Tài liệu Báo cáo khoa học: Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction doc

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

membrane.
Several reports have suggested that lipid rafts are
platforms for signal transduction in T-cells [5]. Lipid
rafts obtained from resting T-cells are enriched in Src-
family kinases, Lck and Fyn [6,7], and the linker for
the activation of T-cells (LAT) [8]. Minor amounts of
CD3f are associated with rafts, but the data concern-
ing other T-cell receptor (TCR)⁄ CD3 constituents
remains contradictory [6,7]. In addition, the partition-
ing or recruitment of CD3e to lipid rafts after TCR
stimulation with antibodies remains uncertain [9,10].
These inconsistent results might be due mainly to the
different methods for isolating lipid rafts. Currently,
Keywords
raft; cholesterol; T-cell signalling;
perfringolysin O
Correspondence
Y. Shimada, Biomembrane Research Group,
Tokyo Metropolitan Institute of Gerontology,
35-2 Sakae-cho, Itabashi-ku, Tokyo 173-
0015, Japan
Fax: +81 3 3579 4776
Tel: +81 3 3964 3241 extn 3063, 3068
E-mail:
(Received 29 June 2005, revised 15 August
2005, accepted 24 August 2005)
doi:10.1111/j.1742-4658.2005.04938.x
We isolated a cholesterol-enriched membrane subpopulation from the
so-called lipid raft fractions of Jurkat T-cells by taking advantage of its
selective binding to a cholesterol-binding probe, BCh. The BCh-bound mem-

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-

scopic observation of GM1- and GM3-enriched rafts
has shown that raft-associated proteins are also distri-
buted asymmetrically in polarized cells [11]. Immuno-
electron microscopy of peripheral blood T-cells shows
distinct clustering and segregation of Lck and LAT on
the inner leaflet of the plasma membrane [12]. These
morphological analyses suggest the existence of raft
subsets. There are some biochemical approaches to
define the heterogeneity of raft-associated molecules by
immunoisolation, providing some information based
on protein–protein interactions in rafts [13–15]. How-
ever, the heterogeneity of lipid rafts is less well under-
stood in biochemical terms because the nature of lipid
rafts remains unclear. For the biochemical characteri-
zation of lipid rafts, a more sophisticated isolation
method, one based on criteria other than detergent
insolubility, is required.
To understand lipid-based raft domains, we have
focused on cholesterol as a major component of lipid
rafts. Previously, we designed the novel cholesterol
probes Ch and BCh [16,17] by modifying h-toxin (per-
fringolysin O), a cholesterol-binding, pore-forming
cytolysin produced by Clostridium perfringens [18]. Ch
and BCh are noncytolytic derivatives of h -toxin that
bind specifically and with high affinity to cholesterol in
membranes [17–20]. Ch is produced by the limited pro-
teolysis of h-toxin [16], and BCh by the biotinylation
of Ch [17]. Their binding to artificial membranes is
highly dependent on the cholesterol content of the
membranes: they bind to liposomes with high choles-

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.

These results suggest that the so-called raft fractions
consist of heterogeneous membrane groups, and that
the cholesterol-enriched membrane domain isolated by
BCh contains a core membrane domain for TCR signal
transduction.
Results
BCh binds to a subpopulation of membranes
in lipid raft fractions of Jurkat cells
Jurkat cells incubated with BCh were treated with 1%
(v ⁄ v) Triton X-100 solution for 15 min on ice and
homogenized. The homogenate was ultracentrifuged in
a sucrose density gradient and fractionated into 12
fractions from the top. Membrane-bound BCh was
predominantly detected in fractions three to five
(Fig. 1). These fractions correspond to one of the
peaks of cholesterol, and contain typical raft marker
proteins such as flotillin and Src-family kinases; thus
they are identified as lipid raft fractions (raft frac-
tions). BCh binding to membranes in the raft fractions
was detected by immunoelectron microscopy (Fig. 2).
BCh preferentially bound to some membrane vesicles,
but not all membranes in the raft fractions, suggesting
that raft fractions comprise at least two kinds of mem-
brane groups as evaluated by BCh binding.
Isolation of the BCh-bound membrane subpopu-
lation from raft fractions and its evaluation
We developed an isolation method for a raft subpopu-
lation that binds BCh. The raft fractions were prepared
from BCh-bound Jurkat cells and mixed with avidin-
magnet beads on ice. BCh is a biotinylated probe. Ves-

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

total cellular cholesterol. The cholesterol ⁄ phospholipid
(C ⁄ P) molar ratio of the BCh-bound membrane frac-
tion is approximately 1.0, which is much higher than
that ( 0.3) of the BCh-unbound membrane fraction
(bead-unbound fraction) (Table 1). This clearly indi-
cates that total raft fractions contain two distinctly dif-
ferent subpopulations of membranes with respect to
cholesterol enrichment.
Approximately 40% of total raft protein was recov-
ered in the BCh-bound membrane fraction (Table 1).
Silver staining shows that the BCh-bound membrane
fraction contains several distinctly different proteins
from the unbound membrane fraction (Fig. 3A). To
determine the protein profiles of raft subpopulations,
these two fractions were analysed by western blotting
(Fig. 3B,C). The raft marker protein flotillin was
recovered almost exclusively in the bead-bound frac-
tion, indicating that this molecule is predominantly
localized in BCh-bound membranes (Fig. 3B). The
majority of GM1 ganglioside, a raft marker lipid, is
also localized in BCh-bound membranes as judged by
the binding of cholera toxin (Fig. 3B, CTX).
It has been reported that several proteins participa-
ting in T-cell signalling are enriched in lipid rafts even
under nonstimulated conditions [9,23]. We found that
Src-family kinases (Lck and Fyn) and LAT recovered
in total raft fractions were also associated with
BCh-bound membranes (Fig. 3B,C). On the other hand,
neither Zap70 nor CD3f were detected in the BCh-
bound membrane fraction, but in the BCh-unbound

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

method contain at least two subpopulations of mem-
branes that are distinct from each other in their molecu-
lar components.
Triton X-100 concentration does not affect
the partitioning of signalling molecules into
BCh-bound membranes
It has been reported that molecular species and con-
tents recovered in raft fractions depend on detergent
concentrations. We examined the effect of Triton
A
B
C
Fig. 3. Molecular components in isolated BCh-bound and -unbound vesicles. Raft fractions were prepared from BCh-bound Jurkat cells as
described. The BCh-bound membrane fraction was retrieved with avidin-conjugated magnetic beads, and then the total raft fraction, BCh-
unbound membrane fraction and BCh-bound membrane fraction were subjected to SDS ⁄ PAGE. (A) Proteins were analysed by silver staining.
M, Molecular mass markers (kDa). Open and filled triangles show bands that differ between the BCh-bound and -unbound membrane frac-
tions, respectively. (B) Proteins were visualized by western blotting and probed with anti-(h-toxin) Ig or Igs against raft-associated molecules
(+BCh). In parallel experiments, raft fractions were prepared in the absence of BCh and subjected to fractionation with magnet-beads
(–BCh). (C) Blots were probed with antibodies against T-cell signalling-related molecules. The results are representative of seven independent
experiments.
Table 1. Comparison of cholesterol and phospholipid contents of raft fractions prepared with 1% or 0.2% (v ⁄ v) Triton X-100. Jurkat cells
(10
7
cells) were incubated with either 1% or 0.2% Triton X-100 and subjected to sucrose density gradient centrifugation as described in
Experimental Procedures. Raft fractions (total) were separated into BCh-bound (bound) and –unbound (unbound) membrane fractions. Lipids
were extracted by the method of Bligh and Dyer. Cholesterol, phospholipids, and proteins in 1 mL raft fractions (0.83 · 10
7
cells equivalent)
were determined. Data are means ± SD of values from three independent experiments.
1% Triton 0.2% Triton

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

recovered in total raft fractions prepared at lower Tri-
ton X-100 concentration, these molecules were found
exclusively in the BCh-unbound membrane fraction
regardless of Triton X-100 concentration (Fig. 4).
Thus, the Triton X-100 concentration did not affect
such characteristics of BCh-bound membranes as C ⁄ P
ratio and associated molecular species.
Lipid composition of membrane subpopulations
Lipid extracts from total raft fractions, and from
BCh-bound and -unbound membrane fractions were
analysed by TLC (Fig. 5A). In comparison with total
cell lipid extracts, the raft fractions were rich in choles-
terol and sphingomyelin (data not shown). The BCh-
bound membrane fraction contained cholesterol at a
level more than twofold that of the unbound fraction,
a finding consistent with its higher C ⁄ P ratio. It is
noteworthy that the PS ⁄ PI intensity ratio was remark-
ably different between the BCh-bound and -unbound
membranes, at ratios of 10 : 1 and 1 : 2, respectively.
In the former membranes, PS is a major component,
and PI is a minor one. This relationship is reversed in
the latter membranes. Gangliosides were also analysed
by TLC (Fig. 5B). The total raft fractions contained
GM1 and GM3 as the major components, with GM1
enriched in the BCh-bound membrane fraction pre-
pared with 1% (v ⁄ v) Triton X-100 (ratio of GM1 in
the BCh-bound membrane to that in the BCh-unbound
membrane ¼ 2 : 1). When prepared at lower (0.2%)
Triton X-100 concentrations, the amount of ganglio-
sides recovered in the BCh-unbound fraction increased,

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

type and concentration, DRMs have been assumed to
represent lipid rafts in biochemical aspects. But deter-
gent conditions is the most critical factor in influencing
the partitioning of molecules to raft fractions
[6,7,9,23,24]. To analyse the nature of lipid rafts, it is
necessary to establish a particular probe to isolate lipid
rafts regardless of the preparation conditions. In this
study, we tried to isolate a cholesterol-enriched
Fig. 4. Protein partitioning under different detergent conditions.
BCh-bound Jurkat cells were treated with either 1% Triton X-100 or
0.2% Triton X-100, and then raft fractions were obtained by
sucrose density gradient centrifugation as described. BCh-bound
vesicles were retrieved by avidin-magnetic beads. The total raft
fraction, BCh-unbound membrane fraction and BCh-bound mem-
brane fraction were subjected to SDS ⁄ PAGE. Blot membranes
were probed with anti-CD3e, anti-CD3f and anti-LAT Igs. The
results are representative of five independent experiments.
Two raft subsets in T-cells Y. Shimada et al.
5458 FEBS Journal 272 (2005) 5454–5463 ª 2005 FEBS
membrane domain from the so-called lipid raft fractions
using a cholesterol-binding probe, BCh. The choles-
terol-enriched membranes obtained from Jurkat cells by
our method contain particular proteins for T-cell signal-
ling in addition to so-called raft marker molecules. Gen-
erally, membranes are more resistant to lower detergent
concentrations. We examined the effect of detergent
concentration on the features of the cholesterol-enriched
membranes isolated by BCh. Increased amounts of pro-
teins and lipids were partitioned to the total raft frac-
tions prepared under lower detergent concentrations

(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

gests that our method for isolating a particular choles-
terol-enriched domain provides a useful tool for analy-
sing functional membrane domains concerned with
signal transduction.
Our study clearly shows that so-called lipid raft frac-
tions comprise two subpopulations that differ in
cholesterol content or cholesterol distribution. The two
membrane subpopulations separated by using BCh
each has a distinctive lipid composition. As expected,
the BCh-bound subpopulation comprises cholesterol-
rich membranes with a high C ⁄ P ratio of 1.0, and
characterized as PS-rich membranes containing GM1
and GM3. Because BCh was first bound to the cell
surface and then the cells were subjected to fraction-
ation, it is expected that the BCh-bound membrane
vesicles are derived from the plasma membrane.
Generally, PS distributes in the inner leaflet of plasma
membranes in mammalian cells. Although there is
insufficient information about the inner leaflet of lipid
microdomains, the inner leaflet of BCh-bound mem-
brane subdomains could have a PS-enriched environ-
ment. On the other hand, the BCh-unbound
membranes in raft fractions might be derived from any
of the following origins: PM-derived cholesterol-poor
membranes, intracellular cholesterol-poor membranes
or intracellular cholesterol-rich membranes. Because
the BCh-unbound membranes are cholesterol-poor on
average (C ⁄ P ¼0.3), intracellular cholesterol-rich
membranes are expected to comprise a minor popula-
tion, if any. To evaluate the contribution of intracellu-

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

colocalized with these Src-kinases, accumulating in the
BCh-bound membrane fraction. On the other hand,
CD3e, CD3f and Zap70, main components of the TCR
signalling initiation machinery, were mainly partitioned
to the BCh-unbound subpopulation, segregated from
other signalling molecules such as LAT, Lck and Fyn.
However, upon stimulation with the anti-CD3e Ig,
these molecules were recruited to the BCh-bound mem-
branes in raft fractions. The segregation from and
Fig. 6. Recruitment of signalling molecules to BCh-bound mem-
branes upon T-cell stimulation. Jurkat cells were either stimulated
with anti-CD3e for 10 min at 37 °C (+ anti-CD3e) or kept nonstimu-
lated without antibody addition (– anti-CD3e). Cells were then incu-
bated with 10 lgÆmL
)1
BCh in NaCl ⁄ P
i
⁄ BSA on ice, treated with
0.2% (v ⁄ v) Triton X-100 and subjected to sucrose density gradient
centrifugation as described. The raft fractions were collected and
separated into subfractions with avidin-magnetic beads. The total
raft fraction, and the BCh-unbound and BCh-bound membrane
fractions were analysed by western blotting. p-Zap70, Phospho-
Zap70. The results are representative of seven independent
experiments.
Two raft subsets in T-cells Y. Shimada et al.
5460 FEBS Journal 272 (2005) 5454–5463 ª 2005 FEBS
recruitment of these molecules to BC h-bound mem-
branes could contribute to the on ⁄ off switching of
TCR signalling. In addition to signalling machinery

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-

duction.
Experimental procedures
Materials
Anti-(h-toxin) serum was produced as described previously
[20]. Anti-PAG IgG was raised in rabbits using a synthetic
antigen peptide corresponding to the C-terminal 15 residues
(ESISDLQQGRDITRL) with a cysteine residue added to
the N terminus as a site for conjugation to a carrier protein.
Dynabeads M-280 conjugated with streptavidin were from
Dynal (Oslo, Norway). Jurkat cells were obtained
from ATCC. Anti-Lck, anti-Fyn and anti-CD3f IgGs were
from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-Zap70, anti-Csk, anti-moesin, anti-Grb2 and anti-(flo-
tillin-1) IgGs were from BD Bioscience (San Jose, CA,
USA). Anti-(phspho-Zap70) IgG was from Cell Signaling
Technology, Inc (Beverly, MA, USA). Anti-CD3e IgGs
were from Santa Cruz Biotechnology and R & D Systems,
Inc (Minneapolis, MN, USA). Anti-LAT IgG was from
Upstate Biotechnology (Lake Placid, NY, USA). Cholera
toxin B subunit-peroxidase conjugate was from Sigma
(St. Louis, MO, USA).
Preparation of BCh
h-Toxin was overexpressed in Escherichia coli and purified
from the periplasmic fractions using a DEAE–Sephacel col-
umn [30]. A nicked h-toxin (Ch) was obtained by limited
proteolysis with subtilisin Carlsberg [16]. BCh was prepared
from Ch as described previously [17].
Preparation of detergent-insoluble, low density
membrane fractions (raft fractions)
Jurkat cells (1 · 10

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

slight modification. Cholesterol was quantified by a Determi-
ner cholesterol assay kit (Kyowa Medex, Japan). For the
analysis of phospholipid compositions, 2D TLC was carried
out as follows. Samples were applied to an HPTLC plate
(Merck) at the lower left-hand corner. The plate was chroma-
tographed in the first dimension with chloroform ⁄ methanol ⁄
acetic acid ⁄ formic acid ⁄ water (35 : 15 : 6 : 2 : 1, v ⁄ v ⁄ v ⁄ v ⁄ v),
and then with hexane ⁄ diisopropyl ether ⁄ 80% phosphoric
acid (65 : 35 : 2, v ⁄ v ⁄ v) at the same direction. The third
chromatography was performed in the second dimension
with ethyl acetate ⁄ isopropanol ⁄ water (50 : 35 : 15, v ⁄ v ⁄ v) at
a rotation of 90 °C from the first direction. Phospholipids
were visualized by treatment with 3% cupric acetate ⁄ 8%
phosphoric acid solution [20]. For ganglioside analysis, the
upper phase from the Bligh–Dyer separation was applied to
a Bond Elute packed column equilibrated with chloro-
form ⁄ methanol ⁄ water (3 : 48 : 47, v ⁄ v ⁄ v). The column was
washed with excess distilled water, and the bound materials
Y. Shimada et al. Two raft subsets in T-cells
FEBS Journal 272 (2005) 5454–5463 ª 2005 FEBS 5461
were eluted with 3 mL methanol [32]. Sample solutions were
applied to an HPTLC plate and the plate was developed with
acetonitrile ⁄ isopropanol ⁄ 2.5 m ammonium hydroxide con-
taining 10 m m KCl (10 : 65 : 25, v ⁄ v ⁄ v). Gangliosides were
detected by resorcinol ⁄ hydrochloric acid reagent. Each spot
was scanned and quantified by the image analysis program
macscope.
Electron microscopy
The detergent-insoluble membrane fractions were prepared
from BCh-bound Jurkat cells, adsorbed to nickel grids, and

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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