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,
Departments of
*
Pathology,
Medicine, and
Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain (TCR-) homodimer. This results in the membrane
targeting of another PTK, ZAP-70, to phospho-ITAMs via Src homology 2
domain interactions, and its subsequent activation by Lck. ZAP-70 then
phosphorylates several substrates, including the linker for activation
of T cells (LAT). Phospho-LAT acts as a docking molecule for a variety
of enzymes and adapter proteins, such as Vav, phospholipase C
,
phosphatidylinositol-3 kinase (85 kDa subunit), and Grb2
(6). This cascade is required for efficient activation of
second messenger pathways, including calcium mobilization and the
Ras/mitogen-activated protein kinase pathway
(7). In contrast, the spatial-temporal organization of these enzymatic events is less well understood. For instance, the lateral organization of membrane associated proteins such as the TCR/CD3 complex, PTKs, and adaptor molecules within the lipid bilayer is only beginning to be elucidated (8, 9, 10, 11, 12, 13, 14, 15, 16). The plasma membrane contains lipid assemblies enriched in cholesterol and glycosphingolipids, known as lipid rafts, which form distinct microenvironments for preferential enrichment and exclusion of certain molecules (17). Critical signaling molecules of the TCR/CD3 complex such as Src family PTKs (18), LAT (19), phosphoinositides (20), CD4 (21, 22), and the recently cloned phosphoprotein associated with glycosphingolipid-enriched microdomains/Cbp (23, 24) are targeted to lipid rafts. In the transformed Jurkat T cell line, disruption of rafts by cholesterol depletion reduces calcium mobilization (22, 25, 26). Therefore, it has been postulated that these microdomains are required for signal transduction in T cells.
Most of the studies in humans have been limited to the Jurkat T
leukemia cell line. Therefore, we examined the peripheral blood T cell
(PBT), which more closely reflects a physiological response. In this
study, we report that the integrity of lipid rafts containing the
kinase Lck is essential for the initiation of PBT signal transduction
through the TCR. Furthermore, while both Lck and LAT are targeted to
lipid rafts, we demonstrate heterogeneity in raft composition in the
native plasma membrane of PBT, causing the physical sequestration of
these critical signaling proteins in a resting T cell. However, after
activation, both Lck and LAT coinhabit a localized region 
50–100 nm
within the plasma membrane.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated from healthy donors by Ficoll-Hypaque density
separation. T lymphoblasts were prepared by PHA (0.5%) stimulation for
48 h in the presence of IL-2 (20 U/ml; Chiron, Emeryville, CA) in
RPMI 1640, 10% heat-inactivated FCS, and 2.5% HEPES, and thereafter
carried in IL-2 (20 U/ml) for >8 days to obtain a population of
CD4+CD45RO+ peripheral
blood T lymphoblasts. The following Abs were used: rabbit
anti-LAT, rabbit anti-Lck, and 3A5 mouse anti-Lck mAb,
mouse anti-ZAP-70 (Upstate Biotechnology, Lake Placid, NY),
anti-phosphotyrosine (PY20; BD Transduction Laboratories,
Lexington, KY), rabbit anti-CD4 and HRP-conjugated secondary Abs
(Santa Cruz Biotechnology, Santa Cruz, CA), and mouse TCR- (Zymed
Laboratories, San Francisco, CA), rabbit anti-phospho-ZAP-70
(Y319), and rabbit anti-phospho-Lck (Y394; Cell Signaling
Technology, Beverly, MA). Methyl 
cyclodextrin (MCD) and
poly-L-lysine were purchased from Sigma-Aldrich (St. Louis,
MO). Lysis buffer for lipid raft preparation and immunoisolation
consisted of 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, protease
and phosphatase inhibitor mixtures (Sigma-Aldrich), 1 mM PMSF, and 1%
Brij 58 (Pierce, Rockford, IL). Colloidal gold-conjugated secondary
Abs, 12 nm anti-mouse, and 6 nm anti-rabbit were purchased from
Jackson ImmunoResearch Laboratories (West Grove, PA).
T cell stimulation
PBT, rested overnight in the absence of IL-2 (RPMI 1640, 10%
FCS, 2.5% HEPES) at 37°C, were resuspended (5 x
107 cells/ml) in RPMI 1640, 2.5% HEPES, and a
protease inhibitor mixture with or without 10 mM MCD and incubated
at 37°C for 5 min. Cells were then stimulated via OKT3 Ab (10
µg/ml; Ortho Diagnostics, Raritan, NJ) with cross-linking (sheep
anti-mouse F(ab')2, 10 µg/ml;
Sigma-Aldrich) for 5 min at 37°C, immediately followed by the
addition of 2x Laemmli sample buffer and boiled for 5 min before
Western blot analysis. Unstimulated cells received only the sheep
anti-mouse F(ab')2.
LAT immunoisolation
Rabbit anti-LAT was incubated for 5 h at 4°C with M-280 superparamagnetic polystyrene beads (Dynal Biotech, Lake Success, NY) to which sheep anti-rabbit Abs are covalently attached to the surface. PBT were stimulated as described above, except that reactions were quenched by immediately pelleting the cells at 4°C and resuspending them in 0.5 ml ice-cold lysis buffer. Cells were lysed at 4°C on a rotating wheel for 30 min, after which the postnuclear supernatant was added to the beads bearing rabbit anti-LAT and incubated at 4°C for 90 min with gentle rotation. The M-280 beads were then separated from the cell lysate by magnetic isolation on an MPC-S magnet (Dynal Biotech), and washed four times in ice-cold lysis buffer. After the final wash, the beads were boiled in 50 µl of 1x Laemmli buffer.
Lipid raft fractionation
PBT were resuspended in 0.5 ml ice-cold lysis buffer and lysed at 4°C on a rotating wheel for 30 min. Postnuclear supernatants were gently mixed with an equal volume of cold 85% sucrose (w/v) in lysis buffer without detergent and placed in the bottom of a 4-ml ultracentrifuge tube (Sorvall TST-60.4; Newtown, CT). The sample was overlaid with 2 ml 35% sucrose and 1.2 ml 5% sucrose, all at 4°C in lysis buffer without detergent. Equilibrium centrifugation was performed at 200,000 x g, 4°C, for at least 3 h, as described (27). Fractions were collected as 0.4-ml samples from the top. A fixed volume from each fraction of the sucrose gradient (0.015 ml) was mixed with an equal volume of 2x Laemmli buffer and boiled for 8 min before Western blotting.
Western blotting
Proteins were separated by SDS-PAGE on a 10% gel under reducing conditions and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA) in a transfer buffer consisting of 20 mM Tris-HCl, 150 mM glycine, and 20% methanol. Membranes were incubated at 4°C overnight in blocking buffer (5% nonfat milk, 0.1% Tween 20 in PBS). Primary and secondary Abs were diluted as recommended by the manufacturer in blocking buffer and incubated with the membranes for 1 h at room temperature with six washes in between. Detection of HRP-conjugated Abs was performed using SuperSignal (Pierce). Phosphotyrosine analysis was performed as follows. Membranes were blocked for 1 h at room temperature in blocking buffer (3% BSA, 10 mM Tris (pH 7.2), 100 mM NaCl, 1% Tween 20). HRP-conjugated PY20 Ab was diluted 1/2500 in blocking buffer and incubated with the membranes for 1 h at room temperature. Chemiluminescence of all membranes was detected using Hyperfilm ECL (Amersham, Piscataway, NJ).
Transmission electron microscopy (TEM)
PBT were either stimulated via OKT3 Ab (10 µg/ml) with cross-linking sheep anti-mouse (10 µg/ml) for 5 min at 37°C, as described above, or incubated with only the sheep anti-mouse F(ab')2 at 37°C. Reactions were stopped by placing the samples on ice. The stimulating Abs were then washed away and cells were resuspended in RPMI 1640 at 4°C. Plasma membrane sheets were prepared as described by Sanan and Anderson (28) and Wilson et al. (29). PBT were settled onto poly-L-lysine-coated glass coverslips at 4°C for 45 min. The coverslips were then inverted onto nickel electron microscopy grids that had been previously floated on poly-L-lysine, washed in dH2O, and air-dried. Light finger pressure was distributed over a 15-mm rubber cork placed on the coverslip for 10 s. The grid was plucked off, allowing the apical portion of the adherent PBT plasma membrane to remain attached to the grid and leaving the rest of the cell on the coverslip. The grid containing the membrane sheet was immediately washed in 25 mM HEPES (pH 7.2), 25 mM KCl, and 2.5 mM magnesium acetate for 5 s, to remove any cytosol and contaminants, before fixation in 2% paraformaldehyde for 10 min, followed by three 5-min washes in PBS. The grids were then blocked in 0.5% BSA-c (Electron Microscopy Sciences, Fort Washington, PA) in PBS for 30 min. To prevent cross-reactivity with any mouse OKT3 that may still be attached to the membrane, all samples (including controls) were incubated for 30 min in goat anti-mouse IgG Fab fragments (Jackson ImmunoResearch Laboratories). Controls showed that this completely prevented secondary colloidal gold-conjugated Abs from binding any available OKT3 that may be present in the preparation. Samples were washed for 30 min in 0.5% BSA-c/PBS, with three exchanges between all Ab labeling steps. Primary Ab labeling of LAT (1:100 of rabbit anti-human LAT) and Lck (1:100 of mouse 3A5 anti-human Lck) were performed sequentially, with secondary colloidal gold-conjugated Abs immediately following their respective primary Ab. All Ab incubations were performed in the presence of 0.5% BSA-c/PBS at room temperature for 30 min. Following the final washing steps, samples were fixed overnight in 2% glutaraldehyde/PBS at 4°C. Samples were washed three times in PBS before a 10-min staining in 1% aqueous osmium tetroxide, followed by five 5-min washes in dH2O. Samples were subsequently processed in 1% aqueous tannic acid for 10 min, followed by two 5-min washes in dH2O, and then 10 min in 2% aqueous uranyl acetate, followed by two 1-min washes in dH2O. Samples were air-dried before analysis on a JEOL 1200CX TEM (Peabody, MA). Gold particle distributions were quantified as previously described (29). Singlet and clusters of either 6 or 12 nm gold particles were counted over 40–100 µm2 of membrane in both resting and activated T cells and scored for colocalization or not.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We hypothesized that if lipid rafts were essential for signaling
through the TCR in PBTs, disruption of raft integrity via cholesterol
extraction with MCD would inhibit the very earliest events in
TCR-initiated signal transduction. Resting PBT exhibit a very low level
of basal protein tyrosine phosphorylation (Fig. 1
, lane 1). Upon addition of
10 mM MCD to PBT for 10 min at 37°C, there is no induction of
tyrosine phosphorylation (Fig. 1, lane 2). As expected, when
PBT are stimulated through the TCR via CD3 cross-linking for 5 min at
37°C, there is a pronounced increase in total protein tyrosine
phosphorylation (Fig. 1, lane 3). This robust induction of
the membrane proximal signaling cascade is strongly inhibited when the
PBT are treated with 10 mM MCD for 5 min at 37°C, before the 5-min
stimulation (Fig. 1, lane 4). To confirm that the heavily
phosphorylated protein in the 36–38 kDa range was in fact LAT, we
immunoisolated LAT from control and TCR-stimulated cell lysates.
Conventional protein A-agarose was not a viable option for this
experiment, because the stimulating OKT3 Ab is capable of binding
protein A (A. E. Schade and A. D. Levine,
unpublished results), leading to erroneous conclusions. Therefore, we
used an immunomagnetic approach in which sheep anti-rabbit Abs are
covalently attached to the surface of superparamagnetic polystyrene
beads and then rabbit anti-LAT was incubated with the beads.
Lysates from TCR-stimulated cells or controls were incubated with the
immunomagnetic beads and the bound LAT proteins were extracted by
magnetic isolation and analyzed via SDS-PAGE and Western blotting
initially for total phosphotyrosine and subsequently for LAT (Fig. 2). When PBT are stimulated through the
TCR via CD3 cross-linking for 5 min at 37°C, there is a pronounced
increase in LAT tyrosine phosphorylation (Fig. 2, lane 2).
However, phosphorylation of LAT is strongly inhibited when the PBT are
treated with 10 mM MCD for 5 min at 37°C, before the 5-min
stimulation (Fig. 2, lane 4). These results demonstrate that
disrupting lipid rafts in PBT inhibits membrane proximal signal
transduction through the TCR, consistent with the reduction of calcium
flux, a signaling event downstream from the tyrosine phosphorylation
(22, 25, 26).
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Recognizing the crucial role of the PTK Lck in the initiation of
TCR signaling and the essential function of LAT in linking the TCR
proximal signaling with the more generalized secondary signaling
cascades, we assessed the membrane partitioning of Lck and LAT via
biochemical fractionation after lysing PBT in 1% Brij-58 (Fig. 3). Both Lck and LAT are enriched in
detergent-resistant membranes (DRMs), which represent the microdomains
of the native plasma membrane (rafts) exhibiting liquid-ordered phase
behavior. This property is in large part responsible for rendering
these lipid rafts resistant to nonionic detergent disruption and
therefore, capable of being isolated as low density membrane vesicles.
As shown in the leftmost panel of Fig. 3, in PBT, both Lck and LAT are
indeed associated with lipid rafts, although not to the same extent.
Although Lck is distributed between lipid rafts and the more prevalent
nonraft plasma membrane, LAT is exclusively partitioned to lipid rafts.
Upon TCR stimulation (Fig. 3, third panel), both proteins
remain raft-associated. However, when PBT are treated with 10 mM MCD
under the same conditions that inhibit the induction of immediate early
protein tyrosine phosphorylation, there is a prominent redistribution
in the mass of Lck from lipid rafts to the nonraft plasma membrane.
This change in the raft association for Lck is the same in both
unstimulated and stimulated cells (Fig. 3, second and
fourth panels). Approximately 80% of raft-associated Lck is
displaced from rafts after cholesterol extraction (Table I). In contrast, >50% of LAT
remains associated with lipid rafts in the presence of MCD (Fig. 3, Table I). Importantly, to prevent any systematic error in quantitating
the immunoblots, Ab staining for both proteins was performed on the
same nitrocellulose membrane. The partitioning of both Lck and LAT to
lipid rafts was previously proposed to be essential for successful
activation and propagation of TCR signal transduction (19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40). However, this is the first report to link the
inhibition of membrane proximal TCR signaling with the concurrent loss
of Lck from lipid rafts in PBT. Furthermore, the relative lack of LAT
redistribution after cholesterol depletion, weighed against an almost
complete loss of Lck from lipid rafts, suggests that these proteins
associate with biochemically distinct lipid raft microdomains in the
plasma membrane, a concept we call heterogeneity.
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Although we attribute the loss of proximal TCR signaling to
dissolution of the Lck containing lipid rafts, the significant decline
in total cellular protein and LAT phosphorylation upon MCD treatment
may also be due to an event downstream of Lck. Therefore, we examined
the tyrosine phosphorylation profile of Lck and ZAP-70, key PTKs whose
activation precedes, and is necessary for, LAT phosphorylation (Fig. 4). After TCR engagement, one the
earliest identifiable events of Lck activation is phosphorylation at
tyrosine 394, leading to a conformational change in the catalytic
domain that substantially increases kinase activity
(41, 42, 43). Using an Ab that recognizes phospho-Y394
activated Lck, we demonstrate that Lck is prominently activated upon
TCR cross-linking for 5 min at 37°C (Fig. 4A, lane
3). Dramatic inhibition (18-fold reduction normalized to total Lck
protein) of stimulation-induced Lck kinase phosphorylation (Fig. 4A, lane 4) is observed after cholesterol
extraction under the same conditions that lead to a global decrease in
protein tyrosine phosphorylation, a decrease in LAT phosphorylation,
and significant displacement of Lck from lipid rafts.
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B,
lane 3). Pretreatment with MCD produces a 9-fold
reduction in phospho-ZAP-70 after stimulation via TCR cross-linking
(Fig. 4B, lane 4). Because Lck activation is
necessary for ZAP-70 activation, and ZAP-70 activation is proximal to
and required for LAT phosphorylation, it is unlikely that the reduction
in LAT phosphorylation results from a portion of that protein being
redistributed to the nonraft membrane, and is more likely due to an
inhibition in the upstream signaling cascade.
Rafts containing CD4 and TCR- are both sensitive to cholesterol
extraction
To determine whether the association of LAT with an
MCD-resistant raft is unique and to further explore lipid raft
heterogeneity in the plasma membrane, we examined the sensitivity to
MCD treatment of two additional transmembrane proteins, CD4 and
TCR-, involved in the initial events of TCR signal transduction.
Approximately 30% of CD4 is raft-associated in resting PBT (Fig. 5, upper panel), and upon
treatment with 10 mM MCD, there is a 70% loss of CD4 from rafts
(Fig. 5, upper panel). TCR- is an essential component on
the TCR/CD3 signaling complex, yet its association with lipid rafts in
PBT has not been reported. In this study, we show that slightly >30%
of TCR- associates with lipid rafts and upon cholesterol extraction
is completely displaced (Fig. 5, lower panel). These
findings confirm that several TCR signaling proteins reside in
MCD-sensitive rafts in the native plasma membrane, while LAT is
sequestered into distinct membrane microdomains. The significance of
raft-associated CD4 and TCR- proteins, compared with the more
pronounced nonraft populations of these molecules, is currently under
investigation.
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To directly demonstrate the presence of lipid raft heterogeneity
within the native plasma membrane, we examined the inner leaflet of the
PBT membrane using TEM at a resolution not approachable with
conventional optical microscopy. Using immunogold-conjugated Abs to
detect Lck (12 nm colloidal gold) and LAT (6 nm colloidal gold), we
demonstrate for the first time that these proteins of the TCR signaling
cascade associate with mutually exclusive microdomains in the plasma
membrane of a resting PBT (Fig. 6). We
consistently observe a strong agreement between the TEM analysis of
native membranes and biochemical fractionation of DRMs, predicting the
partitioning of Lck and LAT between the raft and nonraft domains of the
T cell plasma membrane. For instance, Lck fractionates into both the
detergent-resistant (raft) and detergent-soluble (nonraft) membrane
domains after density gradient ultracentrifugation. Similarly,
systematic analysis of transmission electron photomicrographs reveals
that Lck (Fig. 6, squares) partitions both in a clustered distribution
in osmiophilic (lipid rich) regions of the native membrane (75% of 12
nm particles in 105.5 µm2 of membrane) and to a
lesser extent, in a more diffuse random pattern (25% of 12 nm
particles in 105.5 µm2 of membrane). In
contrast, LAT (Fig. 6, circles) is predominately clustered in
microdomains of the native plasma membrane (>92% of 6 nm particles in
85.5 µm2 of membrane).
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We hypothesize that the heterogeneity of lipid rafts regulates T
cell activation and predict that after TCR engagement, relevant
signaling molecules must colocalize. To document this redistribution,
we analyzed membrane sheets from PBT that had been stimulated through
the TCR/CD3 complex. Within 5 min of cross-linking the TCR, there is a
pronounced shift in the membrane distribution of Lck and LAT relative
to one another (Fig. 7). Upon
stimulation, some of the dispersed heterogeneous clusters sequestering
Lck or LAT converge into common domains (39% of 12-nm labeled Lck and
46% of 6-nm labeled LAT in 42.7 µm2 of
membrane), characterized as osmiophilic membrane patches from 50 to 100
nm, which are likely to facilitate initiation of signal transduction
(Fig. 7, hexagons).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, raft-bound LAT is not affected by cholesterol extraction
to the same extent as Lck in the same population of cells. One possible
difference is that Lck is membrane-associated via posttranslational
acyl modifications and LAT is a transmembrane protein that is
palmitoylated on membrane proximal cytoplasmic cysteine
residues. It is conceivable that a more stable association of the
integral membrane protein LAT with the plasma membrane confers this
greater resistance to the perturbing effects of cholesterol depletion.
To address this possibility, we analyzed the membrane
compartmentalization of two additional transmembrane proteins involved
in the earliest aspects of signaling through the TCR/CD3 complex, CD4
and TCR-. Both proteins associate with lipid rafts and both exhibit
a sensitivity to cholesterol extraction similar to that of Lck,
suggesting that the relative affinity of a protein for association with
lipid rafts is not simply dependent on being either a peripheral or
integral membrane protein. We consider the unique biophysical
properties of the LAT-associated rafts (that is, the relative
resistance to cholesterol extraction) to be evidence that distinct
heterogeneous populations of lipid rafts exist in the plasma membrane
of PBT, and this may be another mechanism by which the T cell regulates
the initiation of signal transduction.
TEM is a powerful tool to characterize the distribution and composition
of microdomains within the native plasma membrane of T cells. This is
significant because optical techniques that lack the resolving power of
electron microscopy, such as confocal microscopy, have failed to
distinguish between spatially distinct regions for Lck and LAT
compartmentalization, potentially leading to the erroneous conclusion
that these proteins are localized to a common microdomain. The
identification of distinct clusters of Lck and LAT proteins at
nanometer resolution in resting PBT membranes not exposed to detergents
provides a strong confirmation of the biochemical data that lipid raft
heterogeneity exists in vivo. Similarly, studies in resting RBL-2H3
mast cells have reported that Thy-1 does not colocalize with Lyn or the
Fc
RI (29) and that LAT rarely colocalizes with FcRI
(46). Furthermore, TCR stimulation-induced reorganization
of the membrane microdomains, resulting in Lck and LAT residing in
common domains, suggests a functional role for these specialized
membrane compartments in T cell activation.
The physiological role of lipid raft heterogeneity in T cells remains to be uncovered, but we hypothesize that maintaining signaling molecules in distinct heterogeneous microdomains within the plasma membrane is a mechanism by which T cells orchestrate a rapid, controlled, and dynamic response to Ag exposure. In simple terms, lipid raft heterogeneity isolates an enzyme in lipid raft A from a substrate in lipid raft B in the nonactivated T cell. Upon TCR engagement, changes in the organization of the plasma membrane and its constituents, possibly mediated by the cytoskeleton, facilitate the rearrangement and redistribution of raft-associated proteins, thereby eliminating previous spatial constraints, thus catalyzing the initiation of the enzymatic cascade. In further support of this concept, Gomez-Mouton et al. (47) suggest that protein redistribution in polarized T cells involves raft partitioning. Heterogeneity in the composition, cholesterol-dependence, and localization of lipid rafts in the native membrane of the T cell may be a mechanism by which discrete signaling pathways can be differentially activated during an immune response, thereby modulating cytokine production patterns and T cell proliferation (48). Changes in the lateral mobility of enzymes, substrates, and adaptor proteins within the plasma membrane can regulate these functional differences within an activated T cell, eliciting dramatically different immunological effector functions.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alan D. Levine, Department of Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4952. E-mail address: adl4{at}po.cwru.edu
3 Abbreviations used in this paper: PTK, protein tyrosine kinase; DRM, detergent-resistant membrane; ITAM, immunoreceptor tyrosine activation motif; LAT, linker for activation of T cells; MCD, methyl cyclodextrin; PBT, peripheral blood T cell; TCR-, TCR- chain; TEM, transmission electron microscopy.
Received for publication October 9, 2001. Accepted for publication December 31, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and the Ras pathway. Immunity 9:617.[Medline]
-cyclodextrin does not affect cell surface transport of a GPI-anchored protein. J. Biol. Chem. 274:34459.RI signaling from the inside of the mast cell membrane. J. Cell Biol. 149:1131.1 and Ras activation. EMBO J. 18:1832.[Medline]
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