From the Dipartimento di Fisiopatologia e Medicina Sperimentale, Università degli Studi di Siena, Viale Aldo Moro No. 1, 53100-Siena, Italy
Received for publication, October 22, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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The constitutive/inducible association of the T
cell receptor (TCR) with isolated detergent-resistant, lipid
raft-derived membranes has been studied in Jurkat T lymphocytes.
Membranes resistant to 1% Triton X-100 contained virtually no CD3 The plasma membranes
(PM)1 of many cell types
contain domains rich in cholesterol and sphingolipids, which have
come to be referred to as lipid rafts (1, 2). It is thought that rafts may form because of the segregation of their components from the bulk
of the glycerol-based phospholipid PM because of the orientation and
tight packing of the long, largely saturated acyl chains of the
sphingolipids. This phase separation of the membrane results in patches
of molecules, which form the rafts, existing in the liquid-ordered
phase (lo) but surrounded by and co-existing
with the phospholipids in the bulk PM that are in the liquid disordered phase (ld). In many cell types rafts are
organized into structurally distinct invaginations of the PM called
caveolae (3, 4). However in other types, including lymphocytes, the
rafts are thought to exist as islands of tightly packed sphingolipid
and cholesterol-based structures that, like caveolae, can be isolated
from the rest of the PM by purification methods based on their
detergent insolubility at low temperatures (5, 6). These PM domains
have been therefore called detergent-resistant membranes (DRMs),
detergent-insoluble glycolipid-enriched complexes, and Triton-insoluble
floating fractions (reviewed in Refs. 2 and 7).
The comparative analysis of the detergent-insoluble domains, caveolae,
and the bulk PM has provided an inventory of proteins apparently
residing in each (6, 8). However, doubts have been raised as to whether
the detergent treatment itself may modify the lipid rafts or
destabilize certain proteins resident therein (7). On the other hand, a
recent report (9) strongly suggests that use of different detergents
can result in different DRMs, which contain different proteins and
likely correspond to different cholesterol-based PM lipid rafts.
In T lymphocytes, many proteins involved in signal transduction have
been constitutively or inducibly recovered in DRMs (reviewed in Ref.
10). Among these are glycosylphosphatidylinositol-anchored proteins,
the Src family protein-tyrosine kinases Lck and Fyn (10, 11), the
transmembrane adapter protein linker for activation of T cells (12),
and a variety of co-stimulatory and co-receptor proteins (Refs. 10 and
13-16 and the references therein).
A large body of evidence supports a crucial role for rafts in the
signaling events activated by the T cell receptor (TCR) engagement (see
Ref. 17 for a recent review). Uncertainties, however, still exist
concerning the constitutive or inducible association of TCR to
rafts/DRMs, as well as to the mechanisms underlining the possible
recruitment of the receptor complex to rafts upon T cell stimulation.
Neither a constitutive nor an inducible (after treatment with
antibodies to CD3) association of the TCR/CD3 complex to DRMs was found
in Jurkat T cells (18, 19). No constitutive association to DRMs of
TCR Here we have preliminarily assessed a suitable experimental protocol to
prepare cholesterol-based DRMs in Jurkat T lymphocytes. Taking
advantage of this assessment, we have then investigated on the possible
constitutive/inducible association of TCR/CD3 with DRMs. As a main
result, we report that TCR/CD3 can be recruited to DRMs/rafts upon T
cell stimulation. Moreover, we provide evidence that clustering of
TCR/rafts can be a determinant of the recruitment, independently of
cell signaling activation.
Cells--
Jurkat cells, Jurkat-derived JCaM 1.6 cells
(purchased from the American Type Culture Collection, Manassas, VA) and
the Epstein-Barr virus transformed human B cells (EBV-B, kindly
supplied by Chiron-Biocine, Siena, Italy) were grown in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum
(Invitrogen), 2 mM L-glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin (Sigma). In the case of EBV-B
cells, 50 µM 2-mercaptoethanol was also included in the
culture medium. The cells were harvested 48-60 h after transplantation.
Cell Treatments--
To stimulate Jurkat cells with
antigen-presenting cells, 12.5 × 106 Jurkat cells (in
0.5 ml of RPMI 1640 at 37 °C) were mixed with 12.5 × 106 EBV-B cells (in 1 ml of RPMI 1640 at 37 °C), the
mixture was rapidly centrifuged at 400 × g, and
pelletted cells (in the presence of supernatant) were incubated for 30 min at 37 °C. Prior to adding to Jurkat T cell suspension, EBV-B
cells were preincubated (5 × 106/ml, in RPMI 1640)
with or without 1 µg/ml of staphylococcal enterotoxin E (SEE) for
1.5 h at 37 °C and then washed twice with RPMI 1640 to remove
free SEE. Phytohemagglutinin (PHA) stimulation was performed by
treating cells with 10 µg/ml of the lectin in RPMI 1640 supplemented with 0.1% fetal calf serum for 30 min at 37 °C. Treatment with anti-CD3 antibodies was performed by incubating cells (10 × 106/ml, in RPMI 1640 supplemented with 0.1% bovine serum
albumin) in the presence of 5 µg/ml of the anti-CD3 antibody, TR66,
for 5 min at 37 °C. To cross-link CD3-TR66 complexes (18),
TR66-treated cells (washed and resuspended in fresh RPMI 1640) were
subsequently treated (10 min at 37 °C) with an antibody to TR66
(anti-mouse IgG, 8 µg of protein/ml). To induce GM1
cross-linking, cells (10 × 106/ml in RPMI 1640 supplemented with 0.1% bovine serum albumin) were treated with cholera
toxin B subunit (CTB; 0.1 µg/ml) for 1 min at 37 °C, washed twice
with RPMI 1640, and subsequently treated with an anti-CTB antibody for
5 min at 37 °C.
Preparation of Detergent-resistant Membrane Fractions
(DRMs)--
Cells (25 × 106) were washed twice
with ice-cold RPMI 1640 and homogenized in 1.5 ml of ice-cold MBS (0.15 M NaCl, 25 mM Mes, pH 6.5) containing the
detergent (i.e. 1% Triton X-100, 0.2% Triton X-100, or 1%
Brij 58) and a mixture of protease inhibitors (1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride).
The homogenates were incubated for 1 h on ice under gentle
shacking and then centrifuged for 5 min at 400 × g to
remove nuclei and debris. The supernatants were then adjusted to 45% sucrose by the addition of an equal volume of 90% sucrose/MBS, placed
in the bottom of ultracentrifuge tubes, and overlaid with 5 ml of 35%
sucrose and 4 ml of 5% sucrose (24). The gradients were centrifuged at
187,000 × g in a SW41 rotor (Beckman) for 20 h at
4 °C. Ten fractions (1 ml each) were collected from the top of the
gradients (fractions 1-10), and the residual volume of the centrifuge
tube (1.5-1.7 ml) was recovered as fraction 11. The protein content of
fractions was determined by a modified Lowry assay (Bio-Rad). Aliquots
(50-100 µl) of the sucrose gradient fractions were withdrawn to
measure light scattering and cholesterol content (see below). Because
fractions 9-11 contained the bulk of solubilized cell materials, they
were subsequently pooled for futher analysis. The proteins contained in
fractions 1-8, as well as in the pooled fractions 9-11, were
recovered by trichloroacetic acid/deoxycholic acid precipitation
as reported in Ref. 25. The proteins were then dissolved in SDS-PAGE
buffer, and half (fractions 1-8) or 1/12 (pooled fractions
9-11) of the solutions were loaded onto 5-15% gradient
polyacrylamide gels and blotted onto nitrocellulose. The immunoblots
were probed with the different antibodies and analyzed by enhanced
chemiluminescence (Amersham Biosciences). Scanning densitometry was
performed within the linear range of preflashed x-ray film with a
Bio-Rad VERSADOC mod.1000 imaging densitometer.
Tyrosine Phosphorylation Assay--
The cells were lysed for 30 min at 4°C in 1% Nonidet P-40 buffer (20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM
MgCl2, 1 mM EGTA) in the presence of protease
and phosphatase inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin,
1 mM phenylmethylsulfonyl fluoride, 50 mM NaF,
10 mM Na4P2O7, and 1 mM NaVO4). The samples were centrifuged (at
13,000 × g for 10 min) and postnuclear supernatants
were subjected to SDS-PAGE and anti-phosphotyrosine immunoblotting.
Light Scattering Assay--
Aliquots (0.1 ml) of the sucrose
gradient fractions (see above) were diluted with 1.9 ml of 10 mM K-Pipes, pH 7.0, containing 1 mM EGTA. Light
scattering intensity of each fraction was measured at 400 nm at right
angles to the incoming light beam (whose intensity was 80% reduced
with the aid of a grid) using a fluorimeter (Perkin-Elmer model
650-10S) equipped with a temperature-controlled cuvette holder
(22 °C) (26, 27).
Cholesterol Determination--
The cholesterol content of
sucrose gradient fractions was measured enzymatically, essentially as
reported in Ref. 28. Briefly, 50-100 µl of the fractions were
reacted (for 30 min at 37 °C in the dark) in 1.5 ml of KPi buffer
(0.1 M, pH 7.4) containing 2 mM sodium cholate,
0.66 mg/ml of p-hydroxyphenilacetic acid, 0.1 UI/ml of
cholesterol oxidase, and 1 UI/ml of horseradish peroxidase. Parallel
samples without cholesterol oxidase were also run as blanks. The final
product of the coupled reactions, oxidized
p-hydroxyphenilacetic acid derivative, was measured
fluorimetrically (excitation and emission wavelengths, 325 and 415 nm, respectively).
[Ca2+]i Measurements--
The cells were
loaded with fura-2 (acetoxymethyl ester), and cytosolic free
Ca2+ concentration ([Ca2+]i) was
measured as described in Ref. 29. To minimize the leakage of
intracellular fura-2, the assay temperature was 30 °C, and 200 µM sulfinpyrazone was included in the medium (29).
Microscopical Analysis--
Cells suspensions (2 × 106 cell/ml in serum-free RPMI 1640) were treated with 0.15 µM BODIPY FL-labeled C5-ganglioside for 2 min at
22 °C. The cells were rapidly harvested by centrifuging at 1000 × g for 15 s, resuspended in 0.1 ml of serum-free RPMI 1640, placed on a coverslip, and immediately observed with a real time
confocal microscope (Bio-Rad DCV 250 mounted on a Nikon Eclipse 300 inverted microscope). The images were acquired with a cooled CCD camera
(Princeton Inst.) and a Metamorph® imaging system.
Materials--
Triton X-100, PHA, polyclonal antibodies to
cholera toxin B subunit and horseradish peroxidase (type 4A) were
obtained from Sigma. Brij 58 was obtained from Fluka.
4-Amino-5-(4-cholrophenyl)-7-(t-butyl)pirazolo[3,4-d]pyrimidine (PP2)
and cholesterol oxidase were from Calbiochem. Fura-2 (acetoxymethyl ester) and BODIPY FL-labeled C5-ganglioside GM1 were from
Molecular Probes. Polyclonal antibodies to CD3 Characterization of DRMs Prepared under Different Detergent
Solubilization Conditions--
A classic method to prepare DRMs is
based on the disruption of cells with 1% Triton X-100 at 0-4 °C.
However, alternative detergents/conditions have been also used to
prepare DRMs, particularly in lymphocytes (13, 20, 22, 23, 30-37). As
a prelude to examining the constitutive or inducible association of
CD3/TCR to DRMs, we have characterized DRMs prepared from
(unstimulated) Jurkat cells treated with 1% Triton X-100, 0.2% Triton
X-100, or 1% Brij 58 at 0-4 °C and separated by sucrose gradient
ultra centrifugation. Fig. 1 shows the
distribution of particulate membranes (evaluated by light scattering),
total proteins, and cholesterol content across the density gradient in
the three experimental conditions of membrane solubilization.
Particulate membranes were largely recovered in fractions 4 and 5 (Fig.
1A) in all cases, indicating that these fractions contain
DRMs. Actually fractions 4 and 5 contained an opaque band, which
equilibrated by flotation at 10-25% sucrose (data not shown),
independently of the detergent treatment employed. A relatively high
protein (Fig. 1B) and cholesterol (Fig. 1C)
content was also found in fractions 4 and 5. Notably, in the cases of
both 0.2% Triton X-100 and 1% Brij 58, the amount of protein and
cholesterol in the DRM-containing fractions was significantly higher
than in the case of cell solubilization with 1% Triton X-100 (Fig. 1,
B and C). Also the content in particulate membranes was apparently higher in DRMs prepared from cells treated with the lower concentration of Triton X-100 or Brij 58. However, light-scattering intensity may be influenced by factors other than the
concentration of membranes, such as, for example, the size of the
membrane particles (26, 27). The fact that not only the protein but
also the cholesterol content is higher in DRMs prepared with 0.2%
Triton X-100 or Brij 58, indicates that these DRMs can be considered
cholesterol-based and can be regarded as raft-derived. Consistently,
either the transferrin receptor or CD45 proteins, which are assumed to
be located in the conventional lipid environment of the PM (22, 36,
38), were found to be virtually undetectable in the DRM-containing
fractions (Fig. 1D). On the other hand, the Lck protein that
is known to be largely localized in DRMs from Jurkat cells (10, 11) was
found to be associated with the DRM-containing fractions at a very
similar extent in all of the solubilization protocols employed (Fig.
1D).
A Minor Portion of TCR/CD3 Is Constitutively Associated
with 0.2% Triton X-100- and Brij 58-resistant Membranes--
In the
case of cell membrane solubilization with 1% Triton X-100, virtually
no CD3 Recruitment of TCR/CD3 to 0.2% Triton X-100 and Brij
58-resistant Membranes--
To investigate whether or not TCR/CD3 can
be dynamically recruited to DRMs as a consequence of T cell
stimulation, Jurkat cells have been treated with the mitogenic lectin
PHA or EBV-B cells prepulsed with SEE as a model for antigen-presenting
cells. Both treatments did result in a marked increase in the amount of
CD3 Recruitment of TCR/CD3 to 0.2% Triton X-100-resistant
Membranes Does Not Require Activation of Cell
Signaling--
Challenging T cells with antigen-presenting cells or
PHA results in the ligation of TCR and of a variety of co-stimulatory proteins as well. A downstream key event is the activation of the Src
kinase Lck, which in turn causes tyrosine phosphorylation and
Ca2+ signaling. To investigate on the role of cell
signaling in the recruitment of TCR/CD3 to DRMs, we employed the
Jurkat-derived cell line JCaM 1.6, which lacks the activity of the Lck
tyrosine kinase (39). As shown in Fig.
4A, PHA treatment of JCaM 1.6 cells resulted in a recruitment of the CD3
Src kinases other than Lck, such as Fyn, may also be involved in T cell
signaling (40-42). In further experiments, we therefore investigated
whether or not the CD3
In the experiments shown in Figs. 3-5, the cells were stimulated with
PHA for 30 min (see "Experimental Procedures"). However, comparable
amounts of CD3
In all of the experimental conditions described above, no
immunodetectable CD3 Antibody-mediated Multiple Cross-linking of GM1 and CD3
Can Determine the Recruitment of TCR/CD3 to 0.2% Triton
X-100-resistant Membranes--
It is well known that clusters of TCR
are formed at the site of contact between T cells and
antigen-presenting cells (44), as well as on the PM surface of
lectin-treated T cells (45). It also has been shown that cross-linking
of either the raft component GM1 or TCR results in the
co-clustering of the ganglioside and the receptor (18). Therefore, in
subsequent experiments, we investigated whether or not the
cross-linking of either GM1 or CD3 can induce the
recruitment of TCR/CD3 to DRMs.
To induce GM1 cross-linking, the cells were treated with
CTB as a ligand for GM1, and then the GM1-CTB
complex was cross-linked with an antibody to CTB (18, 46). This
treatment resulted in a marked increase in the amount of the CD3
Cross-linking of TCR/CD3 was performed by treating the cells with the
antibody to CD3, TR66, and subsequently with antibodies to TR66. As can
be seen in Fig. 6B, cross-linking of CD3 resulted in a
marked increase in the amount of the CD3
In the two experimental conditions as above, virtually no
immunodetectable CD3 Recruitment of TCR/CD3 to 0.2% Triton X-100-resistant
Membranes Is Associated with Clustering of PM Rafts--
In a final
set of experiments, we investigated whether or not the recruitment of
CD3 to DRMs (resistant to 0.2% Triton X-100) is paralleled by lipid
raft clustering by microscopical observation of PM rafts probed with a
fluorescent analogue of GM1. Indeed, previous microscopic
observations have shown that fluorescent GM1 probes
uniformly label the PM of resting (unstimulated) Jurkat cells but
selectively stain PM patches in cells treated with cross-linking antibodies to GM1 or CD3 (18). The logical explanation is
that the GM1 analogue inserts in PM lipid rafts; lipid
rafts in resting cells, however, are too small (
As demonstrated in Fig. 7, clusters of
rafts were formed in the experimental conditions, in which we observed
the recruitment of the CD3 Although previous microscopical evidence suggest that the TCR is
present in PM rafts (18), a variety of biochemical studies gave
conflicting results with respect to the constitutive/inducible association of the receptor with DRMs (18-23). The present data show
that a relatively low amount of the TCR component CD3 These data were gained by using 0.2% Triton X-100 or 1% Brij 58 to
solubilize "conventional" nonraft membranes. On the other hand, we
observed that DRMs prepared with the "classic" concentration of
Triton X-100, i.e. 1%, did not contain any detectable CD3
amount. It could be argued that 1% Triton X-100, but not the less
hydrophobic detergent Brij 56 or a lower concentration of Triton X-100
itself, simply extracts CD3/TCR from rafts. However, solubilization
with either Brij 58 or the lower concentration of Triton X-100 also resulted in a higher recovery of membranes as well as of cholesterol and proteins in the DRM-containing fractions. Therefore, a logical explanation is that the CD3/TCR complex is contained in a subset of
cholesterol-enriched membranes that are not resistant to 1% Triton
X-100 but are resistant to a lower Triton X-100 concentration or to
Brij 58. The idea that heterogeneity in cholesterol-based DRMs and/or
PM raft domains exists is not unprecedented. For example, the
co-existence within a membrane domain, such as the apical plasma
membrane, of different cholesterol-based lipid rafts has recently been
proposed (9). Moreover, evidence for structural diversity of the PM
domains occupied by functionally different glycosylphosphatidylinositol-anchored proteins has been previously forwarded (49).
It should be noted that a variety of previous studies on the
association of signaling proteins to rafts/DRMs in T cells has been
based on the use of detergents other that Triton X-100 (13, 14, 20, 22,
23, 31-35) or of Triton X-100 concentrations lower than 1% (14, 30,
36, 37). The aim of these studies, however, was not related to the
possible heterogeneity in DRMs/rafts; presumably, the more
convenient/efficient detergent type/concentration was merely used. With
respect to the previous conflicting results on the association of TCR
with DRMs/rafts, the use of different solubilization protocols, in
addition to other experimental differences such as the T cell type
investigated, may account for by the discrepancies. For example, no CD3
has been found in DRMs by using 1% Triton X-100 (19), whereas some CD3
was recovered in membranes resistant to a lower concentration (0.5%)
of Triton X-100 (36), as well as to 1% Brij 58 (35). While this work
was being completed, evidence for the constitutive association of TCR
to a subset of DRMs, prepared with 1% Brij 98 at 37 °C, was
reported (23).
The recruitment of TCR to PM rafts may favor a role for these domains
as platforms coordinating activation/polarization of signaling
pathways. It is known that TCR is recruited to the site of contact
between T cells and antigen-presenting cells (so-called immunosynapse)
(43, 50), as well as to the capped PM regions after treatment with
mitogenic lectins (45). It is possible, therefore, that these polarized
PM regions contain raft domains including the recruited TCR. Indeed,
both at the immunosynapse level (51) and in the capped PM regions
(present study), the raft marker GM1 is concentrated. On
the other hand, the direct activation of TCR signaling by antibodies to
receptor components (e.g. OKT3 or TR66) can promote
(unpolarized) signaling events, which are apparently independent of the
recruitment of CD3/TCR to rafts (Fig. 6B). This is
consistent with our very recent data (52) showing that cell protein
phosphorylation and Ca2+ signaling, induced by direct
stimulation of the TCR, are not inhibited by T cell raft disassembly.
Cell signaling events, such as protein phosphorylation,
Ca2+ mobilization/influx, cytoskeleton rearrangement, and
phosphorylation of TCR components, might be necessary determinants for
the recruitment of CD3/TCR to DRMs. The present data, however, indicate
that the recruitment of CD3/TCR to DRMs can occur in the absence of
signaling events, such as increase in tyrosine phosphorylation and
Ca2+ mobilization/influx. Moreover, we did not observe any
CD3 recruitment in Jurkat cells stimulated with the anti-CD3 antibody
TR66 (Fig. 6B), a treatment that induces a marked increase
in tyrosine phosphorylation and cytosolic free Ca2+ levels
(52).
On the other hand, multiple cross-linking of either the TCR or of the
raft component GM1 can result in a recruitment of the receptor to DRMs/rafts. This is the case of the exposure of cells pretreated with the anti-CD3 antibody TR66 to antibodies to TR66 (Fig.
6B). Clustering of GM1 (by treating Jurkat cells
with CTB and anti-CTB antibodies) also resulted in an evident
recruitment of CD3/TCR to DRMs (Fig. 6A). As mentioned
above, GM1 and TCR clusters have been shown to be present
both in the PM caps induced by PHA treatment (Ref. 44; see also Fig. 7)
and in the PM of the T cells at the immunosynapse level (43, 50, 51).
Moreover, GM1 clusters can be formed independently of cell
signaling activation (Fig. 7), which is consistent with previous
observation by others in JCaM 1.6 cells (46).
The fact that multiple cross-linking of TCR results in raft coalescence
and in recruitment of the receptor to DRMs/rafts is consistent with a
variety of previous results discussed in Ref. 7. For example, it has
been proposed (7) that clustering of a protein that has an affinity for
rafts could either cause small, dispersed rafts containing the protein
to coalesce into larger rafts or increase the overall raft affinity of
the protein cluster enough to recruit it to rafts. The fact that
multiple cross-linking of GM1 results not only in raft
coalescence but also in the attendant recruitment of the (unligated)
receptor to DRMs/rafts is consistent with the previous observation that GM1 clustering results in co-clustering of CD3/TCR (18). A
clear mechanistic reason for this phenomenon cannot be presently
forwarded; one could argue that if CD3 is loosely associated with lipid
rafts, then the aggregation of small rafts into larger ones increases and stabilizes them in the raft domain, making them more resistant to
detergent extraction. We should also consider that the overall picture
is likely more complex, because of the heterogeneity in the
physicochemical structure of rafts. In addition, several co-stimulatory molecules in the T cell PM have been reported to become dynamically associated with DRMs/rafts upon multiple cross-linking of the component
itself as well as of other (raft) components. Examples are CD2 (15),
CD26 (53), and CD28/GM1 (48).
In any event, the multiple cross-linking of molecules in the PM of the
T cell facing the PM of the antigen-presenting cell may result
per se in a local recruitment of the TCR to raft
structures/clusters. This mechanism, however, does not exclude the
participation of (subsequent) cell signaling events in the formation
and/or in dynamic evolution of the immunosynapse. Consistently, it has
been observed that in the immunosynapse some protein distribution
patterns may arise directly from the physicochemical properties of
molecules bound to ligands on an opposing cell membrane (54), although synapse formation also requires participation of the actin cytoskeleton and signaling from the initial pool of engaged TCR (54-56).
,
part of the TCR complex, irrespective of cell stimulation. On the other hand, membranes resistant either to a lower Triton X-100 concentration (i.e. 0.2%) or to the less hydrophobic detergent Brij 58 (1%) contained (i) a low CD3
amount (approximate 2.7% of total) in resting cells and (ii) a several times higher amount of the TCR component, after T cell stimulation with either antigen-presenting cells or with phytohemagglutinin. It appeared that CD3/TCR was constitutively associated with and recruited to a raft-derived membrane
subset because (i) all three membrane preparations contained a similar
amount of the raft marker tyrosine kinase Lck but no detectable amounts
of the conventional membrane markers, CD45 phosphatase and transferrin
receptor; (ii) a larger amount of particulate membranes were resistant
to solubilization with 0.2% Triton X-100 and Brij 58 than to
solubilization with 1% Triton X-100; and (iii) higher cholesterol
levels were present in membranes resistant to either the lower Triton
X-100 concentration or to Brij 58, as compared with those resistant to
1% Triton X-100. The recruitment of CD3 to the raft-derived membrane
subset appeared (i) to occur independently of cell signaling events,
such as protein-tyrosine phosphorylation and Ca2+
mobilization/influx, and (ii) to be associated with
clustering of plasma membrane rafts induced by multiple cross-linking
of either TCR or the raft component, ganglioside
GM1. We suggest that during T cell stimulation a
lateral reorganization of rafts into polarized larger domains can
determine the recruitment of TCR into these domains, which favors a
polarization of the signaling cascade.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
has been also found both in Th1 and Th2 lymphocytes,
whereas TCR
was recruited to DRMs only in Th1 cells challenged with
antigen-presenting cells (20). In a murine T cell hybrydoma, it has
been demonstrated that the TCR complex is excluded from DRM domain
before and after TCR stimulation, although a portion of the TCR
component appeared to be constitutively associated with DRMs (21).
Another report (22) has shown the recruitment of TCR/CD3 to DRMs, which
is dependent on both receptor engagement and the activity of Src family
kinases. While this work was being completed, evidence was been
provided for the constitutive presence of a minor portion of TCR/CD3 in
a subset of DRMs prepared from splenic and thymic T lymphocytes (23).
On the other hand, microscopical evidence has been provided for the
recruitment of TCR to PM rafts as a consequence of raft clustering
(18).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and monoclonal
antibodies to Lck and CD45 were obtained from Santa Cruz Biotechnology.
Monoclonal antibodies to transferrin receptor and the
anti-phosphotyrosine fragment, directly conjugated with horseradish
peroxidase (RC20:HRP), were from BD Transduction Laboratories. SEE was
obtained from Toxin Technology. Anti-CD3 (clone TR66) and CTB were a
gift from Chiron-Biocine (Siena, Italy). All other chemicals were of
analytical grade.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of DRMs prepared under
different detergent solubilization conditions. Jurkat cells
(25 × 106 cells) were lysed in a medium containing
1% Triton X-100, 0.2% Triton X-100, or 1% Brij 58, the lysates were
then fractionated by sucrose density centrifugation, and 11 fractions
were collected from the top of the gradients as described under
"Experimental Procedures." Light-scattering intensity
(A), protein (B), and cholesterol content
(C) of each fraction were determined as detailed under
"Experimental Procedures." D, proteins derived from
12.5 × 106 or 2 × 106 Jurkat cells,
in the case of fractions 3-8 or the solubilized materials (pooled
fractions 9-11), respectively, were analyzed by SDS-PAGE and Western
blotting and probed with antibodies recognizing the indicated proteins;
the positions of molecular mass markers (in kilodaltons) are shown;
fractions 1 and 2, which showed no immunoreactivity, are not shown for
clarity. In B, protein levels represented by
continuous and dotted lines correspond to the
wider and smaller abscissa scales, respectively.
In A-C data are the means ± S.E. of four to six
different experiments. In D a representative experiment of
three is shown. TX-100, Triton X-100; Ab,
antibody.
protein was detected in DRMs (Fig.
2A), which is in agreement
with previous observations (19). However, a minor portion of total
CD3
protein was immunorevealed in DRMs prepared from Jurkat cells
upon solubilization with 0.2% Triton X-100 and Brij 58 (Fig. 2). The
percentages of the CD3
protein in DRM-containing fractions
(fractions 4 and 5) were 2.8 ± 0.4 and 2.7 ± 0.4 (mean ± S.E.), in the case of cell solubilization with 0.2% Triton X-100
and 1% Brij 58, respectively. It was previously observed that cell
membrane solubilization with 1% Brij 58 at 0-4 °C (22) or with 1%
Brij 98 at 37 °C (23) resulted in the recovery of a portion of cell
CD3/TCR in DRMs.
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Fig. 2.
Associations of CD3
with DRMs prepared by different detergent treatments of resting
Jurkat cells. The cells (25 × 106 cells) were
lysed in a medium containing 1% Triton X-100, 0.2% Triton X-100, or
1% Brij 58, and the lysates were then fractionated by sucrose density
centrifugation as described under "Experimental Procedures."
A, proteins derived from 12.5 × 106 or
2 × 106 Jurkat cells, in the case of fractions 3-8
or the solubilized materials (pooled fractions 9-11), respectively,
were analyzed by SDS-PAGE and Western blotting and probed with
antibodies recognizing the CD3
protein; fractions 1 and 2, which
showed no immunoreactivity, are not shown for clarity. B,
Western blots like those shown in A were quantified using
scanning densitometry. Normalized densitometry data are presented as
the percentages of total intensity, and they represent the means ± S.E. of four different experiments; the values for the pooled
fractions 9-11 are not shown for clarity. TX-100, Triton
X-100; Ab, antibody.
protein associated with DRMs prepared with 0.2% Triton X-100
(compare Fig. 3 with Fig. 2). The
percentages of the CD3
protein present in DRMs of cells stimulated
with PHA or SEE-pulsed EBV-B cells (fractions 4 and 5) were 18.7 ± 2.2 and 12.6 ± 0.9 (means ± S.E.). The amount of CD3
protein recovered in DRMs of Jurkat cells treated with control EBV-B
cells (without SEE treatment) was very similar to that of resting
Jurkat cells (compare Fig. 3 with Fig. 2). Experiments with Brij 58, which were performed in the case of PHA stimulation (not shown), gave
analogous results; the percentage of CD3
protein associated with
DRMs (fraction 4 + fraction 5) was 19.7 ± 1.7 (mean ± S.E.,
n = 4). In both above stimulatory conditions, virtually
no immunodetectable CD3
protein was found in DRMs prepared by cell
membrane solubilization with 1% Triton X-100 (data not shown).
View larger version (18K):
[in a new window]
Fig. 3.
Recruitment of CD3
to DRMs in Jurkat cells stimulated with SEE-prepulsed EBV-B cells
or with PHA. Jurkat cells were treated with control EBV-B cells,
SEE-prepulsed EBV-B cells or PHA, and lysed in a medium containing
0.2% Triton X-100, and the lysates were then fractionated by sucrose
density centrifugation as described under "Experimental
Procedures." A, proteins derived from 12.5 × 106 or 2 × 106 Jurkat cells, in the case
of fractions 3-8 or the solubilized materials (pooled fractions
9-11), respectively, were analyzed by SDS-PAGE and Western blotting
and probed with antibodies recognizing the C CD3
protein.
B, Western blots like those shown in A were
quantified using scanning densitometry. Normalized densitometry data
are presented as the percentages of total intensity, and they represent
the means ± S.E. of three to five different experiments; the
values for the pooled fractions 9-11 are not shown for clarity.
Ab, antibody.
protein to DRMs (prepared with 0.2% Triton X-100), at an extent that was comparable with that
observed in PHA-treated Jurkat cells (Fig. 3A). As expected, PHA stimulation caused virtually no increase in tyrosine
phosphorylation and Ca2+ signaling in JCaM 1.6 cells (Fig.
4, B and C), whereas it resulted in a robust
increase in tyrosine phosphorylation and Ca2+ signaling in
Jurkat cells (Fig. 4, B and C). Ca2+
signaling was presumably due to both mobilization of cell
Ca2+ stores and influx of extracellular Ca2+,
because it was evaluated in the presence of (1 mM)
extracellular Ca2+ (29).
View larger version (22K):
[in a new window]
Fig. 4.
Recruitment of CD3
to DRMs (A), cell protein-tyrosine
phosphorylation (B), and Ca2+ signaling
(C) in JCaM 1.6 cells stimulated with PHA.
A, JCaM 1.6 cells were treated with PHA and lysed in a
medium containing 0.2% Triton X-100, and the lysates were then
fractionated by sucrose density centrifugation, as described under
"Experimental Procedures." proteins derived from 12.5 × 106 or 2 × 106 cells, in the case of
fractions 3-8 or the solubilized materials (pooled fractions 9-11),
respectively, were analyzed by SDS-PAGE and Western blotting and probed
with antibodies recognizing the CD3
protein. B, total
lysated of JCaM 1.6 and Jurkat cells were analyzed by SDS-PAGE and
Western blotting and probed using an anti-phosphotyrosine antibody, as
described under "Experimental procedures"; the positions of
molecular mass markers (in kilodaltons) are shown. C,
variations in [Ca2+]i induced by PHA addition (10 µg/ml) to JCaM 1.6 and Jurkat cells were measured as described under
"Experimental Procedures." Ab, antibody.
protein is recruited to DRMs obtained from
Jurkat cells pretreated with the selective inhibitor of Src kinases,
PP2 (43). As shown in Fig. 5A,
an evident PHA-induced recruitment of the CD3
protein to DRMs was
also present in PP2-treated Jurkat cells. As expected, PHA stimulation
caused little or no increase in tyrosine phosphorylation and
Ca2+ signaling in PP2-treated cells (Fig. 5, B
and C). In addition, the CD3
protein was recruited to
DRMs irrespective of its phosphorylation status. Indeed, the CD3
protein associated with DRMs (after PHA stimulation) was apparently not
phosphorylated in PP2-treated cells, whereas it was phosphorylated in
PHA-treated control cells (Fig. 5A).
View larger version (38K):
[in a new window]
Fig. 5.
Recruitment of CD3
to DRMs (A), cell protein-tyrosine
phosphorylation (B), and Ca2+ signaling
(C) in Jurkat cells pretreated with the Src kinase
inhibitor PP2 and stimulated with PHA. A, cells were
pretreated with 10 µM PP2 (for 10 min at 37 °C),
stimulated with PHA, and lysed in a medium containing 0.2% Triton
X-100 (also including 50 mM NaF, 10 mM
Na4P2O7 and 1 mM
NaVO4, to allow phosphotyrosine detection), and the lysates
were then fractionated by sucrose density centrifugation, as described
under "Experimental Procedures"; proteins derived from 12.5 × 106 or 2 × 106 Jurkat cells, in the case
of fractions 3-8 or the solubilized materials (pooled fractions
9-11), respectively, were analyzed by SDS-PAGE and Western blotting;
the blot membranes were probed with an anti-phosphotyrosine antibody
(antibody to PY) and then reprobed with antibodies to the CD3
protein. B, total lysated of Jurkat cells were analyzed by
SDS-PAGE and Western blotting and probed using an anti-phosphotyrosine
antibody, as described under "Experimental Procedures"; the
positions of molecular mass markers (in kilodaltons) are shown.
C, variations in [Ca2+]i induced by
PHA addition (10 µg/ml) to PP2-pretreated Jurkat cells were measured
as described under "Experimental Procedures." Ab,
antibody.
protein were found associated with DRMs either in
control Jurkat cells or in PP2-treated Jurkat and JCaM 1.6 cells, at
later times of PHA treatment (60-90 min) of PHA stimulation (data not
shown). This suggests that the stability of the association of CD3/TCR
with DRMs over time does not require cell signaling activation, at
least in the case of PHA stimulation.
protein was found in DRMs prepared with 1% Triton X-100 (data not shown).
protein associated with DRMs (Fig.
6A). In the cells treated with
CTB alone, the amount of the CD3
protein associated with DRMs was
comparable with that observed in control (unstimulated) cells (Fig.
2A).
View larger version (33K):
[in a new window]
Fig. 6.
Multiple cross-linking of GM1
(A) or CD3 (B) results in the
recruitment of CD3 to DRMs. A,
Jurkat cells were pretreated with the GM1 ligand, CTB, and
then treated with or without antibodies to CTB to cross-link
GM1/CTB complexes as described under "Experimental
Procedures." B, Jurkat cells were pretreated with the
antibody to CD3, TR66, and then treated with or without antibodies to
TR66, as described under "Experimental Procedures." The cells were
lysed in a medium containing 0.2% Triton X-100, and the lysates were
then fractionated by sucrose density centrifugation, as described under
"Experimental Procedures"; proteins derived from 12.5 × 106 or 2 × 106 cells, in the case of
fractions 3-8 or the solubilized materials (pooled fractions 9-11),
respectively, were analyzed by SDS-PAGE and Western blotting and probed
with antibodies recognizing the CD3
protein. Ab,
antibody
protein associated with
DRMs. In the cells treated with TR66 alone, the amount of the CD3
protein present in DRMs was comparable with that observed in control
(unstimulated) cells (Fig. 2A).
protein was found in DRMs prepared with 1% Triton X-100 (data not shown).
70 nm in
diameter (4-6)) to be resolvable by light microscopy, whereas their
aggregates (patches) are resolvable by light microscopy (18, 46,
47).
protein to DRMs. This appeared to be the
case independently of activation of cell signaling. Indeed, PHA
treatment of JCaM 1.6 or PP2-pretreated Jurkat cells resulted in both
the recruitment of CD3
protein to DRMs (Figs. 4 and 5) and raft
clustering (Fig. 7) but in no evident activation of tyrosine
phosphorylation and Ca2+ signaling (Figs. 4 and 5). On the
other hand, treating Jurkat cells with the antibody to CD3, TR66,
caused neither the recruitment of the CD3
protein to DRMs (Fig.
6A) nor clustering of PM rafts (Fig. 7). Instead, as
expected on the basis of previous reports (22, 48), TR66 stimulation
caused a marked increase in tyrosine phosphorylation and
Ca2+ signaling also in the present experimental conditions
(data not shown).
View larger version (27K):
[in a new window]
Fig. 7.
Clustering of PM rafts upon treatment with
different stimulators and/or inhibitors in Jurkat and JCaM 1.6 cells. The cells were treated with the different compounds as
indicated in the legend to Figs. 3-6. After treatments, the cells were
labeled with 0.15 µM BODIPY FL-labeled C5-ganglioside
(for 2 min at 22 °C), rapidly harvested by centrifugation, placed on
a coverslip, and immediately observed with a real time confocal
microscope, as detailed under "Experimental Procedures."
Ab, antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is constitutively associated with a DRM subset and that this amount can be
largely increased as a result of T cell stimulation in a cell
signaling-independent manner.
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ACKNOWLEDGEMENT |
---|
We are grateful to Antonella Viola for helpful discussion.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Italian Ministry of Instruction, University and Scientific Research Cofin 2001 (to A. B.), University of Siena Progetto Giovani Ricercatori (to E. G.), and the Italian Space Agency (to A. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Leukocyte Biology Section, Biomedical Sciences
Division, Imperial College, School of Medicine, London, UK.
§ To whom correspondence should be addressed. Tel.: 39-0577-234021; Fax: 39-0577-234009; E-mail: benedetti@unisi.it.
Published, JBC Papers in Press, December 22, 2002, DOI 10.1074/jbc.M210758200
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ABBREVIATIONS |
---|
The abbreviations used are: PM, plasma membrane; TCR, T cell receptor; DRM, detergent-resistant membrane; Lck, lymphocyte-specific protein-tyrosine kinase; PP2, 4-amino-5-(4-cholrophenyl)-7-(t-butyl)pirazolo[3,4-d]pyrimidine; SEE, staphylococcal enterotoxin E; CTB, cholera toxin B subunit; PHA, phytohemagglutinin; Mes, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid.
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