring substituents, 1–3, and performed ligation experiments in the presence of phospholipid vesicles as a model membrane. Upon incubation of 500 µM of palmitic acid salicylaldehyde ester with 250 µM sphingosine at 37 °C in PBS (pH 7.4), C16:0 ceramide was formed within hours (SI Appendix, Fig. S1). The salicylaldehyde group is released spontaneously during the reaction, resulting in the traceless formation of ceramide, without the introduction of any extraneous chemical moieties (Fig. 1A). From these initial experiments, we found that compound 3 offered superior hydrolytic stability, showing very little degradation (<2%) over 16 h (Fig. 1B). Therefore, we chose to employ 3 as a sphingosine ligation partner in a method we term traceless ceramide ligation (TCL).

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Fig. 1.

Fatty acid salicylaldehyde esters and sphingosine react chemoselectively in model membranes and live cells. (A) Proposed reaction mechanism for TCL. (B) Sphingosine (sph) and 3 in model membranes react to form ceramide (cer) at 37 °C, pH 7.4 over a period of 16 h. (C and D) The reaction between sph and 3 forms cer chemoselectively in the presence of serine (C) and lyso-sphingomyelin (D). The production of a small quantity of reversibly formed byproduct b1 (*) (B–D) and b2 (**) (D) is also detected (SI Appendix, Fig. S2). (E) Isotopic palmitic acid salicylaldehyde ester 3i reacts with sph within live HeLa cells to form cer detectable in the crude mitochondrial cell fraction.

To investigate the chemoselectivity of this ligation we performed TCL in the presence of two potentially competing biomolecules, serine (Fig. 1C) or lyso-sphingomyelin (Fig. 1D). Serine possesses the same 2-aminoethanol moiety as sphingosine but is water-soluble. Lysosphingomyelin is localized in the membrane but has only a free amine and not the terminal 2-aminoethanol functionality found in sphingosine. Under these conditions, TCL proceeded with no observable decrease in yield or reaction rate, suggesting that both the terminal 2-aminoethanol functionality and localization in the phospholipid membrane are necessary for ligation. To determine if TCL could occur in live cells, we used a ¹³C isotopic derivative of 3, 3i. HeLa cells were incubated in the presence of sphingosine, 3i, or sphingosine and 3i combined for 16 h and then lysed. Cell fractionation was used to obtain crude mitochondrial fractions and enrich for the ceramide product formed in internal membranes (14) (SI Appendix, Fig. S3). MS analysis detected TCL-synthesized ceramide in the crude mitochondrial fraction only when both ligation partners were added to cells but not when they were added separately. Notably, we could not detect any TCL-synthesized ceramide when cells were treated with 3i alone. This suggests that endogenous levels of sphingosine are insufficient for TCL. Additionally, the reaction was performed in the presence of an inhibitor of all six ceramide synthases, fumonisin B1 (FB1), which we determined was nonreactive toward 3 (SI Appendix, Fig. S4). Under these conditions, the isotopic ceramide product was still observed, confirming that it is not a metabolic product of 3i (15) (Fig. 1E). By performing a whole-cell lipid extraction (16), we determined the total level of ¹³C-labeled ceramide produced in HeLa cells by TCL to be 136 ± 23 pmol/10⁶ cells, indicating that physiological levels of ceramide can readily be synthesized in situ (SI Appendix, Fig. S5) (17).

Having confirmed the reaction proceeds in living cells, we investigated whether ceramide-induced apoptosis could be effected by TCL (5, 6). Certain ceramide species, most notably C16:0 ceramide, are known to increase in concentration during apoptosis (18, 19). Evidence suggesting that increases in levels of certain ceramides can directly trigger apoptosis is based on studies utilizing unnatural short-chain ceramides. These nonnative ceramides exhibit substantial differences in structure, solubility, lipid ordering effects, and intracellular diffusion, raising concerns about the validity of these conclusions (20, 21). To test whether we could use TCL to study the apoptotic effects of natural C16:0 ceramide in cells, we incubated cultured HeLa cells in the presence of 100 µM 3 and varying concentrations of sphingosine. Cell viability was not impacted by 3 alone, but incubation with 3 and sphingosine together resulted in significantly lower cell viability compared with the same concentration of sphingosine alone (Fig. 2A). Exogenous addition of 100 µM C16:0 ceramide had no effect on cell viability, highlighting the substantial increase in ceramide delivery through in situ TCL, and providing further evidence that TCL occurs intracellularly. Additionally, to confirm that the decrease in viability is due to TCL and not the metabolism of sphingosine to ceramide, we performed the same cell viability assay in the presence of FB1 and observed the same decrease in cell viability when cells were treated with 3 and sphingosine together compared with sphingosine alone (SI Appendix, Fig. S6). Visual quantification of cell death (Fig. 2B) and fluorescent detection of caspase 3/7 activity (Fig. 2C and SI Appendix, Fig. S7) confirmed that C16:0 ceramide delivered via TCL results in the activation of apoptotic caspases. To determine if TCL is compatible with cell lines other than HeLa cells, we performed the same viability experiments using NIH 3T3 cells (SI Appendix, Fig. S8). Again, we observed significantly lower cell viability in cells treated with 3 and sphingosine compared with 3 or sphingosine alone.

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Fig. 2.

TCL in live cells triggers apoptosis. (A and B) Treatment of cultured HeLa cells with 100 µM 3 and sph results in significantly more cell death than sph alone as monitored by a WST-1 proliferation assay (A) and total live cell count (B). Viability assays were performed in three biological replicates and shown as means ± SD. Statistically significant differences between points at a given sph concentration (A) or condition (B) are indicated: *P < 0.05, ***P < 0.001, ****P < 0.0001. ns, not significant. (C) Treatment of HeLa cells with 100 µM 3 and 40 µM sph results in the activation of caspases necessary for apoptosis. (D) Production of C16:0 ceramide during TCL triggers translocation of Bax-GFP from the cytosol to mitochondria during apoptosis (images in D are maximum intensity Z-projections).

C16:0 ceramide is hypothesized to exert its proapoptotic function through the recruitment of the protein Bax to the mitochondria, which in turn triggers mitochondrial membrane permeabilization and eventual cell death (22⇓–24). This model, however, is based on a correlation between increased levels of cellular ceramide and Bax association with the mitochondria (23), or studies using unnatural short-chain ceramides (25). Therefore, it remains unclear if C16:0 ceramide is an effector of Bax translocation in vivo. To determine whether an intracellular increase in C16:0 ceramide alone is sufficient to trigger Bax recruitment to mitochondria, we transiently expressed a Bax-GFP fusion protein in HeLa cells and observed the localization of Bax-GFP with respect to the mitochondrial staining dye MitoTracker. TCL-driven synthesis of C16:0 ceramide within cells resulted in Bax-GFP transitioning to a punctate distribution which partially colocalized to the mitochondria, consistent with previous reports of Bax localization during apoptosis (26, 27) (Fig. 2D and SI Appendix, Fig. S9). These results demonstrate that increases in intracellular C16:0 ceramide can directly trigger Bax translocation and suggest that this lipid plays a central role in Bax-induced apoptosis.

Cells produce numerous ceramide species with various N-fatty acyl groups (28). Ceramides may exert differing biological effects depending on their acyl-chain functionality (1). Analyzing the roles of specific ceramide species is often confounded by the presence of six individual endogenous ceramide synthases (CerS1–6), which produce multiple ceramide products varying in both chain length and degree of unsaturation (9). Additionally, many ceramide species are produced by more than one ceramide synthase, further hindering their study through traditional biochemical methods (9, 28). TCL offers the capability to deliver individual and specific ceramides to cells, something not possible through manipulation of cellular metabolism. As a proof of concept, we synthesized salicylaldehyde esters with differing fatty acid substituents 4, 5, and 6 and confirmed that they react with sphingosine to yield a range of ceramide products (SI Appendix, Fig. S10). We then generated the different ceramides in living HeLa cells and studied the effect of ceramide structure on cell viability. Delivery of fully saturated C16:0 or C18:0 ceramide using TCL resulted in a significant reduction in cell viability compared with the sphingosine control (Fig. 3 A and B). In contrast, we observed no reduction in cell viability when monounsaturated C18:1 or C24:1 ceramide was delivered (Fig. 3 C and D). Differences in chain length did not affect the intracellular delivery of the precursors, as there was no significant difference in cellular levels of C16:0 and C18:1 ceramide formed after addition of sphingosine and isotopic salicylaldehyde esters 3i and 4i (SI Appendix, Fig. S11). We hypothesized that the observed difference in apoptotic activity of different ceramide species may be due to their ability to promote translocation of Bax to the mitochondria. To test this, we used TCL to synthesize C16:0 and C24:1 ceramide in HeLa cells expressing Bax-GFP. We found that Bax localized to the mitochondria significantly more when cells were treated with C16:0 ceramide (95 ± 2% of cells) than when they were exposed to C24:1 ceramide (10 ± 6% of cells) (Fig. 3 E and F and SI Appendix, Fig. S12). Our results suggest that ceramide saturation plays a determinant role in the apoptotic activity of the molecule and support the hypothesis that ceramide-enriched membrane domains, formed preferentially by saturated ceramides, are required for the recruitment of Bax to the mitochondria and the activation of apoptosis pathways (1, 23, 29).

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Fig. 3.

TCL delivery of specific ceramide species reveals an N-acyl chain saturation-dependent effect on cell viability and Bax localization. (A–D) Viability of cultured HeLa cells after 16-h incubation with TCL precursors as monitored by a WST-1 viability assay. Treatment of cells with 100 µM 3 (A) or 100 µM 5 (B) and sph results in significantly more cell death than sph alone. Cells treated with 100 µM 4 (C) or 100 µM 6 (D) and sph or sph alone show no significant difference in cell viability. Viability assays were performed in three biological replicates and shown as means ± SD. Statistically significant differences between points at a given sph concentration are indicated: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Lines represent a normalized dose–response curve with variable slope. (E and F) Composite images of Bax-GFP (green) and MitoTracker (red) after treatment with 100 µM 3 or 6 and 40 µM sph for 16 h. Generation of C16:0 ceramide within HeLa cells promotes translocation of Bax-GFP to the mitochondria (E), while synthesis of C24:1 ceramide has no effect on Bax-GFP localization (F) (images are maximum intensity Z-projections).

Ceramide formation is stringently controlled in both time and space (30). We sought to achieve a similar level of control using our ligation strategy. Previously, light-reactive photocages have been developed for the protection and photoinitiated deprotection of biological lipids including sphingosine and ceramide (31⇓–33). While photocaged ceramide is an exceptional tool for the study of ceramide in model membranes (33), it has seen limited application in biological systems (10). Modifying a previous synthetic scheme, we successfully synthesized a coumarin-photocaged analog of sphingosine, 9. We found that this compound was stable in HeLa cell lysate (SI Appendix, Fig. S13) and, upon UV exposure, was uncaged to yield sphingosine with a half-life of 10.6 s (SI Appendix, Fig. S14). We used the efficient uncaging and stability of this compound to spatiotemporally control ceramide synthesis in a live cell system. To determine the effect of 9 uncaging in cells, we cultured HeLa cells expressing Bax-GFP as a marker of apoptosis and then exposed them to a range of concentrations of the compound 9 (0–300 µM) and irradiated multiple regions of the cell culture with 405-nm laser and imaged over 10 h (SI Appendix, Fig. S15). We chose to use 50 µM 9 for TCL experiments because it did not result in significant cell death after irradiation but, based on cell viability experiments (Fig. 2A), should provide a level of sphingosine sufficient for TCL. We cultured HeLa cells expressing Bax-GFP and then incubated them in the presence of 100 µM 3 and 50 µM 9. Using laser scanning microscopy, we uncaged 9 in a single cell using the 405-nm laser line and then imaged over a period of 12 h. We found that after 10 h only the cell in which 9 was uncaged underwent apoptosis, evidenced by the shrinkage of the membrane and punctate Bax-GFP localization (Fig. 4). Controls in which cells were incubated with 3 or 9 separately, and single cells irradiated under the same conditions, showed no apoptosis over the same period (SI Appendix, Fig. S16). To determine if this same behavior occurred when 9 was uncaged in multiple cells, we treated HeLa cells under the same conditions as before but irradiated multiple cells in several regions of the chamber slide. As expected, we found that irradiation of cells treated with 3 and 9 resulted in significantly more cell death than irradiation of cells treated with 9 alone (SI Appendix, Fig. S17). Furthermore, we found that when cells were treated with precursors which form the unsaturated C24:1 ceramide (100 µM 6 and 50 µM 9) and then irradiated under the same conditions, there was no significant difference in cell death compared with cells treated with 9 alone (SI Appendix, Fig. S17). Spatiotemporally controlled TCL allows for the delivery of ceramides in a cell-selective manner. This could allow for modulation of specific ceramide levels in defined cells or regions of an organism and help to uncover the roles of ceramide in development (34), neurodegeneration (35), and cancer (6).

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Fig. 4.

Photocontrolled initiation of TCL. (A) Photocaged sphingosine 9 can be uncaged by 405-nm laser light to produce sph and trigger TCL. (B) HeLa cells expressing Bax-GFP were incubated with 100 µM 3 and 50 µM 9. A single cell in the frame was exposed to 405-nm laser within the outlined area (Left) and the sample imaged over a period of 12 h. After 10 h, only the cell in which ceramide synthesis was triggered showed cell death and mitochondrial Bax localization characteristic of C16:0 ceramide-induced apoptosis (Right) (t = 10 h; images are maximum intensity Z-projections).