Leonards, NSW, Australia Leukocytes and other cells show an enhanced intensity of mobile lipid in their1H NMR spectra under a variety of conditions.Such conditions include stimulation, w
Trang 1Detergent-resistant membrane fractions contribute to the total
1
H NMR-visible lipid signal in cells
Lesley C Wright1, Julianne T Djordjevic2, Stephen D Schibeci2, Uwe Himmelreich5, Nick Muljadi3,4, Peter Williamson2and Garry W Lynch3,4
1
Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology & Medical Research;2Institute of Immunology & Allergy Research;3Centre for Virus Research, Westmead Millennium Institute, the Cellular and Molecular Pathology Research Unit, Department of Oral Pathology and Oral Medicine, Westmead Hospital Dental School and4National Centre for HIV Virology Research, University of Sydney, Westmead Hospital, NSW, Australia;5Institute for Magnetic Resonance Research,
Westmead Royal North Shore Hospital, St Leonards, NSW, Australia
Leukocytes and other cells show an enhanced intensity of
mobile lipid in their1H NMR spectra under a variety of
conditions.Such conditions include stimulation, which has
recently been shown to involve detergent-resistant, plasma
membrane domains (DRMs) often called lipid rafts.As there
is much speculation surrounding the origin of cellular
NMR-visible lipid, we analysed subcellular fractions, including
DRMs, by NMR spectroscopy.We demonstrated that
DRMs isolated by density gradient centrifugation from
lymphoid (CEM-T4, stimulated Jurkat cells), and
mono-cytoid (THP-1) cells produced NMR-visible, lipid signals
Large scale subfractionation of THP-1 cells determined that
while cytoplasmic lipid droplets constituted much of the total
NMR-visible lipid, the contribution of DRMs was
signifi-cant.Qualitative and quantitative lipid analyses revealed that
DRMs and lipid droplets differed in their lipid composition
DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets.Both lipid droplets and DRMs con-tained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospho-lipids, contribute to the NMR-visible lipid.The lipid droplets also exhibited different protein profiles and contained 500-fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities.The NMR-visible lipid in DRMs is therefore unlikely to be a contami-nant from lipid droplets.We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intra-cellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains
Keywords: lipid; membrane; domain; NMR; Triton X-100
The origin of prominent 1H NMR signals from lipids in
spectra from many different cell types has been the subject
of controversy for almost two decades.Currently, two
sources for the1H NMR-visible lipid have been suggested;
these are the mobile acyl chains of triacylglycerol and/or
cholesterol ester) localized to either membranes, or to
EM-visible intracellular lipid droplets [1].Ferretti et al.[2]
concluded that these NMR signals originate from both
cytoplasmic lipid droplets and intramembrane amorphous
lipid vesicles
Highly intense lipid resonances have been associated with
activation or proliferation of lymphocytes, macrophages
and neutrophils [3–5], as well as T cell lines, many cancer cells, and cancer tissue both ex vivo and in vivo [6].Other cellular conditions linked with the appearance of NMR-visible lipid include the antiproliferative effects of tetra-phenylphosphonium chloride on a transformed breast cell line [7], unstimulated human neutrophils in the presence
of high levels of free fatty acids [8], treatment of thymic lymphocytes with anti-CD3 antibody [4], and the induction
of apoptosis or activation in Jurkat T-lymphoblasts [9].The conclusion to be drawn from these and many other studies
is that no single event is linked with the appearance of NMR-visible lipid
Recently, much evidence has accumulated for the pres-ence of neutral lipid-containing plasma membrane domains that are resistant to solubilization with nonionic detergents
at low temperatures and have a low buoyant density when subjected to density gradient centrifugation.Such domains have been given the term DRMs (detergent-resistant membranes), DIGS (detergent-insoluble glycolipid-enriched domains), GEMS (glycolipid-enriched membrane domains), rafts, or caveolae when the protein, caveolin, is present [10]
In this article, we refer to these membrane domains as DRMs and rule out the use of the term caveolae as caveolins are not expressed in hematopoietic cells such as the ones used in this study [10].In comparison to the rest of the plasma membrane which is in the liquid-crystalline (lc)
Correspondence to L.C.Wright, Centre for Infectious Diseases and
Microbiology, Institute of Clinical Pathology & Medical Research,
Westmead Hospital, Westmead NSW 2145, Australia.
Fax: + 61 2 98915317, Tel.: + 61 2 98457367,
E-mail: lesleyw@icpmr.wsahs.nsw.gov.au
Abbreviations: DRM, detergent-resistant membrane; DSM, detergent
soluble membrane; CT-B, cholera toxin biotin; PABA,
p-aminoben-zoic acid; PE, phycoerythrin; NaCl/P i (–), phosphate-buffered saline
without calcium and magnesium; STR-HRP, streptavidin-conjugated
horseradish peroxidase; PtdCho, phosphatidylcholine.
(Received 17 December 2002, revised 03 March 2003,
accepted 19 March 2003)
Trang 2phase, DRM structure is more ordered giving it
character-istics of being in the liquid-ordered (lo) phase [10].DRMs
are rich in cholesterol and sphingolipids (including GM1
and GM3) and selectively retain a number of proteins
including CD4, GPI-anchored proteins such as CD48
[11,12], and proteins associated with T cell receptor (TCR)
signalling such as the src-family tyrosine kinase Lck, and
LAT [10].In addition, TCR engagement with antigen or by
anti-CD3 antibody crosslinking leads to an increased
partitioning of TCR subunits and their associated signalling
molecules into DRMs implicating their central role as a
focal point for T cell activation.Interestingly,
DRM-associated proteins, such as caveolins and GPI-anchored
proteins, have been identified in the surface monolayer of
lipid droplets [13], indicating a possible link between lipid
droplets and DRMs [14]
The size of DRMs has been suggested by some to
be > 100 nm [15], and by others to be around 26 nm
[16].The smaller domain size coincides with that suggested
for NMR-visible microdomains [6].In addition, parallels
can be drawn between the diversity of functions associated
with DRMs and the appearance of NMR-visible lipid in
cells.These factors, as well as the presence in lipid droplets
of known components of rafts and caveoli, such as
cholesterol and caveolin, prompted us to investigate
whether DRMs contain NMR-visible lipid.In this study
we show that DRMs containing NMR-visible lipid can be
isolated from several cell lines under conditions where
mobile lipid is evident in the 1H NMR spectra of the
intact cells.Based on their distinctive protein and lipid
composition, these detergent-insoluble domains differ
from lipid droplets, and can be separated from them
on the basis of different buoyant densities on sucrose
gradients
Materials and methods
Antibodies and reagents
Phycoerythrin (PE)-conjugated anti-CD69/anti-CD25 and
biotin-conjugated cholera toxin (CT-B) were purchased
from Becton Dickinson (San Jose, CA, USA) and Sigma
Aldrich (St.Louis, MO, USA), respectively.Monoclonal
anti-Lck and horseradish peroxidase (HRP)-conjugated
antimouse Ig/streptavidin (STR-HRP) were obtained from
Santa Cruz and Amersham Pharmacia Biotech Inc.,
respectively.Antibodies against Hck, protein disulphide
isomerase and tubulin were purchased from Transduction
Laboratories, Stressgen and Sigma Aldrich, respectively
Monoclonal anti-CD48 was provided by T.Henniker
(Westmead Hospital, Sydney, Australia) and polyclonal
anti-CD4 (T4-5) was a gift from R.Sweet (Smith-Kline
Beecham, King of Prussia, PA, USA)
Cell lines
The CEM-T4 human T lymphoblastoid cell line was
obtained from the NIH AIDS Research and Reference
Reagent Program (Rockville, MD, USA).The human
monocytic leukemic THP-1 and the Jurkat T-cell lines
(J32.2) were obtained from the American Type Culture
Collection (ATCC; Rockville, MD, USA)
Cell culture and stimulation All cell lines were maintained in RPMI/10% fetal bovine serum.For Jurkat cell stimulation, cells were seeded into serum-free RPMI/0.1% BSA (106ÆmL)1) and incubated for
24 h (viability > 80%).Harvested cells were then seeded into RPMI/10% fetal bovine serum and were either left unstimulated or were stimulated with PMA (30 ngÆmL)1) and ionomycin (300 ngÆmL)1) for 24 h
Preparation and fractionation of plasma membranes Jurkat and CEM-T4 cell membranes and cytosol, prepared from 1 and 5· 108cells, respectively, were solubilized with 1% Triton X-100 (v/v) and fractionated by 5–40% sucrose gradient centrifugation as described previously [17].THP-1 cells were used for quantitative NMR and lipid analytical work.Membranes from THP-1 cells (1.56· 109) were separated from the cytosol by ultracentrifugation at
105 000 g for 60 min.The membrane-containing pellet was then solubilized with 1% Triton X-100 and fractionated
by sucrose gradient centrifugation.Thirteen fractions of equal volume were collected from the top to the bottom of each gradient
Cholera toxin dot-blotting and SDS/PAGE immunoblotting
Sucrose gradient fractions were examined for GM1 ganglio-side and protein content by cholera toxin dot-blotting and immunoblotting of SDS/PAGE-separated proteins, respect-ively.For Western blotting, proteins were recovered from cell extracts with Strataclean resin (Stratagene, La Jolla, CA, USA), eluted by boiling in Laemmli sample buffer, subjected
to SDS/PAGE (10% or 4–16%) and electrotransferred to nitrocellulose membranes.Membranes were probed with primary antibody to Lck, Hck, CD48, protein disulphide isomerase, tubulin or CD4 followed by HRP-conjugated anti-rabbit/mouse Ig.For cholera toxin dot-blotting, 1 lL of each fraction was spotted directly onto nitrocellulose prior
to incubation with CT-B followed by STR-HRP.Chemi-luminescent bands and spots were detected on X-ray film Flow cytometry
Cells (2· 105) were incubated with CD69 or anti-CD25 for 30 min on ice, washed with NaCl/Piand then fixed with 1% paraformaldehyde prior to FACScan ana-lysis (Becton Dickinson).Data were analysed using CELL QUESTsoftware (Becton Dickinson)
1H NMR analysis of whole cells, cell fractions and sucrose gradient fractions
Sample Preparation Cells (5· 107) were washed three times in NaCl/Pi(–) containing 0.1 mgÆmL)1 BSA, then washed and resuspended in 400 lL NaCl/Pi(–) made up in
2H2O.Homogenates, supernatants, floating lipid droplets and gradient fractions were dialyzed against NaCl/Pi(–), and NaCl/Pi(–)2H2O added to a final concentration of 10% Membrane pellets were washed three times in NaCl/Pi(–) and resuspended in NaCl/P(–)2HO.For quantitation of lipid
Trang 3signals, a known volume (usually 20 lL) of 10 mM
p-aminobenzoic acid (PABA) was added to all fractions
isolated from the cells.Integrals of phase and baseline
corrected NMR spectra were determined usingXWINNMR2.6
(Bruker Biospin, Rheinstetten, Germany).The integrals were
normalized to the PABA resonances at 6.83 p.p.m and
7.83 p.p.m The CH2resonance at 1.3 p.p.m was utilized to
quantify the fatty acid residues.This resonance contains
contributions mainly from the protons on CH2groups that
are adjacent to other CH2groups, rather than those adjacent
to double bonds or the carbonyl group of the acyl chains [7]
From the biochemical fatty acid analysis it was estimated that
the average number of such CH2groups per fatty acid residue
was 9.1 This was used for calculation of the fatty acid residue
concentrations based on the NMR integrals.For most
fractions, only trace amounts of valine, leucine, threonine
and isoleucine contributed also to the 1.3 p.p.m resonance
However, for the THP-1 supernatant fraction, a substantial
contribution from these amino acid residues was noted,
resulting in overestimation of the fatty acid concentration by
up to twofold.Some contribution from amino acids was also
noted in the membrane and DSM fractions from THP-1 cells
NMR spectroscopy For both cells and gradient fractions,
NMR spectra were acquired at 37C with the sample
spinning at 20 Hz, using a Bruker Avance 360 MHz
spectrometer; parameters for NMR spectroscopy of the
cells were essentially as in [8].1D 1H NMR spectra of
gradient fractions were run using a selective excitation field
gradient method of water suppression [18], a spectral width
of 4000 Hz, 256 accumulations, and total acquisition time
per transient of 3.14 s 2D NMR spectroscopy of sucrose
gradient fractions was carried out by acquiring 1H, 1H
COSY NMR spectra in magnitude mode.Remaining water
was suppressed by selective excitation.A total of 2000 data
points were collected in the t2time domain with a spectral
width of 10 p.p.m The evolution time (t1) was incremented
to obtain 200 free induction decays, each with 32 scans for
cells, and for the rafts 160 free induction decays, each with
256 scans.The total relaxation delay per scan was 1.6 s
Sine–bell window functions were applied in the t1dimension,
and Gaussian–Lorentzian window functions were applied
in the t2dimension according to [7].Zero filling was used
to expand the data matrix to 1K in the t1dimension
Lipid extraction and thin layer chromatography (TLC)
Gradient fractions were extracted in chloroform and
methanol, partitioned against water, and the lipid species
in the organic phase separated by TLC as previously
described [5,8].The solvent system for neutral lipids was
petroleum ether (BP 60–80C)/diethyl ether/acetic acid
(90 : 15 : 1 v/v/v) and for polar lipids was CHCl3
/meth-anol/water (65 : 25 : 4 v/v/v).The lipids were visualized
with iodine vapour and also stained with sterol spray
reagent and Coomassie Blue [8].Their identities were
confirmed by comparison with authentic standards
Lipid analyses
Cholesterol and triacylgycerol estimations were performed
using the Roche Modular Analytical System with
CHOD-PAP methodology for cholesterol and GPO/CHOD-PAP metho-dology for triacylgycerols (Roche).Fatty acid analyses were conducted on saponified lipid extracts converted to fatty acid methyl esters by acid methanolysis.These were separated on an HP Series II 5890 gas chromatograph with
an Agilent Ultra 2 capillary column 19091B-102, using the method of MIDI Inc.(Delaware, USA).Results are expressed as percentages of the sum of the areas of all peaks identified
Results Examination of stimulated Jurkat cells by NMR spectroscopy
Stimulation of Jurkat T cells with PMA/ionomycin was assessed using flow cytometry as shown in Fig.1A Augmentation of the fluorescence levels attributable to surface expression of the T-cell activation markers, IL-2 receptor alpha chain (CD25) and CD69, was observed following stimulation
Fig 1 Examination of stimulated Jurkat T cells by flow cytometry and
1
H NMR spectroscopy PMA and ionomycin-stimulated and non-stimulated Jurkat cells were incubated with anti-IgG 1 (isotype control), anti-CD25 or anti-CD69 (all PE–conjugated) and fluorescence was assessed by flow cytometry (A).The shaded peaks represent the stimulated phenotype in each case.1D1H NMR spectra of sti-mulated and nonstisti-mulated Jurkat cells are shown in B, confirming the presence of more NMR-visible CH 2 resonances at 1.3 p.p.m in the stimulated cells.Sample preparation and 1 H NMR spectroscopy for this and subsequent figures were carried out as in the Materials and methods.
Trang 4Stimulated and nonstimulated Jurkat cells were examined
for mobile lipid content using 1H NMR.As shown in
Fig.1B, there was a marked increase in NMR-visible,
mobile lipid on stimulation with PMA plus ionomycin
Protons from the methylene groups of the lipid acyl chains
are at 1.3 p.p.m., and other resonances have previously been
assigned to protons from methyl groups (0.9 p.p.m) and
choline-containing residues (3.2 p.p.m) [4] The CH2/CH3
ratio increased from 1.44 to 3.02 after stimulation, with a
concomitant increase in the CH2/choline peak height ratio
from 0.44 to 2.82, due to a decrease in choline These trends
have also been observed previously [4,9]
Examination of plasma membrane fractions
from stimulated Jurkat cells by NMR spectroscopy
Next we examined the plasma membrane distribution of
mobile lipids by fractionating plasma membranes on the
basis of Triton X-100 solubility and buoyant density using
sucrose gradient ultracentrifugation.The separation of lipid
domains to the lighter, detergent-insoluble gradient
frac-tions (DRMs, 3–6) was indicated indirectly by detecting the
DRM-resident proteins Lck and CD48, and directly by
detecting the glycosphingolipid GM1 with cholera toxin
(Fig.2A–C).The DRM protein markers were also detected
in the high density fractions (9–10) which contain cytosolic
material and Triton-soluble membrane (DSM) components
Lck and CD48 were, however, preferentially associated with
DRMs, which contained less than 2% of the total protein
found within the DSM fractions (determined by
densito-metric scanning of Coomassie Blue-stained gels)
When the DRM and DSM fractions were analysed for
mobile lipid content by 1H NMR, lipid CH2 and CH3
resonances at 1.3 and 0.9 p.p.m were present in the DRM
fractions of stimulated cells (mean CH2/CH3 ratio of
1.9 ± 0.2 SEM, n¼ 3), but lipid was below the level of
detection, or present at a reduced level (mean CH2/CH3
ratio of 1.4 ± 0.06 SEM, n¼ 3), in similar fractions from
the same number of nonstimulated cells (Fig.2D).Lipid
resonances were not detected in the DSM fractions from
either cell type (not shown).Carbohydrate/polyol residues
(CHOH, 3.4–4 p.p.m region), but not choline, were visible
in both the DRM and DSM fractions, but these were found
to be artefacts from residual sucrose and/or dialysis cassette
materials (see later)
Identification of mobile lipid in other cell types and
membrane fractions by1H NMR spectroscopy
CEM-T4 cells and membranes NMR spectra of CEM-T4
lymphoblasts displayed less mobile lipid than the stimulated
Jurkat cells (CH2/CH3 ratio of 2.78), but the choline
resonance was much more prominent (Fig.3A) No
increase in mobile lipid was observed upon stimulation
(data not shown).NMR-visible lipid and carbohydrate (but
not choline) were again present in the DRM fractions (4–5)
isolated from these cells (Fig.3A), but this lipid was not
observed in the DSM fractions (not shown).The distribution
of CD4 protein is shown in Fig.3B and demonstrates that a
portion of CD4 is present in the DRM fractions containing
the NMR-visible lipid.The DRM fractions contained about
2% of the total membrane protein content
THP-1 cells and membranes NMR spectra of intact cells
of the human monocytoid cell line THP-1, were clearly dominated by protons arising from lipid (Fig.4A) The
CH2/CH3peak height ratio was calculated to be 5.2, which was much higher than the equivalent signal observed in stimulated Jurkat cells.In contrast, the intensity of the choline resonance was very low, compared with the unstimulated Jurkat or CEM-T4 cells.The THP-1 cells did not increase their NMR-visible lipid when stimulated (data not shown)
As with CEM-T4 DRM fractions, THP-1 DRM fractions 4–6, which contain less than 2% of the total membrane pro-tein, were found to localize specifically the DRM-associating proteins CD4 and Hck, without contamination by either of the abundant endoplasmic reticulum (protein disulphide isomerase) [19] or cytoskeletal (tubulin) representative protein markers (Fig.4B).All proteins colocalized with the DSM fractions 10–12
Notably, the spectra arising from these DRM fractions were similar to the whole cell spectra in that they were again dominated by lipid (CH2/CH3 ratio of 5.0; Fig 5A) A small peak from choline-containing compounds was visible
at 3.2 p.p.m As with the Jurkat and CEM T-4 gradients, carbohydrate/polylol resonances were distinguished in both the DRM and DSM fractions (Fig.5A,C).These resonan-ces, and one at 5.4 p.p.m., were also found in spectra from dialysed blank sucrose gradient fractions (Fig.5B) which were prepared under the same conditions as the membrane-containing gradients, and were therefore not cell-derived.In comparison, virtually no NMR-visible lipid was detected in the DSM fractions (Fig.5C), which did, however, contain most of the extracted membrane proteins.Blank gradient fractions isolated from the same part of the gradient produced spectra that were identical to that in Fig.5B (results not shown).An NMR spectrum of Triton X-100 is shown in Fig.5D.Resonances from Triton were not found
in DRM, DRM control or DSM fractions (Fig.5A–C), or
in DSM control fractions (not shown)
Two-dimensional 1H,1H correlation spectroscopy (COSY) confirmed that the resonance at 1.3 p.p.m was indeed derived mainly from lipid (Fig.6).The crosspeaks labelled A–G¢ in Fig.6 indicate spin-spin coupling between protons on adjacent carbon atoms.Crosspeaks A–F have previously been assigned to resonances from acyl chain protons found in triacylglycerol and/or cholesterol esters [20,21].These could also arise from phospholipids.G¢
is derived from the glycerol backbone of triacylglycerol [20].The intensities of crosspeaks C and D (– CH2-CH2 -CH¼CH- and ¼CH-CH2-CH¼CH-, respectively) indicate that relatively large amounts of unsaturated fatty acid residues are present.The absence of resonances from lactate, threonine, valine, leucine and isoleucine was confirmed by COSY and spin-echo experiments (TE¼ 135 ms, data not shown)
Distinguishing the NMR-visible lipids of DRMs and cytosolic lipid droplets
Most of the cytosolic lipid droplets floated as a visible, milky layer on the top of the supernatant formed when the plasma membrane fraction was sedimented from the cellular homogenate.The remainder of the lipid droplets were
Trang 5observed floating on top of fraction 1 of the sucrose gradient
used for separation of DRMs from DSMs.The lipid
droplets isolated from THP-1 cells displayed an NMR
spectrum (Fig.7A) which resembled that of the DRM
fraction shown in Fig.5A, except that the CH/CH ratio
was only 2 (compared with 5 for DRMs) and a resonance at
3 p.p.m was more prominent than the choline peak at 3.2 p.p.m Comparison of NMR spectra from THP-1 cells (Fig.4A) and their sedimented plasma membranes (Fig.7B) shows a marked reduction in NMR-visible lipid
in the membranes, indicating removal of cytoplasmic lipid droplets.The relative reduction in the intensity of the choline resonance at 3.2 p.p.m confirmed previous obser-vations that most of the choline signal visible in whole cells derives from intracellular metabolites such as free choline, choline phosphate and glycerophosphocholine [4,7]
A large-scale fractionation of THP-1 cells was carried out
to quantify the contribution of the lipid droplets and DRMs
to the total cellular NMR-visible lipid.Relative concentra-tions of the NMR-visible lipid in all subcellular fracconcentra-tions were calculated by comparing the integrals of the lipid methylene resonances at 1.3 p.p.m to those of a known concentration of the internal standard, PABA (Table 1) The only fraction not included was the hard pellet at the bottom of the gradients, which presumably contains aggregated proteins and cytoskeletal elements, as it is Triton-insoluble, but does not float on a density gradient
We calculated that approximately 12.4% of the total
Fig 2 Identification and examination of Jurkat DRMs by1H N MR
spectroscopy Proteins in sucrose gradient fractions from
nonstimu-lated Jurkat cells were fractionated by SDS/PAGE, electrotransferred
to nitrocellulose and immunoblotted with antibodies to DRM protein
markers such as Lck (A) or CD48 (B) (1 lgÆmL)1) followed by ECL
detection as in the Materials and methods.Sucrose gradient fractions
were also spotted onto nitrocellulose and probed with CT-B
(2 lgÆmL)1) followed by ECL detection of GM1, a DRM lipid marker
(C).The1H NMR spectra of dialysed DRMs from 108stimulated and
nonstimulated Jurkat cells are compared in D, indicating the presence
of NMR-visible CH 2 resonances at 1.3 p.p.m in the former, and low
amounts of these resonances (sometimes below the limits of detection,
as shown here) in the latter.
Fig 3 Examination of CEM-T4 cells and DRMs by 1 H N MR spectroscopy The1H NMR spectra of CEM-T4 cells and dialysed DRM fractions obtained from 5 · 10 8 cells are shown in A.The presence of CH 2 resonances at 1.3 p.p.m can be seen in both cells and DRMs.The sucrose gradient distribution of the DRM marker, CD4,
is shown by immunoblotting with anti-CD4 (1 lgÆmL)1) followed by ECL detection in B.
Trang 6cellular NMR-visible lipid signal was derived from the
DRM fractions, about 2.1% from DSM fractions, and
about 62.5% from lipid droplets The latter figure was
calculated as the sum of the signal from the supernatant
(assumed to be contaminated with lipid droplets) plus that
from the isolated cytoplasmic lipid droplet fraction.The rest
of the signal was found in the membranes and low speed
pellet (which includes unbroken cells and organelles).The
contribution of the insoluble membrane pellet, including
the cytoskeleton, was not calculated, as the pellet could not
be resuspended uniformly
Lipid composition of DRMs and lipid droplets
The THP-1 cells contained high levels of cholesterol and
triacylglycerol.The DRM and lipid droplet fractions
differed considerably in their content of these two neutral
lipids (Table 1).As expected, the DRM fractions were
enriched in total cholesterol relative to triacylglycerol
(cholesterol/triacylglycerol ratio of 3.0) when compared
with the plasma membrane fraction from which they were
derived (ratio of 1.5), and the DSM fractions (ratio 1.0) The
DRM fraction contained about 35% of the total cellular
cholesterol and 23% of the total cellular triacylglycerol
Conversely, the lipid droplet fraction was enriched in
triacylglycerol (cholesterol/triacylglycerol ratio 0.6), as was
the lipid droplet fraction floating on the top of the sucrose
gradient (ratio 0.5, not shown) The lipid droplet fraction contained 15.4% of the total cellular triacylglycerol, and this fraction plus the supernatant fraction (which presumably also contains lipid droplets) contained 40.3% of the cellular triacylgycerols
The major neutral and polar lipid components of DRM and DSM fractions from CEM-T4, THP-1 and Jurkat cells were examined qualitatively by TLC.The dominant neutral lipid in the Jurkat DRM fractions (from both stimulated and nonstimulated cells) was free cholesterol, with a small amount of triacylglycerol and free fatty acid.CEM-T4 DRM fractions contained mainly free cholesterol and cholesterol ester, whereas THP-1 DRMs, by contrast, contained predominantly free cholesterol, triacylglycerol,
Fig 5 Examination of THP-1 gradient fractions by1H N MR spectro-scopy Spectra of dialysed DRM fractions (4–6) isolated from 1.56 · 10 9
cells, and the corresponding dialysed fractions from a blank (control) sucrose gradient are shown in A and B, respectively.The spectrum of the DRMs is dominated by CH 2 resonances at 1.3 p.p.m., which are absent in the control, and present at a much reduced level
in the dialysed DSM fractions shown in C.DSM control fractions resembled those in B (not shown).The absence of detergent contami-nation in A, B and C can be confirmed by comparison with the spectrum of Triton X-100 [1% (v/v) in NaCl/P i (–)2H 2 O], shown
in D.PABA (20 lL of a 10-m M solution) was added as an internal standard in A, B and C.
Fig 4 Examination of THP-1 cells by1H NMR spectroscopy and the
distribution of proteins following sucrose gradient centrifugation The 1D
1
H NMR spectrum of intact THP-1 cells, dominated by CH 2
reso-nances at 1.3 p.p.m., is shown in A After fractionation of THP-1
membranes, proteins in each sucrose gradient fraction were subjected
to SDS/PAGE, electrotransferred to nitrocellulose and
immunoblot-ted with antibodies to DRM markers, CD4 and Hck, and antibodies to
protein disulphide isomerase (PDI, endoplasmic reticulum) and
tub-ulin (cytoskeleton), followed by ECL detection, shown in B.Lysates
were also loaded as controls.
Trang 7and small amounts of cholesterol ester, ether-linked
triacyl-glycerol and free fatty acids.Phosphatidylcholine (PtdCho),
sphingomyelin and other phospholipids were present in the
polar lipid component of all DRMs
DSM fractions from CEM-T4 cells contained mainly free
cholesterol and PtdCho, and those from THP-1 cells
contained mainly cholesterol ester, triacylglycerol and
PtdCho.Small amounts of other phospholipids were
detected also.Apart from GM1, the glycolipid content of
DRM and DSM fractions was not determined
The fatty acid composition of the total lipids extracted
from the various fractions of the THP-1 cells is shown in
Table 2.Surprisingly, the DRM fractions were not enriched
in saturated fatty acids, relative to the cell homogenate and
the total membrane fraction, except for a very small increase
in myristic acid (14:0).Rather there was a small increase in
palmitoleic acid (16:1), 18:2 + 18:3, and arachidonic acid
(20:4) at the expense of palmitic, stearic and oleic acids (16:0,
18:0, 18:1, respectively).The DSM fractions contained
higher levels of 18:0, 18:2 + 18:3 and 18:1 at the expense of
16:0, 14:0 and 16:1, whereas there was a marked increase in
the amount of 16:0 and a decrease in the amount of 16:1 and
17:1 in the lipid droplet fraction (Table 2).The fatty acid
analyses of fractions taken from blank gradients revealed no
contamination from Triton X-100
We next compared the levels of the sphingolipid, GM1, in
THP-1 DRMs and lipid droplets by dot-blot analysis using
biotinylated cholera toxin (Fig.8A).After accounting for fraction volumes, the enrichment of GM1 in DRMs relative
to lipid droplets was found to be greater than 250-fold
Fig 6 The 2D COSY spectrum of the THP-1 DRM fraction The 1D
spectrum is shown on top.Crosspeaks identified previously as arising
from protons associated with lipid [20] are indicated by A–G¢,
indi-cating that the DRM spectrum is dominated by resonances arising
from lipid.Lys, lysine; CHOH, polyol and/or carbohydrate residues.
Fig 7. 1H NMR spectra of THP-1 cytoplasmic lipid droplets and plasma membranes Lipid droplets (A) were isolated by high speed centrifugation (105 000 g) of cellular, organelle-depleted homogenates and represented the milky layer at the top of the supernatant.This layer was dialysed against NaCl/P i (–) and NaCl/P i (–) 2 H 2 O was added
to a final concentration of 10%.Intense CH 2 resonances were visible at 1.3 p.p.m in the lipid droplets, but were obviously less prominent in the membrane-containing pellet (B) obtained after removal of the lipid droplets and supernatant and resuspended in NaCl/P i (–) 2 H 2 O.PABA (20 lL of a 10-m M solution) was added as internal standard to both samples.
Table 1 Cholesterol, triglyceride and NMR-visible lipid content of THP-1 cell fractions Results are expressed as lmol of CH 2 equivalents (NMR-visible lipid) or lipid species (cholesterol and triacylglycerols) per 1.56 · 10 9
cells.The total cellular content is the sum of the top four fractions.Percentage compositions are shown in brackets.The method for calculation of the amount of NMR-visible lipid is described in the Materials and methods section.
Fraction
NMR-visible lipid Cholesterol Triacylgycerols Low speed pellet 2.47(13.7) 0.90(10.4) 0.42(9.7) Membranes 4.30(23.8) 3.41(39.4) 2.17(50.0) Supernatant 8.46(46.8) 3.93(45.5) 1.08(24.9) Lipid droplets 2.84(15.7) 0.40(4.6) 0.67(15.4) Total cellular content 18.07 8.64 4.34 DRM fractions 2.24(12.4) 3.00(34.7) 1.00(23.0) DSM fractions 0.38(2.1) 0.31(3.6) 0.31(7.1)
Trang 8Protein content of DRMs and lipid droplets
The protein profiles of DRMs and lipid droplets were
compared by SDS/PAGE (Fig.8B).Prior to analysis, lipid
droplets were subjected to sucrose density gradient
centri-fugation to remove potentially contaminating cytoplasmic
proteins, and the resulting milky layer at the top of fraction 1
was collected.We confirmed that these droplets also do not
stain positive for GM1 (results not shown).Proteins were
visualized by Coomassie Blue staining, and densitometric
scanning revealed that the DRM fraction contained 500
times more protein than the lipid droplet fraction.This was
calculated after taking into account that protein from 30%
and 1.25% of the total lipid droplet and DRM fractions,
respectively, was loaded onto the gel.Although there were
protein bands of similar size in both fractions between 45 and
55 kDa, there were clearly bands unique to each fraction (see
arrows).This finding, together with the relatively low level of
the GM1 glycolipid in lipid droplets, and the differences in
fatty acid and neutral lipid content, is further evidence that
lipid droplets and DRMs are distinct subcellular fractions
Discussion
Microscopically, detergent-resistant plasma membrane
domains have proved difficult to detect [15].However, we
can now visualize by 1D and 2D1H NMR spectroscopy the
mobile lipid component of such domains, which we have
found to have a protein and lipid composition characteristic
of DRMs or rafts, as described by others.While the
membrane fractionation procedure may have altered the
physical state of the lipids from that in the whole cells,
the resonances appearing in DRM spectra are probably
derived from the lipid acyl chains of triacylgycerols and
cholesterol esters, as well as small amounts of free fatty
acids.Some contribution could also come from the acyl
chains of PtdCho or sphingomyelin; however, neither the
choline headgroups of PtdCho and sphingomyelin (except
for a small peak in THP-1) nor the sphingosine chain of
sphingomyelin were visible in the NMR spectra of DRMs
In bilayer environments, the choline headgroups are
relat-ively immobile and therefore of low visibility in NMR
spectra [7].This suggests that headgroup mobility might
also be restricted in DRMs from some cells
Although DRMs from three different cell types produced NMR spectra dominated by resonances from lipid acyl chains, we found differences in the composition of the
Fig 8 Analysis of THP-1 DRMs and lipid droplets for GM1 and total protein (A) THP-1 DRMs were isolated from total membranes by sucrose gradient fractionation as described in Materials and methods Lipid droplets, prepared as in the legend to Fig.7, were subjected to sucrose density gradient fractionation to remove any cytoplasmic proteins and were collected from the top layer (fraction 1).Pooled DRMs (fractions 4–8) and lipid droplets (0.5 lL of each) were spotted onto a nitrocellulose membrane which was subsequently probed with CT-B (2 lgÆmL)1).GM1 was detected by ECL.(B) Proteins from 1.25% and 30% of the total DRM and lipid droplet fractions, respectively, were captured with Strataclean resin and separated by SDS/PAGE.Proteins were visualized by Coomassie Blue staining Molecular mass markers are shown in the left lane, and bands unique
to each fraction are indicated by arrows.
Table 2 The fatty acid composition of THP-1 cell fractions Results are
expressed as percentages of the total area under all peaks measured.
Low concentration components with <1% of the area have been
omitted from the calculations.
Fatty acid Homogenate Membranes DRM DSM
Lipid droplets 14:0 6.08 5.68 7.19 3.29 6.88
16:0 32.28 29.39 28.23 25.59 44.76
16:1x7c 18.27 18.75 22.07 14.88 12.06
17:1 5.57 5.64 6.62 7.06 1.88
18:2 + 18:3 1.57 1.59 2.63 2.91 1.79
18:1x9c 17.66 17.85 15.01 20.83 18.44
18:1x7c 7.78 7.86 6.36 8.99 8.84
18:0 6.59 9.05 5.45 11.06 6.05
20:4 4.19 4.18 6.41 5.37 2.29
Trang 9neutral lipids, which could potentially contribute to these
spectra.Notably, the DRMs extracted from the monocytoid
cell line, THP-1, contained high levels of triacylgycerols,
whereas those from both of the lymphoid cells, CEM-T4 and
Jurkat, were comprised mainly of cholesterol ester and free
cholesterol/triacylgycerols, respectively.This indicates that
just as there are cell-specific differences in the proteins found
in DRMs, with cell-specific functions, there is cell-specific
variability in the neutral lipid content of DRMs.While no
quantitation was performed on the DRM lipids from
stimulated and nonstimulated Jurkat cells, it appeared that
the same components were present, therefore either an
increase in the amount of DRMs present, or in the amount of
lipid in the DRMs from the stimulated cells may explain their
more intense lipid spectra.The fact that DRMs were difficult
to detect in both stimulated and nonstimulated Jurkat cells
may be due to the qualitatitive observation that little neutral
lipid was present in this cell line and its DRMs, and also the
use of much smaller cell numbers used to prepare gradients
(108), compared with the THP-1 cells (1.56· 109).While
some neutral lipids, as well as phospholipids, were present in
DSM fractions, their high protein to lipid ratio may account
for the observation of very little NMR-visible lipid
Although triacylgycerols and cholesterol esters have been
observed in intact cells and pure plasma membrane fractions
by1H NMR spectroscopy and chemical analysis [5,8,20,21],
triacylgycerols, as described for the THP-1 membrane
fractions, have not previously been identified as a
compo-nent of DRMs.The authenticity of our DRM fractions was
confirmed not only by the presence of typical DRM protein
markers, but also by the presence of other DRM lipid
components described in the literature, e.g free cholesterol,
sphingomyelin and gangliosides.There is precedence for
intercalation of triacylgycerols and cholesterol esters, at
least temporarily, into bilayers of some cell types.They form
the cores of lipoproteins, which must be translocated
through membranes for secretion [22].Phospholipid
bilay-ers can accommodate about 3% triacylgycerol and 5%
sterol ester on a molar basis [23,24] before the neutral lipids
phase-separate to form spherical domains sandwiched
between the bilayer leaflets [25].Such a mechanism has
been invoked for the formation of lipid droplets in the
endoplasmic reticulum [26] and in the secretion of milk [27]
Intracellular lipid droplets are surrounded by a
mono-layer of amphipathic phospholipids, glycolipids and/or
sterols that encircles the hydrophobic core of neutral lipids,
such as triacylgycerols, diacylglycerols or sterol esters
[13,14,25].In THP-1 cells we have shown that lipid droplets
have similar NMR spectra to DRMs (compare Figs.5A
and 7A), but contain different proportions of cholesterol
and triacylgycerols, and almost no GM1.Lipid droplets
were also enriched in saturated fatty acids, whereas DRM
lipids were more unsaturated.We now propose that the
mobile lipid visible in the NMR spectra of intact cells is
derived from at least two pools – a large, intracellular, lipid
droplet pool, and a smaller pool, specifically localized to
detergent-insoluble, plasma membrane domains
The figure given for the contribution of acyl chains from
DRM lipids (12.4%) to the THP-1 cell spectrum is a
minimum percentage.Although no nonlipid contribution to
the lipid signal at 1.3 p.p.m could be detected in the low
speed pellet, lipid droplets and DRM fractions, there was
(as expected) a large contribution from nonlipid compo-nents in the supernatant fraction (see Materials and methods).The supernatant contains only 25% of the cellular triacylgycerol, but contributes 46.8% towards the mobile lipid signal (Table 1), therefore this latter figure would be a considerable overestimate, leading to an overestimation of the total cellular lipid signal.Because in THP-1 cells much of the lipid signal would derive from triacylgycerol, a better estimate may be obtained from the triacylgycerol content of the DRMs, namely 23% of the total, and for the supernatant plus lipid droplet fraction a figure of 40% might be more accurate (Table 1).Thus we would estimate the contribution of the DRMs to the cellular NMR-visible lipid in the range of 12–23%, with lipid droplet contribution at around 40%.Interestingly, the contribution of the total membrane fraction to the NMR-visible lipid is around 24%, and as the amount of triacylgycerol in this fraction is 50% of the total, almost all of the NMR-visible triacylgycerol in membranes must reside in the DRM fraction
It could be argued that the NMR signal detected in DRMs is merely contamination from cytoplasmic lipid droplets.This is unlikely, for the following reasons.Firstly, during method development, a lipid droplet preparation from THP-1 cells (rich in NMR-visible lipid) was incubated with membranes from CEM-T4 cells (poor in NMR-visible lipid).After washing, no increase in membrane lipid signal was observed, indicating that contamination by adherence
of cytoplasmic lipid droplets to membrane components during preparation is unlikely (the small amount of droplets remaining are floated to the top of the sucrose gradients during fractionation).Secondly, the protein, lipid and fatty acid composition of droplets and DRMs are quite different, indicating that they are physically separate entities.If contamination with droplets did occur, it would have to be highly selective for the Triton-insoluble fraction of the membranes, because the Triton-soluble fraction (Fig.5C) contains little NMR-visible lipid.Thirdly, the contamin-ation of the DRMs would also need to be very large, since the triacylgycerol content of DRMs is 23% of the total and the isolated lipid droplet fraction contains only 15.4% of the total
The relationship between the two lipid pools (DRMs and lipid droplets) is unclear at present.While the differences we have shown in lipid and protein content of lipid droplets and rafts from THP-1 cells suggest unrelated functions, both are believed to originate in the endoplasmic reticulum/Golgi apparatus, allowing the possibility of some physical inter-action between them.In addition, it has been suggested that free cholesterol may be channelled from triacylgycerol-containing lipid droplets in the endoplasmic reticulum to areas of tight contact between the droplet surface and the plasma membrane, where the free cholesterol could then be incorporated into the bilayer [14].Both metabolic and spatial relationships between neutral lipids (triacylgycerols and sterol esters) and membrane phospholipids have been found, with growing evidence of direct physical continuities between lipid droplets and bilayer membranes [25,27].This deserves further investigation, especially the possibility of translocation of lipid from droplets to DRM domains during stimulation of some cells (e.g Jurkats), with subsequent effects on cell function
Trang 10We would like to thank Ms Leanne Hicks, Department of Infectious
Diseases, for the fatty acid analyses, and the Department of
Biochemistry (Core Pathology), Westmead Hospital for the neutral
lipid analyses.At the time of re-submission of this manuscript, we
discovered that A.Ferretti et al.have quite independently and
simultaneously come to similar conclusions, namely that NMR-visible
lipid is present in DRMs.Their work is now available in the European
Biophysical Journal online, DOI 10.1007/s00249–002-0273-8 as of
January 2003.
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