A new, long-wavelength borondipyrromethene sphingosine for studying sphingolipid dynamics in live cells.

Sphingolipids function as cell membrane components and as signaling molecules that regulate critical cellular processes. To study unacylated and acylated sphingolipids in cells with fluorescence microscopy, the fluorophore in the analog must be located within the sphingoid backbone and not the N-acyl fatty acid side chain. Although such fluorescent sphingosine analogs have been reported, they either require UV excitation or their emission overlaps with that of the most common protein label, green fluorescent protein (GFP). We report the synthesis and use of a new fluorescent sphingolipid analog, borondipyrromethene (BODIPY) 540 sphingosine, which has an excitation maximum at 540 nm and emission that permits its visualization in parallel with GFP. Mammalian cells readily metabolized BODIPY 540 sphingosine to more complex fluorescent sphingolipids, and subsequently degraded these fluorescent sphingolipids via the native sphingolipid catabolism pathway. Visualization of BODIPY 540 fluorescence in parallel with GFP-labeled organelle-specific proteins showed the BODIPY 540 sphingosine metabolites were transported through the secretory pathway and were transiently located within lysosomes, mitochondria, and the nucleus. The reported method for using BODIPY 540 sphingosine to visualize sphingolipids in parallel with GFP-labeled proteins within living cells may permit new insight into sphingolipid transport, metabolism, and signaling.

have the advantages of lower phototoxicity than existing fl uorescent sphingosine analogs and the capability to visualize it in parallel with GFP, which would facilitate assessing sphingolipid colocalization with proteins of interest. For this reason, here we report the synthesis and validation of BODIPY 540 sphingosine ( I ), which has maximum excitation at 540 nm and emission that does not overlap with GFP. In this study, we use fl uorescent organelle-specifi c markers to characterize the distribution of BODIPY 540 sphingosine and its fl uorescent metabolites within living cells. We confi rm that mammalian cells metabolized BODIPY 540 sphingosine to BODIPY 540 sphingolipids and catabolized these fl uorescent sphingolipid metabolites. On the basis of these results, we anticipate that this new fl uorescent sphingosine analog will be a valuable tool for investigating the metabolism, traffi cking, and signaling of acylated and unacylated sphingolipid species in living cells.

Synthesis of BODIPY 540 sphingosine
All solvents and commercially available reagents were used without further purifi cation unless otherwise stated. Tetrahydrofuran was distilled from sodium/benzophenone and dichloromethane from calcium hydride under argon. Air-and moisture-sensitive reactions were carried out in oven-dried or fl ame-dried glassware that was septum-capped and maintained under argon at atmospheric pressure. Flash chromatography was performed with silica gel 60, 230-400 mesh from Silicycle. Proton ( 1 H) and carbon ( 13 C) NMR spectra were recorded on 400 or 500 MHz Varian Unity instruments. ESI-HRMS was carried out on a FTICR instrument. NMR and mass spectrometry data for the compounds described below are provided in the supplementary information.

(4-(Dodec-11-en-1-yloxy)phenyl)boronic acid (III)
Under an argon atmosphere, n -butyllithium (1.6 M in hexane; 2.5 ml) was added over 5 min to 1-bromo-4-(dodec-11en-1-yloxy)benzene ( II ) (1.12 g; 3.3 mmol) in dry tetrahydrofuran (10 ml) at Ϫ 78°C (dry ice/acetone bath). The mixture was stirred at Ϫ 78°C for 1 h, trimethyl borate (0.55 ml, 4.9 mmol) in dry THF (3.5 ml) was added at Ϫ 78°C, and the reaction mixture was stirred for 2 h while warming to rt. Water (20 ml) was added to the reaction, and the solution was stirred for 1 h at rt. The mixture was extracted with diethyl ether (2 × 100 ml). The combined organic layer was washed with 1 M HCl (150 ml), concentrated sodium bicarbonate (aq, 150 ml), and brine (150 ml). The organic layer was dried with anhydrous magnesium sulfate, fi ltered, and concentrated in vacuo. The residue was purifi ed by with a scraper, and pelleted by centrifugation. Lipids were extracted using the Bligh and Dyer method ( 15 ). The glycolipids were selectively hydrolyzed by alkaline treatment with sodium hydroxide; this treatment did not hydrolyze the sphingolipids.

TLC
Lipids extracts were dissolved in chloroform-methanol (20 l; 2:1 v/v), spotted on TLC plates (Silica 60 F254 plates, EMD Chemicals), and developed in a TLC chamber using butanolacetic acid-water (4.5:1:1 v/v/v) as the solvent. Digital imaging of the TLC plates was performed on a Typhoon 9400 imager (GE Healthcare) using 532 nm laser excitation and a 580 nm emission fi lter to detect the fl uorescent spots. Unlabeled lipids were visualized by iodine staining. The BODIPY 540-labeled sphingomyelin, ceramide, and sphingosine were identifi ed by their comigration with BODIPY 540-labeled sphingolipid standards. Because this solvent system primarily separated the lipid species according to their head groups, which was confi rmed by the comigration of the BODIPY 540-labeled sphingomyelin and ceramide with unlabeled sphingomyelin and ceramide, respectively, in this solvent system, the remaining fl uorescent lipid species were identifi ed according to their comigration with unlabeled lipid standards.

Glycerolipid quantifi cation
For each labeling interval (2 h, 1 day), the total lipid extract obtained from a single labeling experiment was dissolved in chloroform-methanol (2:1 v/v) and divided into two equal portions. One portion of the total lipid extract was subjected to alkaline hydrolysis to remove the glycerolipids; the resulting fraction is referred to as the sphingolipid extract because it contained primarily sphingolipid species. For each labeling time, the total lipid extract and corresponding sphingolipid extract were applied to a TLC plate and separated using butanol-acetic acid-water (4.5:1:1 v/v/v) as the solvent system. For each labeling interval, the amount of BODIPY 540-labeled phosphoglycerolipids on the TLC plate was assessed by visualizing the spots with a Typhoon 9400 imager (GE Healthcare) using 532 nm laser excitation and a 580 nm emission fi lter and quantifying their fl uorescence intensities. To quantify the fraction of the fl uorescence that corresponded to glycerolipids, the sum of the fl uorescence intensities measured for each spot that corresponded to fl uorescent phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol was measured using ImageQuant software (GE Healthcare) and divided by the total fl uorescence intensities of all of the fl uorescent lipid species.

Cell culture
NIH3T3 fi broblast cells were grown in DMEM with 10% calf serum and 1% penicillin/streptomycin in a 5% CO 2 incubator at 37°C. For colocalization studies, organelle-specifi c fl uorescent fusion proteins were expressed in HEK293 cells due to their ease of transfection. HEK293 cells were grown in DMEM with 10% FBS and 1% penicillin/streptomycin in a 5% CO 2 incubator at 37°C. For microscopy experiments, cells were cultured in 35 mm glass bottom dishes (MatTek, Ashland, MA) or 35 mm plastic dishes for high-end microscopy (ibidi GmbH, München, Germany). For all other experiments, cells were grown in 150 mm cell culture dishes.

Pulse-chase labeling
Cells were incubated with BODIPY 540 sphingosine in Op-tiMEM supplemented with 2% calf serum (NIH3T3 cells) or 2% FBS (HEK293 cells). BODIPY 540 sphingosine was dissolved in ethanol and added to the labeling medium such that the fi nal concentration of BODIPY 540 sphingosine was 1 or 1.25 µM. For pulse labeling, cells were incubated in labeling medium that contained 1 or 1.25 µM of BODIPY 540 sphingosine for 30 min. After labeling, the medium was removed, and the cells were washed with PBS and cultured in unlabeled growth medium for the specifi ed chase time. For studies of BODIPY 540 sphingosine metabolism, cells were incubated in growth medium that contained 1 µM of BODIPY 540 sphingosine for the specifi ed time.

Lipid extraction
Cells were harvested when they reached 90% to 100% confl uence. Cells were washed with cold PBS, removed from the dish eluent in 40% yield. This reaction produced the protected BODIPY 540 sphingosine ( XI ) in 71% yield. Deprotection of XI afforded BODIPY 540 sphingosine ( I ) in 37% yield after purifi cation.
Characterization of the absorption and fl uorescence of BODIPY 540 sphingosine in ethanol (1 M) demonstrated that its absorption and fl uorescence emission maxima were 540 and 568 nm, respectively ( Fig. 1 ). Thus, the excitation and emission properties of the analog are similar to BODIPY TMR despite replacing the phenoxy methyl in BODIPY TMR with a sphingosine moiety. The long wavelength emission that is indicative of dimer formation appeared to be negligible in labeled cells.

Cells uptake and metabolize BODIPY 540 sphingosine
We fi rst confi rmed that BODIPY 540 sphingosine was internalized by cells and metabolized to more complex fl uorescent sphingolipids. Mouse fi broblast cells (NIH3T3) were pulse labeled by incubation with 1.25 M of BODIPY 540 sphingosine for 30 min at 37°C and transferred to unlabeled medium at 37°C for various chase times before imaging at room temperature. No adverse effect on cell viability was observed during or after labeling. Fig. 2 shows that the pulse-labeled cells exhibited strong fl uorescence, confi rming they internalized the BODIPY 540 sphingosine. After a 30 min chase in unlabeled medium, fl uorescent tubule-like structures and vesicles of various sizes were observed inside the NIH3T3 cells ( Fig. 2A ). The edges of these cells did not exhibit strong fl uorescence, which indicates that the plasma membrane contained low levels of fl uorescent lipid species ( Fig. 2A ). Fluorescent vesicles were also observed in NIH3T3 cells when the duration of the chase in unlabeled medium increased to 20 h ( Fig. 2B ). Although the plasma membrane still appeared to be dim relative to the highly fl uorescent intracellular vesicles, which were especially abundant in NIH3T3 cells, the clearer fl uorescent outline of the cell's perimeter indicates that BODIPY 540 lipid species had incorporated into the plasma membrane ( Fig. 2B ). Overall, these observations suggest that the BODIPY 540 sphingosine was metabolized to fl uorescent sphingolipids, which were transported to the plasma membrane.
To verify that the fl uorescent sphingosine was metabolized fl uorescent sphingolipids, NIH3T3 cells were cultured in the presence of 1 M of BODIPY 540 sphingosine for various time intervals, and their lipids were extracted. The glycerolipids in each total lipid extract were removed by base hydrolysis ( 21 ), and the resulting fraction, referred to as the sphingolipid extract because it primarily contained sphingolipids, was separated by TLC. The BODIPY 540-labeled sphingolipids on the TLC plate could be detected with high sensitivity according to their fl uorescence. Four fl uorescent sphingolipid species, which included BODIPY 540 sphingosine, were detected after a labeling time as short as 2 h ( Fig. 2C ). Comigration with BODIPY 540-labeled sphingolipid standards confi rmed that the NIH3T3 cells metabolized the fl uorescent sphingosine to BODIPY 540-labeled ceramide and sphingomyelin. Because the BODIPY 540-labeled ceramide and sphingomyelin

Confocal laser scanning microscopy
Labeled cells were transferred to phenol red-free OptiMEM supplemented with 2% calf serum (NIH3T3 cells) or 2% FBS (HEK293 cells). The labeled cells were observed on a Carl Zeiss LSM 700 confocal microscope with 63 × 1.4 NA oil immersion objective lens at rt using a 555 nm laser to excite BODIPY 540, a 488 nm laser to excite GFP and FITC, and a 405 nm laser to excite Hoechst 33342.

Synthesis and characterization of BODIPY 540 sphingosine
Our strategy for synthesizing BODIPY 540 sphingosine ( I ) was based on the route developed by Peters et al., which used a cross-metathesis reaction to form the double bond in the sphingosine backbone ( Scheme 1 ) ( 8 ).
Synthesis was completed by cross-metathesis reaction ( 8 ) of the -alkenyl BODIPY 540 fl uorophore ( IX ) with the Garner-allylic alcohol ( X ) that was derived from the Garner aldehyde ( 13 ) and purifed by silica gel column chromatography using hexane-ethyl actate (8:1 v/v) as the TLC plate (22), was observed for a labeling period of 1 day. More complex BODIPY 540-labeled glycosphingolipids, such as BODIPY 540 gangliosides, were not visible on the TLC plate (supplementary Fig. I). Because unlabeled gangliosides also were not detected on iodine-stained TLC plates, the inability to detect BODIPY 540 gangliosides likely refl ects a low level of gangliosides in the NIH3T3 cell extracts and not a lack of BODIPY 540 sphingosine incorporation into complex glycosphingolipid species.
Native sphingolipid catabolism yields phosphoethanolamine and the fatty aldehyde trans-2-hexadecenal, which is oxidized to a fatty acid that can be incorporated into glycerolipids (22)(23)(24)(25). Therefore, if the BODIPY 540 sphingolipids were degraded via this pathway, the resulting BODIPY 540-labeled hexadecenal could be oxidized and used to biosynthesize fl uorescent glycerolipids. To assess this possibility, sphingolipid extracts and total cellular lipid extracts were isolated from NIH3T3 cells that were cultured in the presence of 1 M of BODIPY 540 sphingosine for 2 h, and the lipid components were separated with TLC. In addition to the BODIPY 540-labeled sphingolipid species observed by TLC separation of the sphingolipid extract, the total cellular lipid extract exhibited three additional fl uorescent spots that corresponded to fl uorescent glycerophospholipids ( Fig. 2C ). Comparison of these migration patterns to those of unlabeled comigrated with the analogous unlabeled sphingolipid in this solvent system, we identifi ed the remaining fl uorescent sphingolipid as BODIPY 540 glucosylceramide due to its comigration with the unlabeled standard. In addition to these four sphingolipid species, a fi fth species, identifi ed as lactosylceramide based on its relative location on SCHEME 1. Synthesis of BODIPY 540 sphingosine. catabolism we observed is a characteristic of sphingosine pulse labeling and was not induced by the presence of the fl uorophore.

Subcellular distribution of BODIPY 540 sphingolipids
The studies described above demonstrate that NIH3T3 cells metabolized the BODIPY 540 sphingosine to fl uorescent sphingolipids and degraded these fl uorescent sphingolipids in a manner that was consistent with native sphingolipid catabolism. We performed colocalization studies to confi rm that BODIPY 540 sphingosine and its fl uorescent metabolites were traffi cked through the organelles where sphingolipid biosynthesis primarily occurs. For these experiments, HEK293 cells were used because their high transfectability facilitated the expression of the organelle-specifi c fl uorescent proteins required to assess colocalization. HEK293 cells that expressed the ER or Golgi apparatus markers, GFP-Sec61 ␤ ( 16 ) or CellLight TM Golgi-GFP, respectively, were pulse labeled with 1 µM of BODIPY 540 sphingosine for 30 min, followed by a 30 min or 1 day chase. Confocal microscopy imaging confi rmed the fl uorescent sphingolipid analogs were located in the ER ( Fig. 3A ) and Golgi apparatus ( Fig. 3B ) after the 30 min chase. Low colocalization occurred between the fl uorescent sphingolipid analog and the ER or Golgi apparatus markers for the 1 day chase ( Fig. 3C and 3D, respectively). The transient colocalization of the BODIPY 540 with the ER and Golgi apparatus is consistent with the conversion of BODIPY 540 sphingosine to more complex fl uorescent sphingolipids in these organelles and the subsequent transport of the newly synthesized fl uorescent sphingolipids to the plasma membrane ( 27 ).
Lysosomes are the subcellular compartment where complex sphingolipids are catabolized to sphingosine ( 28,29 ), which can be reused for the biosynthesis of new phospholipid standards indicated that two of these components were BODIPY 540-labeled phosphatidylcholine and phosphatidylethanolamine; the third spot was consistent with either BODIPY 540-labeled phosphatidylserine or phosphatidylinositol. Based on the relative intensities of the TLC spots corresponding to unlabeled lipid species, which were visualized by staining the same TLC plate with iodine, the BODIPY 540 fluorophore was incorporated into the major sphingolipid species but was less abundant in the major glycerolipid species after a 2 h labeling period (supplementary Fig. I). However, after labeling with 1 M of BODIPY 540 sphingosine for 1 day, the BODIPY 540-labeled lipids reflected the major lipid species present in the cell extracts (supplementary Fig. I). These results suggest that fluorescent sphingolipid degradation followed the native sphingolipid catabolism pathway.
We estimated the fraction of BODIPY 540 that had been incorporated into glycerolipids after labeling NIH3T3 cells with 1 µM of BODIPY 540 sphingosine for 2 h and 1 day by measuring the fraction of the total fl uorescence on the TLC plate that was produced by spots corresponding to glycerolipid species. After a 2 h labeling period, BODIPY 540 glycerolipids produced approximately 25% of the fl uorescence measured in the total lipid extract; after labeling for 1 day, approximately 43% of the fl uorescence measured in the total lipid extract was produced by BODIPY 540 glycerolipids. Thus, the BODIPY 540 glycerolipids that resulted from BODIPY 540 sphingolipid catabolism were detectable after a pulse as short as 2 h, and their abundance increased with time. Previous studies have demonstrated that exogenous [ 3 H] sphingosine is also rapidly degraded by cells and that the amount of the radioactive degradation products increases with time ( 26 ). This suggests that the time-dependent sphingolipid 540 sphingosine or its BODIPY 540-labeled metabolites could be detected in the nucleus, we performed colocalization studies between the DNA label, Hoechst 33342, and BODIPY 540 in labeled NIH3T3 cells. After labeling the DNA with Hoechst 33342, the cells were pulse labeled for 30 min with BODIPY 540 sphingosine, followed by a chase in unlabeled medium for approximately 2 days. From the en face confocal microscopy image acquired with the plane of focus at approximately the center of the cell ( Fig. 6A, D ), small amounts of the BODIPY 540 sphingolipids, or degraded to nonsphingolipid species ( 23,24,30 ). We investigated whether BODIPY 540 sphingosine or its fl uorescent metabolites accumulated in lysosomes by assessing colocalization between the lysosome marker, FITC-dextran ( 31 ), and BODIPY 540. NIH3T3 cells were labeled with FITC-dextran, pulse labeled for 30 min with BODIPY 540 sphingosine, and subjected to a 30 min or 16 h chase. Confocal microscopy imaging of these cells demonstrated that low levels of colocalization occurred after a 16 h chase time ( Fig. 4 ). The lack of BODIPY 540 accumulation in the lysosomes confi rms that the fl uorescent sphingolipids that reached the lysosomes were promptly degraded, and the resulting fl uorescent products were exported from the lysosomes.
Finally, we investigated whether BODIPY 540 sphingosine or its fl uorescent metabolites could be detected in the mitochondria or nucleus. We focused on these organelles because sphingolipids and their metabolites have been previously detected within them, and the sphingolipid metabolism that occurs within these organelles plays a major role in modulating cellular function ( 2,(32)(33)(34)(35). For example, internalized fl uorescent ceramides are rapidly detected in mitochondria ( 35 ), where ceramide is catabolized to bioactive metabolites that promote the mitochondrial pathway of apoptosis ( 33 ). Fig. 5A demonstrates that the fl uorescent sphingolipid analogs were colocalized with the mitochondria marker, CellLight TM mitochondria-GFP, in transfected HEK293 cells that were pulse labeled with BODIPY 540 sphingosine for 30 min and then chased for 30 min in unlabeled medium. Consistent with previous reports ( 35 ), colocalization was not observed after the 1 day chase. Sphingolipids also perform regulatory and structural functions in the nucleus, but their presence and distribution in subnuclear compartments have primarily been identifi ed by biochemical analysis of nuclear extracts ( 2 ). To assess whether the BODIPY Fig. 3. Intracellular distribution of BODIPY 540 sphingosine and its fl uorescent metabolites. Transfected HEK293 cells that expressed GFP-Sec61 ␤ or CellLight TM -Golgi were pulse labeled for 30 min in medium containing 1 M of BODIPY 540 sphingosine and transferred to unlabeled medium for the chase time indicated to the right of the last column. Confocal microscopy imaging was performed on living cells at room temperature. The GFP signal (green) locates the ER (A and C) or Golgi apparatus (B and D). The BODIPY 540 signal (red) shows the subcellular distribution of BODIPY 540 sphingosine and its fl uorescent metabolites. The fl uorescent outline of each cell, which signifi es the incorporation of BODIPY 540 lipids into the plasma membrane, is especially distinct in these images because these HEK293 cells have few highly fl uorescent intracellular vesicles. The overlay images show that colocalization between BODIPY 540 and each organelle-specifi c GPF fusion protein (yellow) occurred when the chase time was 30 min (A and B) but not 1 day (C and D). Bars = 20 m. Fig. 4. Localization of BODIPY 540 sphingolipid analogs in lysosomes. The lysosomes in NIH3T3 cells were labeled by incubating the cells for 30 min in medium containing 2 mg/ml of FITC-dextran and then transferring the cells to label-free medium for 5 h. The lysosome-labeled NIH3T3 cells were incubated for 30 min in culture medium containing 1 M of BODIPY 540 sphingosine and transferred to label-free medium for the chase time indicated to the right of the last column. Confocal imaging of the living cells at room temperature revealed the intracellular distribution of the FITC-dextran (green) that labeled the lysosomes and BODIPY 540 sphingosine and its metabolites (red). The overlay images (last column) show that colocalization between BODIPY 540 and FITCdextran (yellow) did not occur after a 30 min chase but was detectable at low levels after a 16 h chase. Bars = 20 m. chromatin ( 36 ), the BODIPY 540-labeled molecule associated with the DNA segment is likely BODIPY 540 sphingomyelin. To our knowledge, this is the fi rst time that a fl uo rescent sphingolipid has been detected in the nucleus of a living cell.

DISCUSSION
We have described the synthesis of a new fl uorescent sphingosine analog, BODIPY 540 sphingosine, and verifi ed its effi cacy for studying the transport, distribution, and metabolism of sphingolipids within living cells. BODIPY 540 sphingosine enabled imaging unacylated sphingolipids, which cannot be accomplished with the existing sphingolipid analogs that contain a fl uorescent fatty acid side chain. Furthermore, unlike previously reported fl uorescent sphingosine analogs (7)(8)(9), BODIPY 540 sphingosine can be visualized in parallel with GFP without the use of UV excitation. By exploiting these capabilities, we verifi ed that mammalian cells rapidly internalized BODIPY 540 sphingosine, transported it to the secretory pathway where it was metabolized to more complex fl uorescent sphingolipids, and eventually catabolized these fl uorescent sphingolipids. The similarity in the pattern of BODIPY 540 sphingosine metabolites and their subcellular distribution to those of the native sphingolipids indicates that the fl uorophore did not signifi cantly hinder lipid metabolism or traffi cking.
fl uorophore appeared to be in close proximity to a DNA segment in the nucleus. The reconstructed cross-sectional z-stack images acquired along ( Fig. 6B, E) and perpendicular to ( Fig. 6C, F) the DNA segment also indicate that the BODIPY 540 fl uorophore was inside the nucleus and adjacent to the DNA segment. Based on the previously reported fi nding that sphingomyelin is associated with  sphingolipid, sphingosine-1-phosphate, stimulates signaling by binding to G protein-coupled receptors on the cell surface ( 1,24 ). Recently, the hypothesis that fundamental cellular processes are regulated by sphingosine-1-phosphate binding to intracellular targets was verifi ed. Specifi cally, sphingosine-1-phosphate binding to histone deacetylases in the nucleus regulates gene transcription ( 34 ), whereas its binding to the proapoptotic effector molecule, BAK, in mito chondria promotes apoptosis ( 33 ). In both cases, the sphingosine-1-phosphate was generated in the organelle where its target resided, and this location determined the effects of this binding on cell function. Efforts to identify additional intracellular targets of unacylated sphingolipids and their mechanisms of action may be facilitated by visualizing the subcellular distributions of BODIPY 540 sphingosine metabolites and GFP-labeled sphingolipid metabolizing enzymes in parallel.
The detection of BODIPY 540 sphingosine metabolites associated with DNA in the nucleus demonstrates the potential to discover new phenomena by metabolic labeling with BODIPY 540 sphingosine. To our knowledge, this is the fi rst observation of fl uorescent sphingosine metabolite association with DNA in the nucleus of a living cell, although further studies are required to confi rm the BODIPY 540labeled molecule was BODIPY 540 sphingomyelin. Since their detection in the nucleus decades ago ( 42,43 ), the identities and subnuclear distributions of sphingolipids have been primarily probed by biochemical analysis of isolated nuclei and visualization of sphingolipid-specifi c functionalized affi nity labels ( 2,44,45 ). Additionally, structural and regulatory roles performed by various types of sphingolipids in the nucleus have been identifi ed ( 2 ). Sphingomyelin associates with chromatin and stabilizes DNA and newly synthesized RNA ( 45,46 ), GM1 regulates nuclear calcium concentrations ( 44 ), and nuclear ceramide is involved in proliferation and apoptosis, although the precise mechanisms remain to be established (47)(48)(49). As mentioned above, nuclear sphingosine-1-phosphate regulates gene expression by binding to histone deacetylases, thereby inhibiting histone deacetylation ( 34 ). Despite these discoveries, many aspects of nuclear sphingolipid function remain poorly understood. BODIPY 540 sphingosine may potentially be used to visualize nuclear sphingolipids, which may allow for a better understanding of how various stimuli infl uence nuclear sphingolipid distribution and the consequences for cell function.
BODIPY 540 sphingosine can be used to monitor the traffi cking and metabolism of acylated and unacylated sphingolipids in living cells. Assessment of colocalization between BODIPY 540 sphingolipids and with GFP-labeled proteins of interest will facilitate identifying the subcellular locations where sphingolipids are metabolized or encounter their binding targets. Such studies can provide new insight into the broad roles of sphingolipids in modulating cellular function.
The authors thank Lauren Macur Brousil for her assistance with the TLC separation and Prof. Hyunjoon Kong and Steven Caliari at the University of Illinois for their discussions of the Hoechst 33342 data .
Our fi nding that BODIPY 540 sphingosine and its metabolites mimicked the analogous native species in living cells is advantageous for using this probe to study dynamic sphingolipid processes but also has implications for its usage. Sphingosine is a bioactive signaling molecule, so consideration must be given to the biological effects that may be induced by labeling with the fl uorescent analog. The BODIPY 540 sphingosine concentrations used in this work were substantially higher than the sphingosine concentrations found in human or bovine serum ( ‫ف‬ 20 nM) ( 37,38 ). However, NIH3T3 cells did not exhibit the changes in morphology or growth rate that are characteristic of sphingosine toxicity during or after BODIPY 540 sphingosine labeling ( 39 ). HEK293 cells were more sensitive to the exogenous sphingosine analog; changes in their morphology occurred during pulse labeling but subsided shortly after transfer to unlabeled chase medium. Previous reports have shown that the effects of exogenous sphingosine depend on the dose per cell and the cell's capacity to metabolize it ( 39 ), so the adverse effects of labeling sensitive cell lines might be reduced by decreasing the BODIPY 540 sphingosine concentration. Alternatively, for labeling more sensitive cell lines, BODIPY 540 sphingosine may need to be synthetically converted to BODIPY 540 sphingomyelin ( 40,41 ), which would have lower bioactivity ( 1 ) and permit visualizing acylated and unacylated sphingolipids in parallel with GFP.
The cells' capacity to degrade the BODIPY 540 sphingolipids in a manner consistent with the native sphingolipid catabolism pathway, which resulted in fl uorophore incorporation into glycerolipid species, including phosphatidylcholine, phosphatidylethanolamine, and either phosphatidylserine or phosphatidylinositol, also has implications for use of BODIPY 540 sphingosine. This property permits observing the entire sphingolipid catabolism pathway, which could shed light on the cellular mechanisms that underlie sphingolipid homeostasis. However, this catabolism complicates using BODIPY 540 sphingosine to visualize the subcellular sphingolipid distribution because some of the fl uorescence observed will be produced by glycerolipids. The same problem is encountered with fl uorescent sphingolipid analogs that contain a fl uorophorelabeled N -acyl fatty acid because they are also catabolized to fluorescent fatty acids that are incorporated into glycerolipids ( 5 ). We observed that the incorporation of BODIPY 540 into glycerolipids increased with increasing labeling time, so the fl uorescence from nonsphingolipid species can be minimized by limiting the labeling time. In addition, the irreversible degradation of sphingosine-1-phosphate to fatty acid precursors, which causes the label scrambling we observed, has been shown to increase with increasing sphingosine concentration in the medium ( 26,39 ). Thus, decreasing the BODIPY 540 sphingosine concentration in the labeling media should reduce fl uorophore incorporation into glycerolipid species.
The ability to track unacylated sphingolipid analogs with BODIPY 540 sphingosine opens the door to identifying the subcellular locations where these bioactive molecules encounter their targets. One such unacylated