Separation of a cholesterol-enriched microdomain
involved in T-cell signal transduction
Yukiko Shimada
1
, Mitsushi Inomata
1
, Hidenori Suzuki
2
, Masami Hayashi
1
, A. Abdul Waheed
1
and Yoshiko Ohno-Iwashita
1
1 Biomembrane Research Group, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo, Japan
2 Center for Electron Microscopy, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo, Japan
Cholesterol is one of the major constituents of the
plasma membrane, and is involved in the formation
of the membrane bilayer. The distribution of choles-
terol in the plasma membrane is not uniform, sug-
gesting that cholesterol is also involved in the
construction of functional membrane domains. One
such functional membrane domain is called lipid
rafts [1,2]. Lipid rafts are lateral lipid clusters formed
of sphingolipids and cholesterol, in which particular
molecules are concentrated to form platforms for
intracellular transport and signal transduction. Cho-
lesterol depletion reduces the association of these
molecules with lipid rafts [3,4], indicating that choles-
terol is necessary for the partitioning of these partic-
ular molecules into functional domains in the plasma

brane subpopulation has a much higher cholesterol ⁄ phospholipid (C ⁄ P)
molar ratio ( 1.0) than the BCh-unbound population in raft fractions
( 0.3). It contains not only the raft markers GM1 and flotillin, but also
some T-cell receptor (TCR) signalling molecules, including Lck, Fyn and
LAT. In addition, Csk and PAG, inhibitory molecules of the TCR signalling
cascade, are also contained in the BCh-bound membranes. On the other
hand, CD3e, CD3f and Zap70 are localized in the BCh-unbound mem-
branes, segregated from other TCR signalling molecules under nonstimulat-
ed conditions. However, upon stimulation of TCR, portions of CD3e, CD3f
and Zap70 are recruited to the BCh-bound membranes. The Triton X-100
concentration used for lipid raft preparation affects neither the C ⁄ P ratio
nor protein composition of the BCh-bound membranes. These results show
that our method is useful for isolating a particular cholesterol-rich
membrane domain of T-cells, which could be a core domain controlling the
TCR signalling cascade.
Abbreviations
C ⁄ P, cholesterol ⁄ phospholipid molar ratio; DRM, detergent-resistant membrane; LAT, linker for activation of T-cells; PAG, phosphoprotein
associated with glycosphingolipid-enriched membrane microdomains; PC, phosphatidylcholine; PE, phosphatidylethanolamine;
PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; TCR, T-cell antigen receptor.
5454 FEBS Journal 272 (2005) 5454–5463 ª 2005 FEBS
detergent-resistant membranes (DRMs) have been
assumed to represent lipid rafts in their biochemical
aspects [2]. However, various biochemical methods and
conditions are used for isolating DRMs, which gives
rise to some conflicts concerning the molecules associ-
ated with DRMs.
The heterogeneity of lipid rafts has recently been
discussed. It has been suggested that several types of
lipid rafts with differing lipid and protein compositions
perform different functions [11,12]. Fluorescent micro-

terol content but scarcely bind to liposomes containing
less than 20 mol% cholesterol [18,21]. In intact cells,
the depletion of cell cholesterol by approximately 30%
abolishes their binding to plasma membranes [17–19].
This is in remarkable contrast to cell binding by filipin,
another cholesterol-binding reagent. Filipin staining is
significantly retained under the same depletion condi-
tions [19]. Thus BCh binds to a specific population of
cholesterol, while filipin binds indiscriminately to cell
cholesterol. We have demonstrated that cell-bound
BCh is predominantly recovered in raft fractions
[18,19,22]. Electron microscopic observations showed
that raft fractions prepared from BCh-bound platelets
contain two populations of membrane vesicles,
BCh-labelled and -unlabelled [19]. This observation
implies that DRMs contain membrane subpopulations
with different cholesterol enrichments, and that BCh
could be a new probe to be used to isolate a particular
lipid domain from raft fractions.
In this study, we used the cholesterol probe BCh to
isolate a cholesterol-enriched membrane domain from
the so-called lipid raft fractions of T-cells. This partic-
ular membrane domain can be prepared irrespective of
the isolation conditions, and selectively retains signal-
ling molecules such as Lck, Fyn, and LAT. The essential
TCR-signalling molecules obtained in raft fractions, for
example CD3f and Zap70, are not concentrated in the
BCh-bound subpopulation under nonstimulated condi-
tions; however, CD3f and Zap70 are recruited to the
BCh-bound subpopulation after TCR stimulation.

icles bound to BCh were retrieved with avidin-magnet
Y. Shimada et al. Two raft subsets in T-cells
FEBS Journal 272 (2005) 5454–5463 ª 2005 FEBS 5455
beads and separated from BCh-unbound vesicles that
were recovered in the bead-unbound fraction. Total
lipid rafts, and avidin-magnet beads-unbound and
-bound fractions were subjected to SDS ⁄ PAGE and
analysed by silver staining and western blotting
(Fig. 3A,B). Almost all BCh was recovered in the
avidin-magnet bead-bound fraction as determined by
detection with antih-toxin antibody, indicating that
most BCh-bound vesicles were recovered in the magnet
bead-bound fraction (Fig. 3B). When raft fractions
were prepared in the absence of BCh, no specific pro-
teins so far tested were retained by the avidin-magnet
beads by western blot analyses (Fig. 3B and data not
shown). This indicates that membrane vesicles are not
retained on the beads by nonspecific adsorption. These
results show that our method is suitable for isolating
membrane vesicles that selectively bind to BCh.
BCh-bound vesicles are cholesterol-enriched
and contain raft-marker proteins and some T-cell
signalling molecules
We analysed the cholesterol and phospholipid contents
of bead-bound and -unbound fractions (Table 1, col-
umns labelled 1% Triton). The total raft fractions con-
tained about 30% of total cellular cholesterol (Fig. 1).
Eighty per cent of the cholesterol in the raft fractions
was retrieved in the BCh-bound membrane fraction
(bead-bound fraction), which corresponds to 24% of

membrane fraction (Fig. 3C). A small amount of CD3e
was partitioned to the raft fractions, all of which was
recovered in the BCh-unbound membrane fraction
Fig. 1. BCh binds to lipid rafts in Jurkat cells. BCh-bound Jurkat
cells (1 · 10
7
) were treated with 1% Triton X-100, homogenized,
and subjected to sucrose density gradient centrifugation. The
resulting gradients were fractionated from the top (0.4 mL each;
total 12 fractions). The distributions of cholesterol and cell-bound
BCh in the gradient fractions were analysed. The BCh detected in
fractions 10 and 11 probably represents a toxin liberated during
membrane homogenization. Total, BCh in the total lysate before
sucrose-density gradient fractionation. The results are representa-
tive of seven independent experiments.
Fig. 2. Immunoelectron microscopic observation of BCh in rafts.
The raft fractions were prepared from BCh-bound Jurkat cells. BCh
was immunolabelled with antibiotin and 10 nm protein-A gold and
observed by negative staining. Arrows indicate BCh-bound vesicles.
Two raft subsets in T-cells Y. Shimada et al.
5456 FEBS Journal 272 (2005) 5454–5463 ª 2005 FEBS
(Fig. 4). The adaptor protein Grb2 was also detected in
the unbound membranes. It is worthy to note that PAG
and Csk, which negatively control TCR signalling, were
found in BCh-bound membranes (Fig. 3C). Thus, mole-
cules participating in T-cell signalling exhibit clear
localizations between BCh-bound and -unbound mem-
branes, suggesting that these two membrane domains
play different roles in T-cell signalling. The results also
show that raft fractions prepared by the conventional

Total Unbound Bound Total Unbound Bound
Cholesterol (nmol) 28.0 ± 3.5 3.0 ± 1.1 23.0 ± 3.5 48.0 ± 9.2 15.0 ± 0.1 33.0 ± 9.1
Phospholipids
a
(nmol) 33.0 ± 1.5 10.0 ± 1.8 23.0 ± 3.2 79.0 ± 9.4 47.0 ± 3.1 32.0 ± 6.2
C ⁄ P ratio 0.80 ± 0.05 0.30 ± 0.04 1.00 ± 0.02 0.60 ± 0.05 0.30 ± 0.03 1.00 ± 0.13
Protein (lg) 47.4 ± 4.6 28.7 ± 1.4 19.3
b
± 2.7 51.0 ± 3.0 38.5 ± 1.5 12.5
b
± 4.5
a
Phospholipids were determined as the amounts of inorganic phosphorus.
b
Amount of proteins in the BCh-bound membrane fraction was
estimated by subtracting the amount in the unbound fraction from that in the total raft fractions.
Y. Shimada et al. Two raft subsets in T-cells
FEBS Journal 272 (2005) 5454–5463 ª 2005 FEBS 5457
X-100 concentration on the partitioning of signalling
molecules into BCh-bound membranes. Total raft frac-
tions prepared with 0.2% (v ⁄ v) Triton X-100 contained
about twofold more membranes than those prepared
with 1% Triton X-100 as judged by lipid content
(Table 1). However, the amount of membranes with a
high C ⁄ P ratio recovered in the BCh-bound membrane
fraction did not increase much by preparation at the
lower Triton X-100 concentration. This is in contrast
to a remarkable increase in membranes with a low
C ⁄ P ratio recovered in the unbound fraction (Table 1).
Although higher amounts of CD3e and CD3f were

while the level in the BCh-bound fraction was
unchanged (ratio of GM1 in the BCh-bound mem-
brane to that in the BCh-unbound membrane ¼ 1 : 2).
Recruitment of Zap70 and CD3d to the choles-
terol-enriched membrane subpopulation after
anti-CD3 stimulation
It has been reported that when activated by stimuli such
as the anti-CD3 Ig, T-cell rafts undergo dynamic chan-
ges in their size and molecular composition [23]. We
analysed initial changes in the components of choles-
terol-enriched subpopulations upon T-cell activation.
After activation, the amounts of CD3e and CD3f
recovered in raft fractions were much increased. We
found that parts of Zap70 and CD3f were recruited to
BCh-bound vesicles upon T-cell activation with anti-
CD3 Ig (Fig. 6). The phosphorylated form of Zap70
was detected in raft fractions from stimulated cells, and
a part of it was associated with BCh-bound vesicles.
Lck and LAT in raft fractions were associated exclu-
sively with BCh-bound vesicles regardless of activation.
These results suggest that the TCR signalling initiation
machinery is formed in cholesterol-enriched membrane
domains. Some proteins, such as moesin, remain associ-
ated with BCh-unbound membranes even after activa-
tion, suggesting that the recruitment is a specific feature
of some signalling molecules.
Discussion
Lipid rafts are defined as lateral clusters of cholesterol
and sphingolipids; however, the biochemical definition
of lipid rafts remains obscure. Regardless of detergent

(Table 1). Obviously, much higher amounts of CD3e
and CD3f were recovered in the total raft fractions
(Fig. 4). However, we found that the detergent concen-
tration scarcely altered the molecular species associated
with BCh-isolated membrane domain. Our study sug-
A
B
Fig. 5. Analysis of lipid compositions of raft
subpopulations. BCh-bound Jurkat cells
were treated with Triton X-100 and subjec-
ted to sucrose density gradient centri-
fugation as described. The raft fractions
were further fractionated with avidin mag-
netic beads. Lipids from the total raft frac-
tion, and the BCh-unbound and BCh-bound
membrane fractions were extracted by the
method of Bligh and Dyer with slight modifi-
cation. (A) After Bligh–Dyer separation, the
lower phase was concentrated and analysed
by HPTLC. Phospholipids were visualized
with 3% cupric acetate ⁄ 8% phosphoric acid
solution. (B) The upper phase was applied
to a Bond Elute packed column. Ganglio-
sides were eluted with methanol, separated
by HPTLC and detected with resorcinol-
hydrochloric acid-reagent. M, Marker lipids.
The results are representative of two
independent experiments.
Y. Shimada et al. Two raft subsets in T-cells
FEBS Journal 272 (2005) 5454–5463 ª 2005 FEBS 5459

lar cholesterol-rich membranes to TCR signalling, we
incubated membranes of total raft fractions with BCh
after detergent extraction and analysed their content.
We found that a majority of CD3 in the raft fractions
was recovered in the BCh-unbound fraction after this
treatment (data not shown), suggesting that CD3
localized in intracellular cholesterol-rich membranes
might represent a small population. As neither endo-
plasmic reticulum nor lysosomal marker proteins (cal-
nexin, nor Lamp-1, respectively) were detected in raft
fractions, it is unlikely that these intracellular organ-
elles contaminate the raft fractions. However, judging
from the observation that the PS ⁄ PI profile of the
BCh-unbound subpopulation is similar to that of the
endoplasmic reticulum of BHK21 cells and rat liver
cells [25], it is possible that the BCh-unbound sub-
population includes membranes of intracellular origin.
Thus our study clearly shows the existence of hetero-
geneous subpopulations with quite different lipid
profiles in raft fractions.
To evaluate the functional meaning of these hetero-
geneous subpopulations in raft fractions, we next
examined the differential distribution of TCR signalling
molecules between the cholesterol-enriched subpopula-
tion (BCh-bound subpopulation) and the cholesterol-
poor subpopulation (BCh-unbound subpopulation).
Under nonstimulated conditions, transducer molecules,
for example Fyn and Lck, were detected in the choles-
terol-enriched subpopulation of lipid rafts. Flotillin and
LAT, which are abundant in raft fractions, were also

molecules, PAG and Csk, which are known to be
negative regulators of TCR signalling [26], were also
detected in BCh-bound membranes. By association
with phosphorylated PAG, Csk negatively regulates
Src-kinases [27], maintaining the ‘off state’ of signalling
under nonstimulated conditions. Experiments in which
b-cyclodextrin is used to remove cholesterol have
provided controversial results [28,29], and the role
of cholesterol in T-cell signalling remains unclear.
However, our results imply that phase separation
of the plasma membrane depending on cholesterol
content might be involved in segregating signalling
molecules from each other to maintain the ‘off’ state of
T-cell signalling.
Taken together, the BCh-bound cholesterol-enriched
subpopulation contains both activator and inhibitor
molecules for TCR signal transduction, and is likely
to play an indispensable role in controlling the on ⁄ off
of the signalling cascade. Using BCh, it is possible to
isolate particular functional membrane domains
regardless of the preparation conditions. At present,
the function of the BCh-unbound raft subpopulation
with a lower cholesterol content is unclear. However,
the BCh-unbound region might also play an import-
ant role in TCR signalling as it contains receptor
molecules for TCR signalling under nonstimulated
conditions. We propose that not total DRMs, but the
BCh-bound cholesterol-enriched subpopulation will
provide an opportunity to elucidate the structure–
function relationship of lipid rafts in signal trans-

7
cells) were incubated with 10 lgÆmL
)1
BCh in NaCl ⁄ P
i
containing 1 mgÆ mL
)1
BSA (NaCl ⁄
P
i
⁄ BSA) for 5 min on ice, washed twice with NaCl ⁄ P
i
, and
incubated with 1% or 0.2% (v ⁄ v) Triton X-100 in TN buf-
fer (25 mm Tris ⁄ HCl pH 6.8, 150 mm NaCl) containing
2mm phenylmethanesulfonyl fluoride, 200 lm leupeptin,
25 lgÆmL
)1
aprotinin and phosphatase inhibitor cocktail
set II (Calbiochem) for 15 min on ice. Then the cells were
homogenized with a Potter–Elvehjem homogenizer, and the
homogenate was mixed with an equal volume of 80%
(w ⁄ v) sucrose and overlaid with 2.4 mL 35% (w ⁄ v) sucrose
and 1.3 mL 5% sucrose in TN buffer. The gradients were
centrifuged at 250 000 g for 18 h at 4 °C in a SW55 rotor.
After centrifugation, fractions (0.4 mL each) were collected
from the top.
Lipid extraction and lipid composition analysis
Total lipids in the detergent-insoluble membrane fraction
were extracted by the method of Bligh and Dyer [31] with

immunolabelled with a rabbit antibiotin IgG and protein-A
coupled to 10-nm colloidal gold particles as described [19].
After negative staining with 1% (w ⁄ v) uranyl acetate, raft
membranes were analysed in a JEM-1200EX electron
microscope (JEOL, Tokyo, Japan).
Western blotting
Proteins were separated by SDS ⁄ PAGE, transferred to an
Immobilon-P membrane and visualized using ECL plus
(Amersham Bioscience, Piscataway, NJ, USA).
Others
Proteins were analysed using a bicinchoninic acid protein
assay kit (Pierce, Rockford, IL, USA). Phosphorus assays
were performed by the method of Fiske and Subbarow [33].
Acknowledgements
We thank Dr H. Waki for technical advice and gifts of
authentic gangliosides. We thank Dr S. Iwashita for
critical reading of the manuscript and helpful discus-
sion. We thank Dr M.M. Dooley-Ohto for reading the
manuscript. This work was supported by a Grant-in-
Aid for Science Research from the Japan Society for
the Promotion of Science (to Y. O I.).
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