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Research Article |
1 Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, London, WC2A
3PX, UK
2 Sackler Institute for Muscular Skeletal Research, Department of Medicine,
University College London, 5 University Street, London, WC1E 6JJ, UK
* Author for correspondence (e-mail: hogg{at}icrf.icnet.uk
Accepted 4 December 2001
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Summary |
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 Top Summary IntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1, and the clustering of 4ß1 is also raft dependent.
Both LFA-1 and 4ß1-mediated adhesion is dependent upon intact
lipid rafts providing proof of the functional relevance of the lipid raft
localisation. Finally we find that non-raft integrins are excluded from the
rafts by cytoskeletal constraints. The presence of integrin in lipid rafts
under stimulating conditions that activate these receptors strongly indicates
that the rafts have a key role in positively regulating integrin activity.
Key words: Integrin, Lipid rafts, Cytoskeleton
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Introduction |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß heterodimeric transmembrane receptors that
mediate cell adhesion (Harris et al.,
2000
; Plow et al.,
2000) and they can exist in several functional states. An increase
in ligand binding activity can be brought about either by conformational
changes that lead to higher affinity forms of the integrin or by lateral
association of integrins into clusters on the plasma membrane, which increases
ligand binding avidity by providing multiple contact sites
(Stewart and Hogg, 1996;
van Kooyk and Figdor, 2000).
The cytoplasmic domains of integrins associate with the cytoskeleton
(Giancotti and Ruoslahti,
1999), and these interactions alter during the course of adhesion.
Cytoskeletal interactions can regulate the clustering of integrins, such as
lymphocyte function-associated antigen 1 (LFA-1)
(Stewart et al., 1998;
van Kooyk et al., 1999).
Ligand binding to integrins results in signals being transmitted into the
cell for which the target pathways are being identified, particularly in
mesenchymal cells (Fashena and Thomas,
2000). It is becoming more certain that integrins on leukocytes
can also signal and one outcome of integrinmediated signalling is the altered
activity of other integrins, a process termed `integrin cross talk'
(Blystone et al., 1994;
Porter and Hogg, 1997). Thus
one subset of integrins can operate to regulate either positively
(Chan et al., 2000;
Leitinger and Hogg, 2000a;
Pacifici et al., 1994;
Porter and Hogg, 1997;
Weerasinghe et al., 1998) or
negatively (Blystone et al.,
1994; Diaz-Gonzalez et al.,
1996; Porter and Hogg,
1997) a second set of integrins on the same cell membrane. The
molecular mechanism of integrin crosstalk is currently not well understood
and, at present, only two kinases have been reported to play a role
(Blystone et al., 1999;
Pacifici et al., 1994).
The integrin I domain contained in the subunit is the principal
ligand binding site of those integrins, including LFA-1, which possess it
(reviewed by Leitinger and Hogg,
2000b). When activation of such integrins occurs, the conformation
and positioning of the I domain alters. Recently, we have removed the I domain
from LFA-1 and expressed the resulting integrin (
I-LFA-1) in Jurkat T
cells (Leitinger and Hogg,
2000a). I-LFA-1 is unable to bind ligand ICAM-1, but has
features of an active integrin in that it exhibits LFA-1 activation-dependent
mAb epitopes. A key feature of T cells expressing I-LFA-1, compared
with T cells expressing wild-type (wt) LFA-1, is that the ß1 integrins,
4ß1 and 5ß1, show increased binding activity to
ligands VCAM-1 and fibronectin. This crosstalk between integrins is associated
with increased clustering of the ß1 integrins and is dependent on an
intact cytoskeleton.
It is increasingly recognised that the lipid bilayer of the plasma membrane
is composed of different subdomains and the cholesterol- and sphingolipid-rich
microdomains known as lipid rafts have attracted much recent interest
(Brown and London, 2000;
Cherukuri et al., 2001;
Simons and Ikonen, 1997).
These lipid domains are platforms for cellular signalling, particularly as
defined for T cells and other leukocytes
(Guo et al., 2000;
Janes et al., 1999;
Montixi et al., 1998;
Viola et al., 1999;
Xavier et al., 1998). Proteins
with glycosyl phosphatidylinositol (GPI)-anchors and many dually acylated
cytoplasmic proteins are enriched in the lipid rafts
(Brown and London, 2000).
Although the rafts are generally deficient in transmembrane proteins, several
such proteins are raft associated potentially through receptor oligomerisation
(Cherukuri et al., 2001). Early
studies suggested that integrins were not localised to lipid rafts
(Fra et al., 1994), but
recently integrins have been found to be raft associated
(Green et al., 1999;
Krauss and Altevogt, 1999;
Skubitz et al., 2000).
However, the relevance of this association with regard to function remains to
be understood.
We demonstrate here a correlation between LFA-1 activity and lipid raft
localisation. In addition, the presence of active LFA-1 in lipid rafts
promotes the movement of 4ß1 integrin to the rafts. Furthermore,
adhesion mediated by LFA-1 or 4ß1/5ß1 and the
increased clustering of activated 4ß1 are all dependent on intact
lipid rafts. Finally we show that inactive integrins, LFA-1 and
4ß1, are tethered away from lipid rafts by cytoskeletal
restraints.
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Materials and Methods |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 mAb 7.2R (CD49d) and anti-DAF mAb 67 (CD55) were prepared by
the ICRF Antibody Production Service. The ß2-integrin activating mAb KIM
185 was a gift from M. Robinson, (Celltech, Slough, UK). Anti-LFA-1 mAb G25.2
(CD11a) was purchased from Becton Dickinson (Oxford, UK) and anti-human
transferrin receptor mAb (CD71) from Roche Diagnostics (Lewes, UK).
Cytochalasin D and methyl-ß-cyclodextrin, cholesterol and fatty acid free
BSA were from Sigma (Poole, UK). Latrunculin A was a gift from R. Treisman,
ICRF.
Cell lines and cell culture
The generation of the human T lymphoma Jurkat cell lines stably expressing
wt LFA-1 or I-LFA-1 has been described
(Leitinger and Hogg, 2000a).
Cells were maintained in RPMI 1640 medium containing 10% FCS (Life
Technologies, Paisley, UK) supplemented with 250 µg/ml Zeocin (Invitrogen,
Leek, The Netherlands). Human T cells were prepared and cultured as previously
(Porter and Hogg, 1997;
Stewart et al., 1998).
Fluorescence microscopy and treatment of cells Raft patching
Lipid raft aggregation or patching was performed according to Janes et al.
(Janes et al., 1999). Aliquots
of 1x106 cells (in 100 µl) were labelled in RPMI 1640
medium with 10 µg/ml TRITC-conjugated cholera toxin B (List Biological
Laboratories, Quadratech, Epsom, UK), which binds to the ganglioside GM1 on
the cell surface, for 30 minutes on ice. After three washes, cells were
incubated with rabbit anti-cholera toxin IgG (Sigma; 1/150 in PBS with 0.2%
BSA) for 30 minutes on ice, followed by a 20 minute incubation at 37°C.
After three washes, cells were fixed in 1% paraformaldehyde for 30 minutes on
ice and stained with anti-integrin mAbs 7.2R or G25.2 (at 10 µg/ml),
anti-human transferrin receptor mAb (at 20 µg/ml) or anti-DAF mAb 67 (at 10
µg/ml), followed by Alexa 488-conjugated goat anti-mouse IgG, (Molecular
Probes, Eugene, OR) at 10 µg/ml for 30 minutes on ice. After three washes,
cells were attached to poly-L-lysine-coated 13 mm round glass coverslips,
fixed in 3% formaldehyde in PBS, and mounted onto slides in Mowiol
(Calbiochem, Nottingham, UK) dissolved in Citifluor antifade solution (UKC
Chemical Laboratory, Canterbury, UK).
Methyl-ß-cyclodextrin treatment
Cells were preincubated with 10 mM (final concentration)
methyl-ß-cyclodextrin (MßCD) in RPMI 1640 for 30 minutes at
37°C. Aliquots of 1x106 cells were then rapidly chilled
and incubated with mAb 7.2R at 10 µg/ml for 30 minutes on ice, then washed
three times in PBS. To prevent antibody-induced clusters, cells were fixed in
1% paraformaldehyde in PBS for 20 minutes on ice before a second incubation
with Alexa 488-conjugated goat anti-mouse IgG, as described above. Viability
of the cells was tested with trypan blue exclusion. No significant cell death
occurred due to cholesterol extraction.
Incubation with integrin activating agonists
Cells were incubated with a final concentration of either 0.5 mM
Mn2+ or 100 nM phorbol 12,13-dibutyrate (PdBu) in 20 mM Hepes, 140
mM NaCl, 2 mg/ml glucose, pH 7.4 for 30 minutes at 37°C. Aliquots of
1x106 cells were then rapidly chilled and incubated with 10
µg/ml TRITC-conjugated cholera toxin B and processed as described above
(see Raft patching).
Confocal microscopy
Fluorescence was analysed using a Zeiss LSM 510 confocal laser scanning
microscope equipped with a 63x, numerical aperture 1.4 objective. Single
channel fluorescence was analysed with an argon laser (wavelength 488 nm). For
double channel fluorescence imaging a second helium neon laser (wavelength 543
nm) was used. Cell surface distribution was evaluated taking horizontal
optical sections at 0.35 µm vertical steps throughout the whole height of
representative cells or at mid section through the cells. Images of optical
sections (512x512 pixels) were digitally recorded. The resulting images
were processed using Adobe (Mountain View, CA) PhotoShop software.
Cell adhesion with/without lipid raft disruption
Cell adhesion to ICAM-1Fc, a chimeric protein containing the five
extracellular domains of human ICAM-1 fused to a human immunoglobulin G1
(IgG1) Fc sequence, was performed as described
(Leitinger and Hogg, 2000a).
Cell adhesion to fibronectin was performed using flat bottom tissue culture
96-well plates (MicrotestTM, Falcon, Becton Dickinson, Oxford, UK) coated
with fibronectin at 2 µg/ml.
Manipulation of plasma membrane cholesterol content using
methyl-ß-cyclodextrin
All treatments were performed in RPMI with 0.1% fatty-acid-free BSA.
Cholesterol depletion and replenishment: cells (at 4x106/ml)
were incubated in either RPMI 1640 (untreated), 10 mM MßCD, or 5 mM
MßCD plus 5 mM MßCD-cholesterol in RPMI 1640 for 15 minutes at
37°C. Cholesterol repletion of cholesterol-depleted cells: after MßCD
incubation as above, cells were washed in RPMI 1640 and incubated with
MßCD-cholesterol inclusion complexes at 0.5 mM cholesterol for 1 hour at
37°C. After the various treatments, cells were directly used for the
adhesion assay, whereby 50 µl aliquots of cells (at
4x106/ml) were added to 50 µl of 2x stimuli.
Preparation of methyl-ß-cyclodextrin-cholesterol inclusion
complexes
MßCD-cholesterol complexes were prepared as described
(Klein et al., 1995). Briefly,
a solution of 25 mg cholesterol, dissolved in 333 µl of methanol/chloroform
(2:1, v/v) was added drop-wise to a stirred solution of 833 mg MßCD in 9
ml PBS on a water bath (80°C). The mixture was stirred until a clear
solution resulted. The MßCD-cholesterol complexes were then lyophilised
and stored at room temperature.
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Results |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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I-LFA-1 but not wt LFA-1 with lipid
raftsI-LFA-1) in the Jurkat T-cell line
J-ß2.7, which lacks endogenous expression of LFA-1
(Weber et al., 1997).
I-LFA-1 is unable to bind ligand ICAM-1, but has the features of a
constitutively active integrin including the expression of many epitopes
associated with LFA-1 activation
(Leitinger and Hogg, 2000a).
By contrast, without prior stimulation, wt LFA-1 does not express these
activation epitopes and has no ligand binding activity. Therefore, wt LFA-1 is
here considered to be `inactive'. Comparison of inactive wt LFA-1 with
I-LFA-1, which resembles active integrin, thus offers the opportunity
to define differences between these two forms of integrin. Krauss and Altevogt
have recently reported that LFA-1 is associated with lipid rafts on the plasma
membrane of murine thymocytes (Krauss and
Altevogt, 1999). We therefore asked whether LFA-1 was similarly
localised on human T cells and whether the state of integrin activation made
any difference to its distribution.
To this end we employed the method used by Janes et al.
(Janes et al., 1999), who
visualised lipid rafts on Jurkat T-cell membranes using fluorescence
microscopy. Although lipid rafts are not usually visible by light microscopy,
it is possible to detect aggregated lipid rafts as distinct patches by
clustering of raft markers with antibodies or other reagents
(Harder et al., 1998). Thus
upon coalescence of the lipid rafts into larger domains, other raft-associated
proteins will colocalise with these patches. Non raft-associated proteins do
not colocalise with the raft patches because of the immiscibility of the
different lipid phases. Using confocal microscopy we detected the lipid rafts
by crosslinking the raft enriched glycosphingolipid GM1 through binding to the
cholera toxin (Ctx) B subunit and patching with anti-Ctx antibodies. The
distribution of I-LFA-1 largely overlapped with the patched Ctx
staining showing a preferential association with the lipid rafts
(Fig. 1A, top). By contrast, wt
LFA-1 appeared less associated with the lipid rafts as staining did not
colocalise with the Ctx patches (Fig.
1A, bottom). GPI-linked proteins are preferentially associated
with lipid rafts (Brown and London,
2000; Brown and Rose,
1992). Therefore, a useful positive control for the localisation
and identification of the lipid rafts was the GPI-linked decay accelerating
factor (DAF; CD55) protein (Fig.
1B). Transferrin receptor (TfR; CD71) does not associate with the
lipid rafts (Harder et al.,
1998; Harder and Simons,
1999; Janes et al.,
1999) and served as a negative control
(Fig. 1C).
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To provide a more extensive analysis of the relative overlap of the patched
Ctx with the different membrane markers, the fluorescent images of 30-40 cells
per experiment were scored into three different categories: good, medium or no
colocalisation (see legend to Fig.
2). The analysis demonstrated that there was a greater tendency
for I-LFA-1, than for wt LFA-1, to be associated with lipid rafts
(Fig. 2A). As expected, the
GPI-linked DAF protein had a similar distribution in both types of LFA-1
expressing cells, being largely raft localised
(Fig. 2B). Conversely, the TfR
was excluded from the same membrane structures in both cell lines
(Fig. 2C). Therefore the two
forms of LFA-1 have different affinities for the lipid rafts, and
I-LFA-1 was more strongly raft associated than wt LFA-1.
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The association of Mn2+ and phorbol ester-activated LFA-1
with lipid rafts
As inactive wt LFA-1 is excluded from the lipid rafts, whereas
I-LFA-1, which resembles active integrin is associated with the rafts,
it was predicted that activation of wt LFA-1 with agonists would mobilise this
integrin from the non-raft compartment to the raft compartment. To test this
hypothesis, the wt LFA-1-expressing Jurkat T cells were exposed to either 0.5
mM Mn2+, which activates integrin by conformationally altering the
integrin ectodomain, or to 100 nM phorbol ester PdBu, which activates integrin
through an intracellular signalling pathway
(Stewart and Hogg, 1996). As
expected, both agonists caused an increase in binding of Jurkat
T-cell-expressed wt LFA-1 to immobilised ICAM-1
(Fig. 3A).
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Next, the effect of these treatments on the colocalisation of LFA-1 with
the lipid raft patches was examined. Wild-type LFA-1 on untreated T cells was
generally excluded from the rafts as was observed in
Fig. 1
(Fig. 3B, unstim). However,
following exposure of wt LFA-1-expressing cells to either Mn2+ or
PdBu, LFA-1 was largely relocated to the raft compartment of the membrane
(Fig. 3B,C). To provide a
quantitative analysis of the degree of overlap between the LFA-1 signal and
the patched Ctx signal, we calculated colocalisation of the two signals using
NIH Image software. Table 1
shows that, relative to unstimulated cells, the overlap between the LFA-1
signal and the lipid raft signal increased on Mn2+ and
PdBu-stimulated cells. These findings correlate well with those shown in
Fig. 3C and thus validate our
semi-quantitative analysis. The correlation between raft association and
ligand binding activity is strong evidence that the mobilisation of LFA-1 into
the lipid raft compartment is a key component in the regulation of the
adhesive activity of this integrin. In addition, the association with the
lipid rafts of both I-LFA-1 and agonist-activated LFA-1 further
confirms that I-LFA-1 does mimic the active ligand binding form of
LFA-1.
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I-LFA-1 crosstalk to 4ß1 integrin
Certain integrins can `crosstalk' to other classes of integrin on the same
cells and either induce or suppress their ligand binding activity
(Porter and Hogg, 1998). A
characteristic of the I-LFA-1-expressing T cells, compared with cells
expressing wt LFA-1, is the constitutively elevated ligand binding activity of
the ß1 integrins, 4ß1 and 5ß1. Therefore we next
asked whether the distribution of 4ß1 was influenced by the
membrane localisation of LFA-1 and, specifically, whether there was any
association of 4ß1 with lipid rafts. Examination of overlap
between Ctx membrane patches and 4ß1 showed that there was good
colocalisation on I-LFA-1-expressing Jurkat cells, but not on wt
LFA-1-expressing cells (Fig.
4A,B). Therefore, expression of I-LFA-1 caused
4ß1 association with the lipid rafts, whereas on wt
LFA-1-expressing cells, neither LFA-1 nor 4 integrins were
predominantly raft associated.
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One possibility was that I-LFA-1 was controlling the behaviour of
4ß1 through physically associating with it on the membrane. The
use of double laser confocal microscopy (but not Ctx crosslinking conditions)
showed that there was no significant colocalisation of 4ß1 and
LFA-1 on Jurkat cells expressing I-LFA-1 or wt LFA-1 (data not shown).
Thus I-LFA-1 and 4ß1 are located within different lipid
rafts that then cocluster with patched Ctx. This emphasises the indirect
effect of I-LFA-1 on crosstalk to 4ß1 integrin.
Integrin-mediated adhesion requires intact lipid rafts
To test whether the presence of integrins in lipid rafts was relevant for
integrin-mediated adhesion, the rafts were disrupted using
methyl-ß-cyclodextrin (MßCD), which depletes the essential
cholesterol component of lipid rafts and has been used to disrupt the rafts in
Jurkat cells (Harder and Kuhn,
2000; Janes et al.,
1999). Jurkat cells were activated by agonists that act either
through an intracellular signalling pathway (PdBu) or by engaging the integrin
ectodomain (Mn2+), and adhered to fibronectin
(Fig. 5A). Adhesion was
dependent on 4ß1 and 5ß1 (data not shown). Following
treatment with 10 mM MßCD, adhesion was reduced to background levels.
Evidence that MßCD was causing cholesterol depletion and not some other
effect was demonstrated by the lack of effect on adhesion when cells were
treated with 5 mM MßCD plus 5 mM MßCD-cholesterol conjugates. This
latter treatment exposed the cells to the same concentration of MßCD as
when cholesterol was depleted but provided the cells with cholesterol in the
form of MßCD-cholesterol conjugates, which facilitate the incorporation
of exogenous cholesterol into membranes
(Klein et al., 1995). Finally
Jurkat T cells were treated first with 10 mM MßCD and then repleted with
MßCD-cholesterol conjugates at 0.5 mM cholesterol. This treatment
completely restored adhesion for Mn2+-treated cells and partially
restored adhesion for PdBu-treated cells, demonstrating that cholesterol
depletion was reversible.
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We next tested whether the adhesion of primary human T cells was also
dependent upon intact lipid rafts. In these experiments the ability of T-cell
LFA-1 to bind to ICAM-1 was assessed, as for the Jurkat cells, following
stimulation with agonists PdBu or Mn2+ as well as the
ß2-integrin-activating mAb KIM 185
(Fig. 5B). In all cases,
treatment with 10 mM MßCD inhibited adhesion; this was due to cholesterol
depletion as treatment with 5 mM MßCD plus 5 mM MßCD-cholesterol
conjugates maintained adhesion at control levels. Repletion experiments with
MßCD-cholesterol conjugates following MßCD treatment also restored
LFA-1 adhesion to ICAM-1. Therefore, for both LFA-1 and 4ß1 (and
5ß1), which are the integrins that are the focus of this study,
there is dependence on intact lipid rafts for adhesion. It is of interest that
this dependence on rafts is independent of the means of integrin
activation.
Depletion of cellular cholesterol inhibits clustering of
4ß1 on cells expressing I-LFA-1
We have previously demonstrated enhanced 4ß1 clustering on
I-LFA-1-expressing Jurkat cells
(Leitinger and Hogg, 2000a)
and in this study we show an association of active 4 integrin with the
lipid rafts. Therefore, we asked whether the clustered form of 4ß1
was dependent upon lipid raft components. Following treatment of Jurkat cells
with 10 mM MßCD, the clustered distribution of 4ß1 on the
I-LFA-1-expressing cells (Fig.
6A) was reduced to background levels
(Fig. 6, compare B with C). The
MßCD treatment had no effect on the distribution of 4 integrin in
wt LFA-1-expressing cells (Fig.
6D). These results indicate that cholesterol, which is required
for lipid raft integrity, is also necessary for the formation of
4ß1 integrin clusters on I-LFA-1-expressing J-ß2.7
cells. Thus, for 4ß1 a link exists between integrin clustering and
lipid rafts.
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The association between the cytoskeleton, integrins and lipid
rafts
Other studies have shown that interactions of proteins with lipid raft
components can be regulated or stabilised by the cytoskeleton
(Holowka et al., 2000;
Oliferenko et al., 1999).
Cytochalasin D is well known to abolish LFA-1-mediated adhesion
(Lub et al., 1997;
Stewart and Hogg, 1996) and
the cytoskeleton is implicated in integrin crosstalk as cytochalasin D
prevented the increased ligand binding activity of 4ß1 in the
I-LFA-1-expressing Jurkat T cells
(Leitinger and Hogg, 2000a).
To understand more about the connection between the cytoskeleton, integrins
and the lipid rafts we investigated the association of LFA-1 and
4ß1 with Ctx crosslinked lipid rafts in Jurkat cells in which the
cytoskeleton had been disrupted. The first observation of the cytochalasin
D-treated lipid rafts was that the rafts formed exceedingly large patches or
`caps' in the cells treated in this manner
(Fig. 7A). Second, treatment
with cytochalasin D caused both I-LFA-1 and wt LFA-1 to associate with
the rafts in an equivalent and extensive fashion. A similar observation was
made for 4ß1, in that, on both I-LFA-1- and wt
LFA-1-expressing Jurkat T cells treated with cytochalasin D, the 4
integrin was associated mainly with the lipid rafts
(Fig. 7B). As expected, the
distribution of the GPI-linked DAF protein also coincided with the lipid raft
patches (Fig. 7C), while the
distribution of the non-raft-associated TfR was unaffected by cytochalasin D
and remained outside the raft membrane compartment
(Fig. 7D). These findings
strongly imply that `inactive' LFA-1 and 4ß1 are restrained by
cytoskeleton tethers so as to be excluded from the lipid rafts and that
release of the constraint allows the integrins to move into the lipid
rafts.
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The effect of cytochalasin D on the cytoskeleton is to cap the barbed ends
of F-actin filaments and prevent their lengthening
(Cooper, 1987). To test the
effects on the lipid rafts of an actin binding drug with a different mode of
action, we investigated latrunculin A, which blocks polymerisation of
monomeric G actin to F actin (Coue et al.,
1987). Similar to the results shown in
Fig. 7, both cytochalasin D and
latrunculin A caused large patches of Ctx crosslinked lipid rafts and
coassociation of integrin 4ß1 from both I-LFA-1-
(Fig. 8A) and wt
LFA-1-expressing (Fig. 8B)
cells. Latrunculin A also had similar effects on the distribution of LFA-1 on
both cell lines (data not shown). This further contributes to the evidence
that following release from cytoskeletal constraint, integrin is mobilised to
lipid rafts.
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Paradoxically, the effects of cytochalasin D and latrunculin A, which
disrupt both LFA-1 and 4ß1 function, cause more lipid raft
association of these integrins. However, the fact that cytochalasin D and
latrunculin A caused raft `capping' suggests that the cytoskeleton must have
an additional role in the normal stabilisation of the lipid raft structure.
These results are in keeping with the finding that there is an enrichment in F
actin on raft membrane patches in Jurkat T cells and that raft-mediated
signalling is dependent upon the integrity of the lipid raft/cytoskeleton
connection (Harder and Simons,
1999).
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 are greatly enriched in the lipid raft compartment of the
T-cell membrane. Thus, activated LFA-1 but not inactive LFA-1 is
preferentially found in lipid rafts. Activated LFA-1 is mimicked by LFA-1 in
which the I domain is deleted (I-LFA-1), and this form is also
preferentially located in the lipid rafts. I-LFA-1-mediated crosstalk
to 4ß1, which activates 4ß1, brings this second
integrin into the lipid rafts. The ß2 integrins LFA-1 and Mac-1
(Krauss and Altevogt, 1999;
Skubitz et al., 2000), as well
as other classes of integrin such as vß3
(Green et al., 1999) and the
ß1 integrins 3ß1 and 6ß1
(Claas et al., 2001;
Thorne et al., 2000) have been
identified in lipid rafts, but a preferential association with the active form
of an integrin has not previously been noted. The fact that adhesion mediated
by both LFA-1 and 4ß1/5ß1 was dependent on intact
lipid rafts provides evidence for their relevance to integrin function.
Finally the raft dependence of adhesion applies not only to Jurkat cells but
also to primary human T cells.
The results further suggest that LFA-1 and 4ß1 have an
intrinsic ability to associate with lipid rafts, which is prevented by their
linkage into the cytoskeleton. The affinity for the rafts may be an inherent
feature of the integrins, as it is shared by integrins activated in several
ways. Thus lipid raft association is observed for high affinity
conformationally altered LFA-1, which has been exposed to the divalent cation
Mn2+ or had its I domain removed; high avidity LFA-1 following cell
exposure to phorbol ester; or 4ß1, clustered as a result of
crosstalk from LFA-1.
How integrins are held within the lipid raft compartment remains to be
resolved. Neither subunit of the integrin ß heterodimer is
modified by palmitoylation, a characteristic of many raft transmembrane
proteins (Brown and London,
2000). It is conceivable that raft localisation is dependent on
complex formation with other membrane proteins. Integrins can complex with
multimembrane spanning proteins such as integrin-associated protein (IAP) and
members of the transmembrane 4 superfamily (TM4SF) (reviewed by
Porter and Hogg, 1998), and
there is increasing evidence that these complexes associate with the lipid
rafts (Claas et al., 2001;
Green et al., 1999). ß2
integrins can form complexes with the TM4SF proteins CD82
(Shibagaki et al., 1999) and
CD63 (Skubitz et al., 2000)
and, in the case of CD63, the ß2 integrin/CD63 complex was isolated from
the raft membrane fraction. However, it is clear that integrin/TM4SF complexes
exist outside the raft compartment (Claas
et al., 2001). There is also increasing evidence that
ligand-induced oligomerisation provides the stimulus for raft association
(reviewed by Cherukuri et al.,
2001). As 4ß1 on I-LFA-1-expressing cells is
clustered through crosstalk, and the integrin activating regimes used in this
study also enhance integrin clustering
(Stewart et al., 1998), it is
an attractive possibility that integrin oligomerisation might trigger the
association with the rafts.
The fact that inactive wt LFA-1 and 4ß1 are largely excluded
from the raft fraction implies that the activation process involves movement
between the two types of membrane compartment. Activation of B cells by
agonists such as phorbol esters causes LFA-1 mobility in the cell membrane
(Kucik et al., 1996). Previous
reports suggest that this mobility comes about as a result of the untethering
of LFA-1 from the cytoskeleton (Lub et
al., 1997; Stewart et al.,
1998). Our results add to this information by demonstrating that,
following activation, a proportion of the T cell's LFA-1 and 4ß1
moves to the lipid raft compartment of the membrane. Moreover, following
cytochalasin D or latrunculin A treatment, the majority of inactive wt LFA-1
and 4ß1 becomes associated with lipid rafts. These findings
provide strong evidence that LFA-1 and 4ß1 are restrained by
cytoskeletal tethers in a manner that causes exclusion from the lipid rafts
and that, following response to activating agonists, a proportion of integrins
are untethered. This finding is in keeping with studies of the interactions of
Fc
RI and CD44 with lipid rafts, which are also regulated by the actin
cytoskeleton (Holowka et al.,
2000; Oliferenko et al.,
1999). In the case of CD44, a similar result to the one presented
here was obtained, in that disruption of the actin cytoskeleton dramatically
increased the proportion of CD44 that was isolated from the lipid raft
fraction (Oliferenko et al.,
1999).
There has been little information about the mechanism of integrin
crosstalk. Here we show that the presence of active LFA-1 in lipid rafts has
consequential effects on 4ß1, causing it to move into the rafts.
The conjecture is that I-LFA-1, which resembles high affinity integrin,
can directly signal the release of 4ß1 from the cytoskeleton. In
this way, the mechanism that induces I-LFA-1 to associate with lipid
rafts will act on other integrins on the same cell surface, and cause their
association with lipid rafts and thus contribute to their activation.
A clustered from of 4ß1 is associated with raft membrane
patches on cells expressing I-LFA-1. Disruption of raft integrity
through depletion of membrane cholesterol with MßCD completely disrupted
4ß1 cluster formation, implying that the lipid rafts are required
for 4 integrin clusters. The precise relationship between the clusters
and rafts remains to be worked out. Little is known about the size of lipid
rafts on living cells. One study calculated the cholesterol-dependent
aggregates of a GPI-linked protein to be <70 nm on living cells
(Varma and Mayor, 1998), which
is too small to be seen by light microscopy, while another study found a
ganglioside and a GPI-linked protein to be confined to domains of about
200-300 nm (Jacobson and Dietrich,
1999). The clusters of 4ß1 integrin fluorescence that
are visualised by confocal microscopy are `hundreds of nm' to `µm' in size.
It has been suggested that the cytoskeleton can regulate raft size by
restraining diffusing small rafts
(Jacobson and Dietrich, 1999).
Therefore, raft size may vary between different cell types.
In summary our observations suggest a model whereby T-cell signals mobilise
LFA-1 by releasing it from the cytoskeleton to the lipid rafts. In this study
we show that LFA-1-mediated adhesion, which is a prerequisite for effective
antigen recognition by T cells (reviewed by
Dustin and Cooper, 2000), is
dependent upon intact lipid rafts. The relocation of LFA-1 has consequences
for other integrins such as 4ß1, causing their movement into lipid
raft domains. Furthermore, the T-cell receptor itself is also found in lipid
raft domains (Janes et al.,
1999) and a model has been suggested whereby the T-cell receptor
is recruited to lipid rafts following antigen stimulation
(Cherukuri et al., 2001;
Montixi et al., 1998;
Xavier et al., 1998). Movement
of activated leukocyte membrane receptors, including integrins, into the lipid
rafts provides foci of signalling for the cell and may be a general mechanism
that ensures effective T-cell function. The recruitment of active integrins to
the raft compartment on leukocytes may provide a model for integrin function
in other cell types.
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionReferences |
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K. Haglund, I. Ivankovic-Dikic, N. Shimokawa, G. D. Kruh, and I. Dikic Recruitment of Pyk2 and Cbl to lipid rafts mediates signals important for actin reorganization in growing neurites J. Cell Sci., May 15, 2004; 117(12): 2557 - 2568. [Abstract] [Full Text] [PDF]
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M. Wadehra, L. Goodglick, and J. Braun The Tetraspan Protein EMP2 Modulates the Surface Expression of Caveolins and Glycosylphosphatidyl Inositol-linked Proteins Mol. Biol. Cell, May 1, 2004; 15(5): 2073 - 2083. [Abstract] [Full Text] [PDF]
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 L. Decker and C. ffrench-Constant Lipid Rafts and Integrin Activation Regulate Oligodendrocyte Survival J. Neurosci., April 14, 2004; 24(15): 3816 - 3825. [Abstract] [Full Text] [PDF]
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H. Katano, L. Pesnicak, and J. I. Cohen Simvastatin induces apoptosis of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and delays development of EBV lymphomas PNAS, April 6, 2004; 101(14): 4960 - 4965. [Abstract] [Full Text] [PDF]
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G. Greicius, L. Westerberg, E. J. Davey, E. Buentke, A. Scheynius, J. Thyberg, and E. Severinson Microvilli structures on B lymphocytes: inducible functional domains? Int. Immunol., February 1, 2004; 16(2): 353 - 364. [Abstract] [Full Text] [PDF]
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 R. G. Sitrin, D. R. Johnson, P. M. Pan, D. M. Harsh, J. Huang, H. R. Petty, and R. A. Blackwood Lipid Raft Compartmentalization of Urokinase Receptor Signaling in Human Neutrophils Am. J. Respir. Cell Mol. Biol., February 1, 2004; 30(2): 233 - 241. [Abstract] [Full Text] [PDF]
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 K. Shibuya, J. Shirakawa, T. Kameyama, S.-i. Honda, S. Tahara-Hanaoka, A. Miyamoto, M. Onodera, T. Sumida, H. Nakauchi, H. Miyoshi, et al. CD226 (DNAM-1) Is Involved in Lymphocyte Function-associated Antigen 1 Costimulatory Signal for Naive T Cell Differentiation and Proliferation J. Exp. Med., December 15, 2003; 198(12): 1829 - 1839. [Abstract] [Full Text] [PDF]
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N. Hogg, M. Laschinger, K. Giles, and A. McDowall T-cell integrins: more than just sticking points J. Cell Sci., December 1, 2003; 116(23): 4695 - 4705. [Abstract] [Full Text] [PDF]
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M. Spaargaren, E. A. Beuling, M. L. Rurup, H. P. Meijer, M. D. Klok, S. Middendorp, R. W. Hendriks, and S. T. Pals The B Cell Antigen Receptor Controls Integrin Activity through Btk and PLC{gamma}2 J. Exp. Med., November 17, 2003; 198(10): 1539 - 1550. [Abstract] [Full Text] [PDF]
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M. R. Tardif and M. J. Tremblay Presence of Host ICAM-1 in Human Immunodeficiency Virus Type 1 Virions Increases Productive Infection of CD4+ T Lymphocytes by Favoring Cytosolic Delivery of Viral Material J. Virol., November 15, 2003; 77(22): 12299 - 12309. [Abstract] [Full Text] [PDF]
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E. Odintsova, J. Voortman, E. Gilbert, and F. Berditchevski Tetraspanin CD82 regulates compartmentalisation and ligand-induced dimerization of EGFR J. Cell Sci., November 15, 2003; 116(22): 4557 - 4566. [Abstract] [Full Text] [PDF]
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H. J. Hathaway, S. C. Evans, D. H. Dubois, C. I. Foote, B. H. Elder, and B. D. Shur Mutational analysis of the cytoplasmic domain of {beta}1,4-galactosyltransferase I: influence of phosphorylation on cell surface expression J. Cell Sci., November 1, 2003; 116(21): 4319 - 4330. [Abstract] [Full Text] [PDF]
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L. Gagnoux-Palacios, M. Dans, W. van't Hof, A. Mariotti, A. Pepe, G. Meneguzzi, M. D. Resh, and F. G. Giancotti Compartmentalization of integrin {alpha}6{beta}4 signaling in lipid rafts J. Cell Biol., September 29, 2003; 162(7): 1189 - 1196. [Abstract] [Full Text] [PDF]
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D. R. M. Graham, E. Chertova, J. M. Hilburn, L. O. Arthur, and J. E. K. Hildreth Cholesterol Depletion of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus with {beta}-Cyclodextrin Inactivates and Permeabilizes the Virions: Evidence for Virion-Associated Lipid Rafts J. Virol., August 1, 2003; 77(15): 8237 - 8248. [Abstract] [Full Text] [PDF]
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Y.-M. Xiong, J. Chen, and L. Zhang Modulation of CD11b/CD18 Adhesive Activity by Its Extracellular, Membrane-Proximal Regions J. Immunol., July 15, 2003; 171(2): 1042 - 1050. [Abstract] [Full Text] [PDF]
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M. R. Marwali, J. Rey-Ladino, L. Dreolini, D. Shaw, and F. Takei Membrane cholesterol regulates LFA-1 function and lipid raft heterogeneity Blood, July 1, 2003; 102(1): 215 - 222. [Abstract] [Full Text] [PDF]
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D. M. Rose, S. Liu, D. G. Woodside, J. Han, D. D. Schlaepfer, and M. H. Ginsberg Paxillin Binding to the {alpha}4 Integrin Subunit Stimulates LFA-1 (Integrin {alpha}L{beta}2)-Dependent T Cell Migration by Augmenting the Activation of Focal Adhesion Kinase/Proline-Rich Tyrosine Kinase-2 J. Immunol., June 15, 2003; 170(12): 5912 - 5918. [Abstract] [Full Text] [PDF]
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R. D. Evans, V. C. Perkins, A. Henry, P. E. Stephens, M. K. Robinson, and F. M. Watt A tumor-associated {beta}1 integrin mutation that abrogates epithelial differentiation control J. Cell Biol., February 5, 2003; (2003) 200209016. [Abstract] [Full Text] [PDF]
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B. J. Holleran, E. Barbar, M. D. Payet, and G. Dupuis Differential recruitment of {alpha}2{beta}1 and {alpha}4{beta}1 integrins to lipid rafts in Jurkat T lymphocytes exposed to collagen type IV and fibronectin J. Leukoc. Biol., February 1, 2003; 73(2): 243 - 252. [Abstract] [Full Text] [PDF]
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H. O. Barazi, Z. Li, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, and D. D. Roberts Regulation of Integrin Function by CD47 Ligands. DIFFERENTIAL EFFECTS ON alpha vbeta 3 AND alpha 4beta 1 INTEGRIN-MEDIATED ADHESION J. Biol. Chem., November 1, 2002; 277(45): 42859 - 42866. [Abstract] [Full Text] [PDF]
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R. Shamri, V. Grabovsky, S. W. Feigelson, O. Dwir, Y. van Kooyk, and R. Alon Chemokine Stimulation of Lymphocyte alpha 4 Integrin Avidity but Not of Leukocyte Function-associated Antigen-1 Avidity to Endothelial Ligands under Shear Flow Requires Cholesterol Membrane Rafts J. Biol. Chem., October 11, 2002; 277(42): 40027 - 40035. [Abstract] [Full Text] [PDF]
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F. Berditchevski, E. Odintsova, S. Sawada, and E. Gilbert Expression of the Palmitoylation-deficient CD151 Weakens the Association of alpha 3beta 1 Integrin with the Tetraspanin-enriched Microdomains and Affects Integrin-dependent Signaling J. Biol. Chem., September 27, 2002; 277(40): 36991 - 37000. [Abstract] [Full Text] [PDF]
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