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

Article abstract of DOI:10.1194/jlr.D029207

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. Copyright

Full text of DOI:10.1194/jlr.D029207

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methods
A new, long-wavelength borondipyrromethene
sphingosine for studying sphingolipid dynamics
in live cells
Raehyun Kim,1 Kaiyan Lou,1,2 and Mary L. Kraft3
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign,
Urbana, IL 61801
Abstract Sphingolipids function as cell membrane compo-
nents and as signaling molecules that regulate critical cellu-
larprocesses. Tostudyunacylatedandacylatedsphingolipids
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 fluores-
cent 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. Visualiza-
tion of BODIPY 540 fluorescence in parallel with GFP-la-
beled organelle-specific proteins showed the BODIPY 540
sphingosine metabolites were transported through the se-
cretory pathway and were transiently located within lyso-
somes, 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 trans-
port, metabolism, and signaling.—Kim, R., K. Lou, and M.
L. Kraft. A new long wavelength borondipyrromethene
sphingosine for studying sphingolipid dynamics in live cells.
J. Lipid Res. 2013. 54: 265–275.
apoptosis, and other critical cellular processes during nor-
mal cell function and disease (1–4). Insight into sphingo-
lipid biosynthesis, transport, and subcellular distribution
has been acquired by observing fluorescent sphingolipid
analogs within living cells (5). Sphingolipid derivatives
that contain a fluorophore-labeled N-acyl fatty acid are
often used to investigate dynamic processes that involve acyl-
ated sphingolipids (5). Fatty acids that contain a polyene
fluorophore are especially attractive for this purpose be-
cause the structure and behavior of polyene-containing
lipids are very similar to the native lipid (6). However, to
study the bioactive, unacylated sphingolipids, sphingosine
and sphingosine-1-phosphate, the fluorophore must be in-
corporated into the sphingosine backbone. Studies have
confirmed that such fluorescent sphingosine analogs can
be metabolized to more complex fluorescently labeled
sphingolipids in living cells (7–10). Despite their potential
utility, only a limited number of fluorophores, such as
pyrene, borondipyrromethene (BODIPY), and nitroben-
zo-2-oxa-1,3-diazole, have been incorporated into the
sphingosine backbone (7–9).
To increase the utility of fluorescent sphingosine ana-
logs for investigating sphingolipid dynamics in living
cells, derivatives with a wider range of fluorescence prop-
erties must be developed. Lacking in particular is a bioac-
tive fluorescent sphingosine that has neither an excitation
maximum that is in the UV range nor emission that inter-
feres with detecting green fluorescent protein (GFP), the
most common genetically encoded fluorescent protein
label (11). A probe with these properties is expected to
Supplementary key words metabolic labeling • fluorescent sphin-
golipid • fluorescent sphingosine • live cell imaging • fluorescence
microscopy • lipid transport • sphingolipid metabolism • sphingo-
lipid catabolism
Sphingolipids and their metabolites serve as structural
components in eukaryotic cell membranes and as bioac-
tive signaling molecules that modulate gene expression,
Abbreviations: GFP, green fluorescent protein; BODIPY, boron-
dipyrromethene; rt, room temperature; ER, endoplasmic reticulum.
1 R. Kim and K. Lou contributed equally to this work.
2 Present address of K. Lou: School of Pharmacy, East China Univer-
sity of Science and Technology, P.O. Box 268, 130 Meilong Road,
Shanghai, China, 200237
M.L.K. holds a Career Award at the Scientific Interface from the Burroughs
Wellcome Fund. This material is based upon work supported by the National
Science Foundation under CHE–1058809.
3 To whom correspondence should be addressed.
e-mail: mlkraft@illinois.edu
Manuscript received 6 June 2012 and in revised form 22 October 2012.
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of supplementary methods
and figures.
Published, JLR Papers in Press, November 4, 2012
DOI 10.1194/jlr.D029207
Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 54, 2013
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flash column chromatography on silica gel using hexane-ethyl
acetate (1:1 v/v) as eluent, yielding III as a white solid (612 mg;
61% yield).
have the advantages of lower phototoxicity than existing
fluorescent sphingosine analogs and the capability to
visualize it in parallel with GFP, which would facilitate
assessing sphingolipid colocalization with proteins of in-
terest. 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 fluorescent
organelle-specific markers to characterize the distribu-
tion of BODIPY 540 sphingosine and its fluorescent me-
tabolites within living cells. We confirm that mammalian
cells metabolized BODIPY 540 sphingosine to BODIPY
540 sphingolipids and catabolized these fluorescent
sphingolipid metabolites. On the basis of these results,
we anticipate that this new fluorescent sphingosine ana-
log will be a valuable tool for investigating the metabo-
lism, trafficking, and signaling of acylated and unacylated
sphingolipid species in living cells.
tert-butyl 2-(4-(dodec-11-en-1-yloxy)phenyl)-1H-pyrrole-1-
carboxylate (V)
To a suspension of tetrakis(triphenylphosphine)palladium
(0) (24 mg; 0.02 mmol) and (4-(dodec-11-en-1-yloxy)phenyl)
boronic acid (III) (310 mg; 1.02 mmol) in methanol (1.2 ml) was
added N-boc-2-bromopyrrole (IV) (300 mg; 1.22 mmol) in tolu-
ene (6.0 ml) and sodium carbonate (2.0 M; 1.0 ml) under argon.
The reaction mixture was refluxed for 14 h and cooled to rt, and
the solvent was removed in vacuo. The residue was extracted with
diethyl ether (2 × 150 ml). The combined organic layer was
washed with 1 M HCl (150 ml), concentrated sodium bicarbon-
ate (aq, 150 ml), and brine (150 ml) and then dried with anhy-
drous magnesium sulfate, filtered, and concentrated via rotary
evaporation. The residue was purified by flash column chroma-
tography on silica gel using a gradient of hexane and methylene
chloride to afford V as a colorless oil (363 mg; 84% yield).
2-(4-(dodec-11-en-1-yloxy)phenyl)-1H-pyrrole (VI)
A solution of tert-butyl 2-(4-(dodec-11-en-1-yloxy)phenyl)-1H-
pyrrole-1-carboxylate (V) (550 mg; 1.29 mmol) and sodium
methoxide (350 mg; 6.48 mmol) in methanol (15 ml) was re-
fluxed for 8 h. The solvent was removed via rotary evaporation,
and the residue was extracted with chloroform (200 ml). The
organic layer was washed by concentrated sodium carbonate (aq)
(150 ml) and brine (150 ml) and dried over anhydrous sodium
sulfate, filtered, and concentrated via rotary evaporation. The
residue was purified by flash column chromatography on silica
gel using a gradient of hexane and ethyl ether to afford VI as a
pale yellow oil (383 mg; 91% yield).
MATERIALS AND METHODS
Synthesis of BODIPY 540 sphingosine
All solvents and commercially available reagents were used
without further purification unless otherwise stated. Tetrahy-
drofuran was distilled from sodium/benzophenone and di-
chloromethane from calcium hydride under argon. Air- and
moisture-sensitive reactions were carried out in oven-dried or
flame-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 (1H) and carbon (13C) NMR spectra were recorded on
400 or 500 MHz Varian Unity instruments. ESI-HRMS was car-
ried out on a FTICR instrument. NMR and mass spectrometry
data for the compounds described below are provided in the
supplementary information.
2-((3,5-dimethyl-2H-pyrrol-2-ylidene)methyl)-5-(4-(dodec-
11-en-1-yloxy)phenyl)-1H-pyrrole (VIII)
Hydrobromic acid (48%; 200 ␮l) was added to a mixture of
2-(4-(dodec-11-en-1-yloxy)phenyl)-1H-pyrrole (VI) (383 mg;
1.18 mmol) and 2,4-dimethylpyrrole-5-carboxaldehyde (VII)
(145 mg; 1.18 mmol, synthesized in 85% yield following the
reported procedure) (12) in ethanol (15 ml) at 0°C. The mix-
ture was stirred at rt for 4 h, and the solvent was removed via
rotary evaporation. The residue was extracted with diethyl ether
(2 × 150 ml), and the combined organic layer was washed with
saturated sodium carbonate (aq) (150 ml) and brine (150 ml). The
organic solution was dried over anhydrous sodium sulfate, fil-
tered, and concentrated to afford crude VIII as a brown solid
(508 mg, nearly quantitative yield) that was used in the next
step without further purification.
1-Bromo-4-(dodec-11-en-1-yloxy)benzene (II)
Under an argon atmosphere, a mixture of 4-bromophenol
(1.00 g; 5.78 mmol), 12-bromododec-1-ene (1.43 g; 5.78 mmol),
and potassium carbonate (1.20 g; 8.68 mmol) was refluxed in
20 ml acetone for 24 h. The reaction mixture was cooled to
room temperature (rt) and filtered. The filtrate was concen-
trated in vacuo, and the residue was purified by flash chroma-
tography on silica gel, eluting with hexane, affording II as a
colorless oil (1.60 g; 82% yield).
(4-(Dodec-11-en-1-yloxy)phenyl)boronic acid (III)
4,4-Difluoro-1,3-dimethyl-5-(4-(dodec-11-en-1-yloxy)
phenyl)-4-bora-3a,4a-diaza-s-indacene (IX)
Under an argon atmosphere, n-butyllithium (1.6 M in
hexane; 2.5 ml) was added over 5 min to 1-bromo-4-(dodec-11-
en-1-yloxy)benzene (II) (1.12 g; 3.3 mmol) in dry tetrahydro-
furan (10 ml) at Ϫ78°C (dry ice/acetone bath). The mixture was
stirred at Ϫ78°C for 1 h, trimethyl borate (0.55 ml, 4.9 mmol)
in dry THF (3.5 ml) was added at Ϫ78°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), con-
centrated sodium bicarbonate (aq, 150 ml), and brine (150 ml).
The organic layer was dried with anhydrous magnesium sulfate,
filtered, and concentrated in vacuo. The residue was purified by
Dipyrromethene (VIII) (508 mg; 1.18 mmol) and 1,8-diazabi-
cycloundec-7-ene (533 ␮l; 3.57 mmol) were dissolved in dry tolu-
ene (20 ml), and BF3·OEt2 (740 ␮l; 6.00 mmol) was added
dropwise. The mixture was heated to 90°C and stirred for 30 min.
After cooling to rt, the reaction was quenched with water and
extracted with diethyl ether (2 × 200 ml). The combined organic
layer was washed with saturated ammonium chloride (aq) and
brine and dried over anhydrous magnesium sulfate, filtered, and
concentrated via rotary evaporation. The residue was purified by
flash column chromatography on silica gel using hexane-methyl-
ene chloride (1:1 v/v) as eluent to afford IX as a red solid (460 mg;
81% yield).
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with a scraper, and pelleted by centrifugation. Lipids were ex-
(S)-4-((R,E)-1-hydroxy-13-[4-(4,4-difluoro-5,7-dimethyl-4-
bora-3a,4a-diaza-s-indacen-3-yl)-phenoxy]tridec-2-en-1-yl)-2,2-
dimethyloxazolidine-3-carboxylic acid tert-butyl ester (XI)
tracted using the Bligh and Dyer method (15). The glycolipids
were selectively hydrolyzed by alkaline treatment with sodium hy-
droxide; this treatment did not hydrolyze the sphingolipids.
The Garner-allylic alcohol (X) (27 mg; 0.10 mmol, synthesized as
reported Ref. 13 in 40% yield after purification by silica gel column
chromatography using hexane-ethyl actate [8:1 v/v] as eluent) and
alkenyl BODIPY 540 (IX) (19 mg; 0.040 mmol) were dissolved in dry
methylene chloride (2 ml), and the solution was degassed. Under
argon atmosphere, a catalytic amount of second generation Grubbs’
catalyst (5 mg; 0.0059 mmol) was added, and the reaction mixture
was refluxed for 4 h. The solvent was removed via rotary evapora-
tion, and the crude material was purified by flash column chroma-
tography on silica gel using a gradient of hexane and ethyl acetate as
eluent to afford XI as a dark red solid (20 mg; 71% yield).
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 butanol-
acetic 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 filter to detect the fluorescent spots. Unlabeled lip-
ids were visualized by iodine staining. The BODIPY 540-labeled
sphingomyelin, ceramide, and sphingosine were identified by
their comigration with BODIPY 540-labeled sphingolipid stan-
dards. Because this solvent system primarily separated the lipid
species according to their head groups, which was confirmed
by the comigration of the BODIPY 540-labeled sphingomyelin
and ceramide with unlabeled sphingomyelin and ceramide, re-
spectively, in this solvent system, the remaining fluorescent lipid
species were identified according to their comigration with
unlabeled lipid standards.
(E)-(2S,3R)-2-Amino-15-[4-(4,4-difluoro-5,7-dimethyl-4-
bora-3a,4a-diaza-s-indacen-3-yl)-phenoxy]pentadec-4-ene-
1,3-diol (I)
Under an argon atmosphere, protected BODIPY-sphingosine
(XI) (20 mg; 0.028 mmol) was dissolved in 4 M HCl in dioxane
(2 ml) and stirred at rt for 20 min. The solvent was removed in
vacuo, and the residue was purified by flash column chromatog-
raphy on silica gel eluting with chloroform-methanol-ammonium
hydroxide (40:10:1 v/v/v) to afford the title compound (I) as a
dark red solid (6 mg; 37% yield).
Glycerolipid quantification
For each labeling interval (2 h, 1 day), the total lipid extract
obtained from a single labeling experiment was dissolved in chlo-
roform-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 pri-
marily 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 filter and quantifying their fluorescence in-
tensities. To quantify the fraction of the fluorescence that corre-
sponded to glycerolipids, the sum of the fluorescence intensities
measured for each spot that corresponded to fluorescent phos-
phatidylcholine, phosphatidylethanolamine, phosphatidylserine,
and phosphatidylinositol was measured using ImageQuant soft-
ware (GE Healthcare) and divided by the total fluorescence in-
tensities of all of the fluorescent lipid species.
Synthesis of BODIPY 540 ceramide and BODIPY 540
sphingomyelin standards
BODIPY 540 ceramide and sphingomyelin standards for
TLC were synthesized by cross metathesis reaction between
4,4-difluoro-1,3-dimethyl-5-(4-(dodec-11-en-1-yloxy)phenyl)-4-
bora-3a,4a-diaza-s-indacene (IX) and ceramide (Sigma-Aldrich)
or sphingomyelin (Sigma-Aldrich), respectively, as previously
reported (14).
Cell culture
NIH3T3 fibroblast cells were grown in DMEM with 10% calf
serum and 1% penicillin/streptomycin in a 5% CO2 incubator at
37°C. For colocalization studies, organelle-specific fluorescent
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% CO2 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 cul-
ture dishes.
Labeling organelles for colocalization study
Pulse-chase labeling
For labeling the endoplasmic reticulum (ER), Golgi appara-
tus, and mitochondria, organelle-specific GFP protein constructs
were used. For ER labeling, HEK293 cells were transfected with
pAc-GFPC1-Sec61␤ (Addgene plasmid 15108) (16) using Trans-
Pass D2 transfection reagent (New England BioLabs, Ipswich, MA).
For Golgi apparatus and mitochondria labeling, CellLightTM Golgi-
GFP and CellLightTM Mitochondria-GFP (Molecular Probes),
respectively, were used according to the manufacturer’s proto-
col. One to two days after transduction, the HEK293 cells were
incubated with 1 ␮M of BODIPY 540 sphingosine for 30 min, fol-
lowed by a chase in unlabeled medium for approximately 1 day.
To label the lysosomes, NIH3T3 cells were incubated with
OptiMEM containing 2 mg/ml of FITC-dextran (MW 10,000;
Sigma-Aldrich) for 30 min. The cells were then cultured for 5 h
in unlabeled medium, followed by 30 min in medium that con-
tained 1 ␮M of BODIPY 540 sphingosine and finally in unla-
beled medium for a 30 min or 16 h chase. To label the nucleus,
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 final
concentration of BODIPY 540 sphingosine was 1 or 1.25 µM. For
pulse labeling, cells were incubated in labeling medium that con-
tained 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 spec-
ified chase time. For studies of BODIPY 540 sphingosine metabo-
lism, cells were incubated in growth medium that contained 1 µM
of BODIPY 540 sphingosine for the specified time.
Lipid extraction
Cells were harvested when they reached 90% to 100% conflu-
ence. Cells were washed with cold PBS, removed from the dish
Long wavelength BODIPY sphingosine for sphingolipid studies
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2 ␮l of Hoechest 33342 (Invitrogen) stock solution (10 mg/ml)
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 purification.
Characterization of the absorption and fluorescence of
BODIPY 540 sphingosine in ethanol (1 ␮M) demonstrated
that its absorption and fluorescence emission maxima
were 540 and 568 nm, respectively (Fig. 1). Thus, the exci-
tation 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 wave-
length emission that is indicative of dimer formation ap-
peared to be negligible in labeled cells.
was added to the growth medium in which the NIH3T3 cells were
cultured. After 30 min, the medium was replaced with unlabeled
medium. After 30 min, the cells were incubated in medium with
1 ␮M of BODIPY 540 sphingosine for 30 min and transferred to
unlabeled medium for a 2 day chase.
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.
Cells uptake and metabolize BODIPY 540 sphingosine
We first confirmed that BODIPY 540 sphingosine was
internalized by cells and metabolized to more complex
fluorescentsphingolipids.Mousefibroblastcells(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 be-
fore 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 fluo-
rescence, confirming they internalized the BODIPY 540
sphingosine. After a 30 min chase in unlabeled medium,
fluorescent 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 fluores-
cence, which indicates that the plasma membrane con-
tained low levels of fluorescent lipid species (Fig. 2A).
Fluorescent vesicles were also observed in NIH3T3 cells
when the duration of the chase in unlabeled medium in-
creased to 20 h (Fig. 2B). Although the plasma membrane
still appeared to be dim relative to the highly fluorescent
intracellular vesicles, which were especially abundant in
NIH3T3 cells, the clearer fluorescent outline of the cell’s
perimeter indicates that BODIPY 540 lipid species had in-
corporated into the plasma membrane (Fig. 2B). Overall,
these observations suggest that the BODIPY 540 sphin-
gosine was metabolized to fluorescent sphingolipids, which
were transported to the plasma membrane.
To verify that the fluorescent sphingosine was metabo-
lized fluorescent sphingolipids, NIH3T3 cells were cul-
tured 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 con-
tained sphingolipids, was separated by TLC. The BODIPY
540-labeled sphingolipids on the TLC plate could be de-
tected with high sensitivity according to their fluorescence.
Four fluorescent 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 confirmed that the
NIH3T3 cells metabolized the fluorescent sphingosine to
BODIPY 540-labeled ceramide and sphingomyelin. Because
the BODIPY 540-labeled ceramide and sphingomyelin
RESULTS
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 dou-
ble bond in the sphingosine backbone (Scheme 1) (8).
Here, the building blocks for the cross metathesis reac-
tion were the allylic garner aldehyde (X) and ␻-alkenyl-
substituted fluorophore (IX). We used BODIPY TMR as
the fluorophore due to its desirable fluorescence proper-
ties (ex/em ف543/569 nm and high quantum yield) and
nonpolar nature (17, 18). However, for synthetic efficiency,
we replaced the phenolic methyl group in BODIPY TMR
with the alkyl tail of sphingosine. Our strategy for synthe-
sizing the ␻-alkenyl-substituted fluorophore (IX) involved
condensing 2-(4-(␻-alkenyloxy)phenyl)pyrrole (VI) and
3,5-dimethylpyrrole-2-carboxaldehyde (VII) and then in-
serting the BF2 bridge (8).
Synthesis of ␻-alkenyl-substituted fluorophore IX began
with ␻-alkenylation of 4-bromophenol with 12-bromodo-
dec-1-ene. This condensation reaction produced 1-bromo-
4-(␻-alkenyloxy)benzene (II), which was converted to the
boronic acid (III) (19). Miyaura-Suzuki cross-coupling of
the boronic acid (III) with N-Boc-2-bromopyrrole (IV) in
the presence of (Ph3P)4Pd and Na2CO3 gave Boc-protected
2-(4-(␻-alkenyloxy)phenyl)pyrrole (V) (20). Deprotection
of V afforded 2-(4-(␻-alkenyloxy)phenyl)pyrrole (VI), a
key building block in the synthesis of the longer wave-
length BODIPY fluorophore. To complete fluorophore
construction, 3,5-dimethylpyrrole-2-carboxaldehyde (VII)
was synthesized in 85% yield following the previously
reported procedure (12) and condensed with pyrrole
building block VI to form VIII. Treatment of VIII with
borontrifluoride and 1,8-diazabicycloundec-7-ene pro-
duced the ␻-alkenyl BODIPY 540 fluorophore (IX) in 81%
yield.
Synthesis was completed by cross-metathesis reaction
(8) of the ␻-alkenyl BODIPY 540 fluorophore (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
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SCHEME 1. Synthesis of BODIPY 540 sphingosine.
comigrated with the analogous unlabeled sphingolipid in
the TLC plate (22), was observed for a labeling period of 1
day. More complex BODIPY 540-labeled glycosphingo-
lipids, 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 reflects 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 phosphoetha-
nolamine and the fatty aldehyde trans-2-hexadecenal,
which is oxidized to a fatty acid that can be incorpo-
rated into glycerolipids (22–25). Therefore, if the BODIPY
540 sphingolipids were degraded via this pathway, the
resulting BODIPY 540-labeled hexadecenal could be
oxidized and used to biosynthesize fluorescent glycero-
lipids. 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 sepa-
rated with TLC. In addition to the BODIPY 540-labeled
sphingolipid species observed by TLC separation of the
sphingolipid extract, the total cellular lipid extract exhib-
ited three additional fluorescent spots that corresponded
to fluorescent glycerophospholipids (Fig. 2C). Compari-
son of these migration patterns to those of unlabeled
this solvent system, we identified the remaining fluores-
cent sphingolipid as BODIPY 540 glucosylceramide due to
its comigration with the unlabeled standard. In addition
to these four sphingolipid species, a fifth species, identi-
fied as lactosylceramide based on its relative location on
Fig. 1. Absorbance and emission spectra of BODIPY 540 sphin-
gosine in ethanol. The emission spectrum was collected using 488
nm excitation.
Long wavelength BODIPY sphingosine for sphingolipid studies
269

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Fig. 2. Subcellular distribution and metabolism of BODIPY 540 sphingosine in NIH3T3 cells. Cells were
incubated in 1.25 ␮M of BODIPY 540 sphingosine for 30 min followed by unlabeled medium for a chase
time of 30 min (A) or 20 h (B). Bars = 20 ␮m. Although the plasma membrane of the cell shown in (B) is
dim in comparison to the highly fluorescent intracellular vesicles that were especially abundant in the
NIH3T3 cells we examined, the more distinct fluorescent outline at the edge of the cell indicates that
BODIPY 540 lipids were present in the plasma membrane. C: Lipid fractions were isolated from cells
that were labeled with BODIPY 540 sphingosine for 2 h or 1 day. The total lipid fractions (Ϫ) and the lipid
fractions subjected to alkaline treatment to remove the glycerolipids (+) were separated by TLC, as described in
the Materials and Methods. Images were taken on a fluorescence imager using 532 nm excitation and a 580 nm
emission filter. The identities of the BODIPY 540-labeled species that are indicated to the right of each spot
were determined by comparison to lipid standards (see Materials and Methods for details). Cer, ceramide;
FA, fatty acids; GluCer, glucosylceramide; LacCer, lactosylceramide; PC, phosphatidylcholine; PE, phosphati-
dylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; Sph, sphingosine.
phospholipid standards indicated that two of these
components were BODIPY 540-labeled phosphatidyl-
choline and phosphatidylethanolamine; the third spot
was consistent with either BODIPY 540-labeled phos-
phatidylserine or phosphatidylinositol. Based on the
relative intensities of the TLC spots corresponding to
unlabeled lipid species, which were visualized by stain-
ing the same TLC plate with iodine, the BODIPY 540
fluorophore was incorporated into the major sphingo-
lipid species but was less abundant in the major glycero-
lipid 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 lip-
ids 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 fluorescence
on the TLC plate that was produced by spots correspond-
ing to glycerolipid species. After a 2 h labeling period,
BODIPY 540 glycerolipids produced approximately 25%
of the fluorescence measured in the total lipid extract;
after labeling for 1 day, approximately 43% of the fluo-
rescence measured in the total lipid extract was produced
by BODIPY 540 glycerolipids. Thus, the BODIPY 540
glycerolipids that resulted from BODIPY 540 sphingo-
lipid catabolism were detectable after a pulse as short as
2 h, and their abundance increased with time. Previous
studies have demonstrated that exogenous [3H] 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
catabolism we observed is a characteristic of sphingosine
pulse labeling and was not induced by the presence of
the fluorophore.
Subcellular distribution of BODIPY 540 sphingolipids
The studies described above demonstrate that NIH3T3
cells metabolized the BODIPY 540 sphingosine to fluo-
rescent sphingolipids and degraded these fluorescent
sphingolipids in a manner that was consistent with native
sphingolipid catabolism. We performed colocalization stud-
ies to confirm that BODIPY 540 sphingosine and its fluo-
rescent metabolites were trafficked through the organelles
where sphingolipid biosynthesis primarily occurs. For
these experiments, HEK293 cells were used because their
high transfectability facilitated the expression of the or-
ganelle-specific fluorescent proteins required to assess co-
localization. HEK293 cells that expressed the ER or Golgi
apparatus markers, GFP-Sec61␤ (16) or CellLightTM 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 confirmed the fluo-
rescent sphingolipid analogs were located in the ER
(Fig. 3A) and Golgi apparatus (Fig. 3B) after the 30 min
chase. Low colocalization occurred between the fluores-
cent 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 conver-
sion of BODIPY 540 sphingosine to more complex fluo-
rescent sphingolipidsintheseorganellesandthesubsequent
transport of the newly synthesized fluorescent sphingo-
lipids 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
270
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Fig. 3. Intracellular distribution of BODIPY 540 sphingosine and its fluorescent metabolites. Transfected
HEK293 cells that expressed GFP-Sec61␤ or CellLightTM-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 indi-
cated 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 fluorescent metabo-
lites. The fluorescent outline of each cell, which signifies the incorporation of BODIPY 540 lipids into the
plasma membrane, is especially distinct in these images because these HEK293 cells have few highly fluores-
cent intracellular vesicles. The overlay images show that colocalization between BODIPY 540 and each or-
ganelle-specific 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.
sphingolipids, or degraded to nonsphingolipid species
(23, 24, 30). We investigated whether BODIPY 540 sphin-
gosine or its fluorescent metabolites accumulated in lyso-
somes 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 oc-
curred after a 16 h chase time (Fig. 4). The lack of BODIPY
540 accumulation in the lysosomes confirms that the fluo-
rescent sphingolipids that reached the lysosomes were
promptly degraded, and the resulting fluorescent prod-
ucts were exported from the lysosomes.
540 sphingosine or its BODIPY 540-labeled metabolites
could be detected in the nucleus, we performed colocal-
ization 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
Finally, we investigated whether BODIPY 540 sphin-
gosine or its fluorescent metabolites could be detected in
the mitochondria or nucleus. We focused on these organ-
elles because sphingolipids and their metabolites have
been previously detected within them, and the sphingo-
lipid metabolism that occurs within these organelles plays
a major role in modulating cellular function (2, 32–35).
For example, internalized fluorescent ceramides are rap-
idly detected in mitochondria (35), where ceramide is
catabolized to bioactive metabolites that promote the mi-
tochondrial pathway of apoptosis (33). Fig. 5A demon-
strates that the fluorescent sphingolipid analogs were
colocalized with the mitochondria marker, CellLightTM
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. Consis-
tent with previous reports (35), colocalization was not ob-
served after the 1 day chase. Sphingolipids also perform
regulatory and structural functions in the nucleus, but
their presence and distribution in subnuclear compart-
ments have primarily been identified by biochemical anal-
ysis of nuclear extracts (2). To assess whether the BODIPY
Fig. 4. Localization of BODIPY 540 sphingolipid analogs in lyso-
somes. The lysosomes in NIH3T3 cells were labeled by incubating
the cells for 30 min in medium containing 2 mg/ml of FITC-dex-
tran 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 col-
umn) show that colocalization between BODIPY 540 and FITC-
dextran (yellow) did not occur after a 30 min chase but was
detectable at low levels after a 16 h chase. Bars = 20 ␮m.
Long wavelength BODIPY sphingosine for sphingolipid studies
271

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chromatin (36), the BODIPY 540-labeled molecule asso-
ciated with the DNA segment is likely BODIPY 540 sphin-
gomyelin. To our knowledge, this is the first time that a
fluorescent sphingolipid has been detected in the nucleus
of a living cell.
DISCUSSION
We have described the synthesis of a new fluorescent
sphingosine analog, BODIPY 540 sphingosine, and veri-
fied its efficacy for studying the transport, distribution, and
metabolism of sphingolipids within living cells. BODIPY
540 sphingosine enabled imaging unacylated sphingo-
lipids, which cannot be accomplished with the existing
sphingolipid analogs that contain a fluorescent fatty acid
side chain. Furthermore, unlike previously reported fluo-
rescent sphingosine analogs (7–9), BODIPY 540 sphingosine
can be visualized in parallel with GFP without the use of
UV excitation. By exploiting these capabilities, we verified
that mammalian cells rapidly internalized BODIPY 540
sphingosine, transported it to the secretory pathway where
it was metabolized to more complex fluorescent sphingo-
lipids, and eventually catabolized these fluorescent sphin-
golipids. The similarity in the pattern of BODIPY 540
sphingosine metabolites and their subcellular distribution
to those of the native sphingolipids indicates that the fluo-
rophore did not significantly hinder lipid metabolism or
trafficking.
Fig. 5. Transfected HEK293 cells that expressed CellLightTM mi-
tochondria-GFP were pulse labeled for 30 min in medium contain-
ing 1 ␮M of BODIPY 540 sphingosine and transferred to unlabeled
medium for 30 min (A) or 1 day (B). The locations of the mito-
chondria (green) and BODIPY 540 sphingolipid analogs (red)
were imaged in living cells at room temperature with confocal mi-
croscopy. The overlay images (last column) show that a high
amount of colocalization occurred between BODIPY 540 and Cell-
LightTM mitochondria-GFP (yellow) 30 min after pulse labeling. In
contrast, colocalization of the two fluorescent components was not
detected when the chase time increased to 1 day. Bars = 20 ␮m.
fluorophore 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 perpendic-
ular to (Fig. 6C, F) the DNA segment also indicate that the
BODIPY 540 fluorophore was inside the nucleus and ad-
jacent to the DNA segment. Based on the previously re-
ported finding that sphingomyelin is associated with
Fig. 6. The DNA in NIH3T3 cells was labeled with Hoechst 33342 for 30 min. After 30 min in unlabeled
medium, the cells were pulse labeled for 30 min with BODIPY 540 sphingosine and chased in unlabeled
medium for approximately 2 days. Confocal imaging of the cell shows the subcellular location of DNA (blue)
and BODIPY 540 sphingolipid analogs (red). A: An en face confocal microscopy image acquired with the
plane of focus at approximately the center of the cell. The horizontal and vertical white dashed lines denote
where cross-sectional images were acquired. B: Reconstructed cross-section acquired at the vertical white
dashed line shown in A. Z-scan step: 0.2 ␮m. C: Reconstructed cross-section acquired at the horizontal white
dashed line shown in A. Z-scan step: 0.2 ␮m. D: Enlargement of the region outlined in yellow in A. E:
Enlargement of the region outlined in yellow in B. F: Enlargement of the region outlined in yellow in C. Bars
in E and F = 1 ␮m.
272
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Our finding that BODIPY 540 sphingosine and its me-
sphingolipid, sphingosine-1-phosphate, stimulates signaling
by binding to G protein-coupled receptors on the cell sur-
face (1, 24). Recently, the hypothesis that fundamental cel-
lular processes are regulated by sphingosine-1-phosphate
binding to intracellular targets was verified. Specifically,
sphingosine-1-phosphate binding to histone deacetylases in
the nucleus regulates gene transcription (34), whereas its
binding to the proapoptotic effector molecule, BAK, in
mitochondria 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 iden-
tify additional intracellular targets of unacylated sphingo-
lipids 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 po-
tential to discover new phenomena by metabolic labeling
with BODIPY 540 sphingosine. To our knowledge, this is the
first observation of fluorescent sphingosine metabolite as-
sociation with DNA in the nucleus of a living cell, although
further studies are required to confirm the BODIPY 540-
labeled 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-specific functionalized af-
finity labels (2, 44, 45). Additionally, structural and regula-
tory roles performed by various types of sphingolipids in the
nucleus have been identified (2). Sphingomyelin associates
with chromatin and stabilizes DNA and newly synthesized
RNA (45, 46), GM1 regulates nuclear calcium concentra-
tions (44), and nuclear ceramide is involved in proliferation
and apoptosis, although the precise mechanisms remain to
be established (47–49). As mentioned above, nuclear sphin-
gosine-1-phosphate regulates gene expression by binding to
histone deacetylases, thereby inhibiting histone deacetyla-
tion (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 influence nuclear sphingolipid distri-
bution and the consequences for cell function.
tabolites 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 fluorescent analog.
The BODIPY 540 sphingosine concentrations used in this
work were substantially higher than the sphingosine con-
centrations found in human or bovine serum (ف20 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 sphin-
gosine labeling (39). HEK293 cells were more sensitive to
the exogenous sphingosine analog; changes in their mor-
phology occurred during pulse labeling but subsided
shortly after transfer to unlabeled chase medium. Previous
reports have shown that the effects of exogenous sphin-
gosine 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 bioac-
tivity (1) and permit visualizing acylated and unacylated
sphingolipids in parallel with GFP.
The cells’ capacity to degrade the BODIPY 540 sphin-
golipids in a manner consistent with the native sphingo-
lipid catabolism pathway, which resulted in fluorophore
incorporation into glycerolipid species, including phos-
phatidylcholine, phosphatidylethanolamine, and either
phosphatidylserine or phosphatidylinositol, also has impli-
cations for use of BODIPY 540 sphingosine. This property
permits observing the entire sphingolipid catabolism path-
way, which could shed light on the cellular mechanisms
that underlie sphingolipid homeostasis. However, this ca-
tabolism complicates using BODIPY 540 sphingosine to
visualize the subcellular sphingolipid distribution because
some of the fluorescence observed will be produced by
glycerolipids. The same problem is encountered with fluo-
rescent sphingolipid analogs that contain a fluorophore-
labeled 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 increas-
ing labeling time, so the fluorescence from nonsphin-
golipid species can be minimized by limiting the labeling
time. In addition, the irreversible degradation of sphin-
gosine-1-phosphate to fatty acid precursors, which causes
the label scrambling we observed, has been shown to in-
crease with increasing sphingosine concentration in the
medium (26, 39). Thus, decreasing the BODIPY 540 sphin-
gosine concentration in the labeling media should reduce
fluorophore incorporation into glycerolipid species.
The ability to track unacylated sphingolipid analogs
with BODIPY 540 sphingosine opens the door to identi-
fying the subcellular locations where these bioactive
molecules encounter their targets. One such unacylated
BODIPY 540 sphingosine can be used to monitor the
trafficking 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 subcellu-
lar locations where sphingolipids are metabolized or en-
counter their binding targets. Such studies can provide
new insight into the broad roles of sphingolipids in modu-
lating 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.
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24. Maceyka, M., S. Milstien, and S. Spiegel. 2009. Sphingosine-1-
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