? Leukocyte Adhesion Laboratory, Cancer Research UK London Research Institute,
Lincoln's Inn Fields Laboratories, Lincoln's Inn Fields, London WC2A 3PX,
UK
* These authors contributed equally to this work
** Author for correspondence (e-mail: nancy.hogg{at}cancer.org.uk)
Accepted 9 April 2003
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Summary |
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Key words: T lymphocyte, LFA-1, Migration, MLCK, Rho kinase
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Introduction |
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LFA-1 is abundantly expressed by leukocytes and is the main receptor used
by T cells for adhesion via the ICAM family of ligands. The ability of LFA-1
to act as a signaling receptor has not been extensively examined, as it has
been chiefly associated with promoting cell-cell contact, thereby optimizing
signaling through other receptors. Clustering of LFA-1 and an increase in
affinity for ICAM-1, resulting from a conformational alteration in the
integrin ectodomain, promote LFA-1-mediated adhesion
(Stewart and Hogg, 1996;
van Kooyk and Figdor, 2000
).
These changes are achieved via intracellular signals generated through
engagement of other cell surface receptors. However, LFA-1 can also be
triggered directly through the use of stimulatory anti-LFA-1 antibodies or by
exposure to ICAM-1 together with divalent cations Mg2+ or
Mn2+ (Stewart and Hogg,
1996
). This method of activation bypasses the need for inside out
signaling and allows the analysis of LFA-1 signaling, initiated by ICAM-1
binding, in isolation from other cell surface receptors.
Cells move by coordinating the generation of lamellipodial protrusions at
the front of the cell leading to new attachments, followed by the detachment
of previous adhesions at the rear of the cell
(Horwitz and Parsons, 1999).
LFA-1 is directly involved in promoting cell contact as T cell attachment to
ICAM-1 is dependent upon the ability of LFA-1 to signal remodeling of the
actin cytoskeleton (Porter et al.,
2002
). LFA-1 is also involved in generating signals, such as
PKC-ß1-dependent microtubule rearrangements, that allow adhesion to be
translated into migration (Volkov et al.,
2001
). Here we describe how LFA-1 induces myosin motor activity
through the activation of two kinases, myosin light chain kinase (MLCK) and
ROCK. The location and activity of both kinases within the cell is restricted,
with MLCK at the leading edge and ROCK concentrated at the trailing edge of
the cell.
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Materials and Methods |
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ICAM-1Fc was produced as a chimeric protein, consisting of the five
extracellular domains of ICAM-1 fused to the Fc fragment of human IgG1
(Berendt et al., 1992). The
fluorescent cell label
2',7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein (BCECF-AM)
was purchased from Calbiochem.
The following peptides linked to biotinylated antennapedia peptide were
synthesized by the Protein and Peptide Chemistry Laboratory of the Cancer
Research UK London Research Institute: MLCK autoinhibitory or LSM1 sequence
(LSKDRMKKYMARR) (Tanaka et al.,
1995; Walker et al.,
1998
), a scrambled version of the LSM1 peptide with Tyr altered to
Ala (RSKDMRAKAKARL) and biotinylated antennapedia peptide alone.
Cell attachment assays
Peripheral blood mononuclear cells were prepared from single donor
leukocyte buffy coats and T cells expanded in culture as previously described
(Porter and Hogg, 1997).
Before use, T cells were washed three times in HEPES buffer (20 mM HEPES, 140
mM NaCl, 2 mg/ml glucose, pH 7.4) and labelled with 2.5 µM BCECF-AM in the
same buffer for 30 minutes at 37°C followed by two further washes.
Flat-bottomed Immulon-1® 96-well plates (Dynatech, Chantilly, VA) were
precoated with 50 µl of ICAM-1Fc (3 µg/ml) in PBS overnight at 4°C.
The plates were blocked with 2.5% BSA in PBS for 2 hours at room temperature,
then washed once with PBS and twice in HEPES buffer. T cells were
pre-incubated with inhibitors for 15 minutes (or overnight at 37°C for C3
exoenzyme) following titration to determine the optimal dose. The inhibitors
were MLCK inhibitor ML-7, ROCK inhibitor Y-27632 (both 0-200 µM), CaM
inhibitor W-7 (0-100 µM) and RhoA GTPase inhibitor C3 exoenzyme (0-20
µg/ml). T cells were preincubated with the antennapedia peptides at
concentrations from 0 to 20 µg/ml for 90 minutes at 37°C and washed
twice with HEPES buffer before use in the experiment. T cells at
2x105 cells/well were added to 96-well plates coated with
ICAM-1Fc and treated with HEPES buffer containing 5 mM Mg2+/1 mM
EGTA (Mg2+/EGTA buffer). The T cells were centrifuged at 100
g for 1 minute, prior to 40 minutes of incubation at 37°C.
Non-adherent cells were removed by washing three times in warmed
Mg2+/EGTA buffer (150 µl/well). T cell attachment was quantified
using a Cytofluor multiwell platereader (PerSeptive Biosystems, Hertford,
UK).
Western blotting
T cells were suspended at 5x107/ml in ice cold lysis
buffer [50 mM Tris pH 8 containing 150 mM NaCl, 2 mM MgCl2, 2 mM
EGTA, 1% Triton X-100, 10 µg/ml phenylmethylsulphonyl fluoride and
CompleteTM protein inhibitor cocktail (Roche Diagnostics, Lewes, UK) used
according to the manufacturer's instructions] and lysed for 20 minutes on ice.
The lysate was microfuged for 15 minutes to remove insoluble material.
Proteins were separated by SDS-PAGE. After transfer to nitrocellulose membrane
and incubation with primary antibodies, the bound antibody was detected with
HRP-conjugated sheep anti-mouse Ig (Amersham Biosciences UK Ltd, Chalfont St
Giles, UK) and ECL western blotting detection reagents (Amersham Biosciences
UK Ltd).
Confocal microscopy
13 mm round glass coverslips were precoated with 300 µl ICAM-1Fc (3
µg/ml) in PBS overnight at 4°C. Coverslips were blocked with 2.5% BSA
in PBS for 2 hours at RT and then washed three times with PBS and once with
HEPES buffer at 4°C. T cells were washed three times in HEPES buffer
before being added to the coverslips (2x105 cells/coverslip)
in 100 µl of Mg2+/EGTA buffer. Cells were incubated for 10
minutes on ice and then a further 30 minutes at 37°C. Unbound cells were
removed with four gentle washes with Mg2+/EGTA buffer. Adherent
cells were then fixed with 3% formaldehyde in PBS for 20 minutes at room
temperature. After two washes with PBS, cells were permeabilized with 0.1%
Triton-X-100 for 5 minutes on ice in order to stain for intracellular
proteins. Cover slips were incubated with MLCK mAb (1:100), ROCK I mAb (2.5
µg/ml), and respective ascites and IgG1 isotype controls or rabbit
anti-myosin II (1:100) for 30 minutes at RT, then followed by Alexa 488-goat
anti-mouse IgG (1:200), goat anti-rabbit IgG (1:200) and 2.5 units/ml Alexa
546-phalloidin for 30 minutes at RT. The images were taken on a Zeiss Laser
Scanning Microscope LSM5101.
Detection of cell spread area by laser scanning cytometry
The Laser Scanning Cytometer (CompuCyte, Mass, USA) combines features of
both flow and image cytometers in that it measures fluorescence and light
scatter from immobilized cells that are moved through a 488 nm laser line on a
motorized stage (Darzynkiewicz et al.,
1999). The spread contours of T cells, mounted on ICAM-1-coated
coverslips as for confocal microscopy, were defined on the basis of their
fluorescence as determined by Alexa 488-phalloidin, and the threshold level
was optimized so that as many single cells as possible could be contoured
without losing fluorescence information. For each cell, the `spread area'
represents the physical area in µm2. 6,000 cells were measured
per coverslip. The data were analyzed using the unpaired Student's
t-test, and a value of P<0.01 was taken as
significant.
Video microscopy
For video-microscopy, 35 mm glass bottom microwell dishes (MatTek Corp.,
Ashland, Mass, USA) were coated at 4°C overnight with 200 µl of
ICAM-1Fc (3 µg/ml) in PBS, then blocked with 2.5% BSA in PBS. T cells at
5x105 per dish were allowed to migrate for 20 minutes at
37°C before being exposed to either 20-25 µM ML-7, 10-20 µM Y-27632
or 50 µM W-7 for an additional 10-70 minutes. Images were taken at 5 second
intervals using a Nikon Diaphot 300 microscope and AQM2001 Kinetic
Acquisition Manager software (Kinetic Imaging Ltd., Bromborough, UK). Over a
70 minute period, cells were tracked using Motion Analysis software (Kinetic
Imaging Ltd.), and the data analyzed using a Mathematica notebook (Wolfram
Research Europe Ltd, Long Hanborough, UK) developed by Daniel Zicha (Cancer
Research UK, London).
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Results |
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ML-7 is an inhibitor of MLCK, a serine/threonine kinase that phosphorylates
myosin light chains (MLC) promoting acto-myosin contraction
(Kamm and Stull, 2001).
Titration of ML-7 showed a saturating dose of 25 µM for inhibition of T
cell attachment (Fig. 1B).
Although ML-7 is an inhibitor of MLCK with a Ki of 300 nM, it can also inhibit
protein kinase A (Ki=21 µM) and protein kinase C (Ki=42 µM)
(Saitoh et al., 1987
). At the
concentrations that effectively interfere with attachment, ML-7 is likely to
selectively block the activity of MLCK because BIM, a broadly specific PKC
inhibitor, had no effect on T cell attachment (data not shown). In order to
confirm the specific effect on MLCK, we treated T cells with a 13-mer MLCK
auto-inhibitory domain peptide or a scrambled version of the peptide, both of
which were fused to antennapedia peptide to promote T cell entry
(Tanaka et al., 1995
;
Walker et al., 1998
). The MLCK
domain peptide inhibited T cell attachment to ICAM-1 at 10-20 µg/ml
(Fig. 1C). The scrambled
version of the peptide had a small effect on T cell attachment that was
attributable to the antennapedia peptide.
ROCK is another kinase that directly phosphorylates MLC and also indirectly
promotes MLC phosphorylation through inhibition of myosin phosphatase
(Ishizaki et al., 1996;
Kimura et al., 1996
). T cell
attachment to ICAM-1 was not affected by the ROCK inhibitor, Y-27632
(Fig. 1B). Inhibition by C3
exoenzyme of RhoA, which is the upstream activator of ROCK, similarly had no
effect on T cell attachment (see supplementary figure 1, available at
jcs.biologists.org/supplemental).
In summary, these data suggest that activation of myosin via MLCK, but not ROCK, has an important role in T cell attachment to ICAM-1.
MLCK and ROCK I expression in T cells
MLCK and ROCK have not previously been identified in T cells. Therefore,
confirmation of protein expression was determined by western blot analysis of
a T cell lysate. Human MLCK is expressed as a 130-150 kDa smooth muscle
form that is present in most cell types and variants of a larger
220 kDa
non-smooth muscle form (Blue et al.,
2002
; Kamm and Stull,
2001
). Two bands were detected in the T cell lysate, one
corresponding to 142 kDa and another at 166 kDa
(Fig. 2). As a positive
control, the small and large forms of 142 and
220 kDa, respectively, were
observed in murine lung cell lysate using anti-MLCK mAb K36. By comparison
with murine lung cells and other cell lines (data not shown), MLCK appears to
be expressed in low abundance in human T cells.
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T cells contained ROCK I at 160 kDa as detected with anti-ROCK I mAb (Fig. 2). The blots of the T cell and lung lysates were also probed with control ascites or IgG1 mAb and showed no positive labelling (data not shown).
LFA-1-mediated T cell attachment to and migration on ICAM-1
T cells are observed to migrate on ICAM-1-expressing endothelium. We
therefore used video microscopy to investigate any alterations in T cell
morphology that occur upon contact with ICAM-1. In the presence of
Mg2+/EGTA, the T cells adhere to ICAM-1, and, by 45 seconds, they
extend membrane projections that appear to govern the subsequent polarization
of the T cell (Fig. 3; see
Movie 1, available at
jcs.biologists.org/supplemental).
Between 90-120 seconds, T cell polarization becomes evident in that a ruffling
leading edge dominates and the opposing edge contracts to form the trailing
edge. By 150 seconds, the T cell becomes motile, indicating that the migratory
machinery is fully engaged. A common feature of T cell migration in the
absence of a chemotactic gradient is a frequent change in direction (see Movie
1 and Fig. 4D). In contrast, in
the absence of Mg2+/EGTA, T cells plated on ICAM-1 were
non-attached (Fig. 4A), and
Mg2+/EGTA-treated T cells were similarly non-attached in the
absence of ICAM-1 (BSA only) (data not shown). Thus attachment of T cells to
ICAM-1 causes a signal that initiates polarization followed by migration.
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A role for MLCK in T cell migration on ICAM-1
Because our data suggested involvement of MLCK in T cell attachment, we
investigated its possible role in T cell migration on ICAM-1. When T cells
made contact with ICAM-1 in the presence of Mg2+/EGTA, attachment,
polarization and migration occurred (Fig.
4B). These activities did not happen when Mg2+/EGTA was
omitted (Fig. 4A) and were also
prevented by preincubation with the MLCK inhibitor, ML-7
(Fig. 4C).
To test the effects of ML-7 on cell migration, T cells were allowed to migrate on ICAM-1 for 30 minutes and then tracked for an additional 70 minutes. Tracking individual cells showed that they followed a random course of migration with an average speed of 10.3±0.1 µm/minute (Fig. 4D). Addition of ML-7 resulted in the average speed decreasing to 0.71±0.03 µm/minute. This showed that blocking MLCK activity prevented T cell migration (Fig. 4E). To investigate the mechanism of the MLCK inhibition in more detail, the behavior of migrating T cells was followed by time lapse microscopy from the time of exposure to ML-7. In the presence of 20 µM ML-7, lamellar extension ceased within 1 minute and the main cell body gradually retracted and collapsed back towards the trailing edge (Fig. 5; see Movie 2, available at jcs.biologists.org/supplemental). These results provide the first evidence that MLCK has a key role at the leading edge of the migrating T cell.
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A role for CaM in T cell migration
A known regulator of MLCK activity is CaM, which activates MLCK by binding
to its regulatory domain (Lukas et al.,
1986). The CaM inhibitor W-7 blocks T cell attachment to ICAM-1
with maximum effect at 50-100 µM (Fig.
6A). Microscopic examination showed that T cells, in contact with
ICAM-1 and Mg2+/EGTA, attached and polarized, but following
preincubation with W-7, the cells were rounded and failed to polarize (data
not shown). To assess the effect on cell migration, T cells were tracked over
60 minutes after exposure to W-7. As previously noted, individual cells
displayed a random course of migration with an average speed of
7.94±0.15 µm/minutes, but following preincubation with 50 µM W-7,
the average speed decreased to 1.53±0.15 µm/minutes
(Fig. 6B). To assess, in more
detail, where CaM might be acting in the migratory process, T cells were
allowed to migrate on ICAM-1 for 20 minutes prior to exposure to 50 µM W-7.
After treatment with W-7, lamellar extension rapidly ceased, and the main cell
body gradually retracted, mirroring the effects of MLCK inhibition
(Fig. 6C; Movie 3,
available at
jcs.biologists.org/supplemental).
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T cell migration on ICAM-1: a role for ROCK in deadhesion
Inhibition of ROCK with Y-27632 had no effect on T cell attachment to
ICAM-1 (Fig. 1B). However, when
migrating T cells were treated with 10 µM Y-27632, the trailing edge of the
T cells was substantially elongated compared with control T cells
(Fig. 7A). Incubation of the T
cells with C3 exoenzyme mirrored this effect, indicating a role for RhoA as
the activator of ROCK (Fig. 7B,
and supplementary figure 1, available at
jcs.biologists.org/supplemental).
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Tail elongation could potentially occur via an extensive protrusion from the cell or, alternatively, by a failure to detach the trailing edge as the cell moved forward. To analyze the effect of ROCK inhibition at the single cell level, we again used time lapse microscopy to visualize the cells. T cells were allowed to attach and migrate for 20 minutes on ICAM-1 before Y-27632 was added. Observation of T cells following Y-27632 treatment revealed that elongation occurred because the leading edge continued to be highly motile, whereas movement ceased at the trailing edge (Fig. 7C, Movie 4, available at jcs.biologists.org/suplemental). This was attributable to the tail remaining firmly anchored to ICAM-1. This lack of detachment had a negative effect on T cell migration, resulting in an average decrease in cell speed from 12.60±0.22 to 4.81±0.13 µm/minute (Fig. 7D). Therefore, inhibition of ROCK did not prevent activity at the leading edge of the T cell, but prevented detachment at the trailing edge necessary for cellular translocation.
Distinct roles of the MLCK and ROCK as assessed by cell
spreading
To gain further insight into the roles of MLCK and ROCK in T cell
migration, we examined their contribution to the spreading of T cells on
ICAM-1. We tested the effect of the inhibitors by quantifying both area of
cell spread and also the length of the migrating T cell. There was a reduction
in area of cell spread of T cells treated with ML-7 (21.1%) and W-7 (22.8%) on
ICAM-1 compared with control migrating T cells
(Fig. 8A). This reduction was
similar to that of non-spreading T cells on control BSA substrate (29.4%).
There was also a loss of cell length induced by ML-7 (35.7%) and W-7 (26.8%)
as expected with the loss of cell spreading and retraction to a rounded shape
(Fig. 8B).
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In contrast, ROCK inhibition produced no significant alteration in cell spread area compared with the control T cells (Fig. 8A). However, the Y-27632-treated T cells showed an average overall increase in cell length of 40% compared with untreated T cells (Fig. 8B). RhoA inhibition increases cell length in a comparable fashion to ROCK inhibition (Fig. 8B).
Subcellular localization of actin, myosin and the two kinases, MLCK
and ROCK
The findings so far have indicated that the kinases MLCK and ROCK are
important for forward propulsion and rear detachment, respectively, during
LFA-1-mediated T cell migration on ICAM-1. It was, therefore, of interest to
localize the two kinases in the migrating T cell and to compare their
distribution with that of F-actin and myosin II. Immunofluorescence revealed
that the F-actin filaments were concentrated predominantly in the
lamellipodial projections at the leading edge of the T cell with much lower
staining in the main body of the cell (Fig.
9). MLCK was most concentrated at the leading edge of the T cells
overlapping with F-actin positive lamellipodial extensions and was not
detectable at the same level elsewhere
(Fig. 9A). In contrast, ROCK
staining was more generally distributed in the tail region
(Fig. 9B). Thus MLCK and ROCK
are spatially separated in the cell.
|
Given that MLCK and ROCK show different distribution patterns, we next investigated the expression pattern of myosin II. There was a high concentration of myosin at the leading edge of the cell where the pattern of staining ranged from either lining the F-actin-positive extensions (Fig. 9C) or overlapping with the F-actin-positive lamelli (data not shown). Myosin was also expressed in the tail region of the polarized T cell where F actin was also localized. In summary, myosin was strongly detected in the leading edge and tail of the T cell, corresponding to the locations enriched in MLCK and ROCK respectively.
MLCK and ROCK both affect T cell migration but by different
routes
Inhibiting either MLCK (with ML-7 or indirectly through the CaM
inhibitor,W-7) or ROCK (Y-27632) reduced the speed of T cell migration. An
average speed of 10.30±1.74 µm/minute was reduced to
0.71±0.03 (ML-7), 1.53±0.15 (W-7) or 4.81±0.13 (Y-27632)
µm/minute (Fig. 10A). In
order to determine how general were the described inhibitory effects of ROCK
and MLCK, we quantified the proportion of attached cells within the T cell
population that were actively migrating, displaying lamellar protrusion and,
finally, displaying firmly anchored tails. In a population of untreated T
cells, 63% were actively migrating, whereas 37% polarized but failed to
migrate (Fig. 10B). All cells
exhibited active lamella. In the presence of Y-27632, the proportion of
migrating cells was reduced with a concomitant increase in the number of cells
with anchored tails. Importantly, as with untreated T cells, these cells
exhibited lamellar protrusion, providing further evidence that the activity of
ROCK was restricted to the trailing edge. However, when MLCK (or CaM) was
blocked, the percentage of cells that were migrating dropped to <10% of the
total, and this was accompanied by a loss of lamellar extension activity.
|
When both ML-7 and Y-27632 were co-incubated with the T cells, the ML-7 effect prevailed with no moderating effect of Y-27632. (see supplementary figure 1, available at jcs.biologists.org/supplemental). These data suggest that MLCK function at the leading edge dominates the ROCK-generated events at the trailing edge in terms of T cell migration. Evidence for the independent control of T cell migration by MLCK and ROCK was demonstrated by coincubating T cells with their respective inhibitors (Movie 5, available at jcs.biologists.org/supplemental). T cells were first exposed to Y-27632 and displayed the attached tail phenotype. Subsequently, following MLCK inhibition, it was observed that the collapse of the leading edge did not affect the firmly anchored tail. This further demonstrates that compartmentized signaling events are required for the attachment and detachment of the migrating T cell.
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Discussion |
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MLCK is located at the leading edge of the T cell where the lamellipodia
are in a continual and rapid process of protruding and retracting as the cell
moves forward. MLCK inhibition prior to T cell contact with ICAM-1 greatly
reduces cellular attachment, showing that it has a direct role in this
activity. Insight into this process was obtained when MLCK activity is
inhibited after cell migration is underway. In this situation, lamellipodial
extensions collapse and the T cell retracts towards the trailing edge. This is
further confirmed by the reduction in cell spreading to levels similar to the
non-motile T cells. These observations indicate that MLCK has an active role
at the front of the migrating T cell in extending the leading edge and
permitting new attachments required for forward movement. A role for MLCK in
cell division and migration has also been described for PTK2 kangaroo rat
epithelial cells (Chew et al.,
2002).
The upstream activator of MLCK is CaM in many cell types
(Lukas et al., 1986). In this
study, the effect of inhibiting MLCK in T cells is mirrored by inhibiting the
activity of CaM. Many downstream effectors are activated by CaM, and it is
therefore of interest that MLCK appears to be its predominant target in the
migrating T cell. The only recognized substrate of MLCK is MLC, which is
phosphorylated on Thr18 Ser19, thereby activating myosin ATPase activity
(reviewed in Bresnick, 1999
).
There is also evidence in smooth muscle cells that the actin-bundling activity
of MLCK, rather than the kinase activity, is dominant in membrane ruffling
(Kishi et al., 2000
;
Ye et al., 1999
). The
inhibitor ML-7 and the MLCK auto-inhibitory peptide used in this study both
target the kinase domain and block catalytic activity of MLCK
(Saitoh et al., 1987
;
Tanaka et al., 1995
). The fact
that these agents prevent T cell attachment and migration provides evidence
that it is the kinase activity of MLCK that is paramount.
A number of forms of human MLCK have been identified, of which the most
common are a `small' 130-150 kDa smooth muscle form and a `large' higher
molecular weight non-smooth muscle form of
220 kDa
(Blue et al., 2002
;
Kamm and Stull, 2001
). T cells
contain a
140 kDa MLCK, which equates to the small form and an
intermediate molecular weight form of
166 kDa. No band could be detected
in T cells that represented the large 220 kDa MLCK. In comparison with murine
lung cells and other cell lines (data not shown), MLCK appears to be expressed
in low abundance in human T cells. Four other variants of the MLCK protein
have been described previously (Lazar and
Garcia, 1999
) and, from the chicken genomic sequence, the
identification of canonical splice consensus sites in each of the 31 exons
raises the possibility that more forms exist
(Birukov et al., 1998
).
In fibroblasts, the large form of MLCK contains five rather than the three
actin-binding motifs found in the small form
(Kudryashov et al., 1999;
Smith et al., 2002
;
Smith et al., 1999
). The
resulting higher affinity for actin (Smith
et al., 2002
) is suggested as an explanation for the targeting of
the large form of MLCK to actin stress fibers and to the cleavage furrow of
dividing cells (Poperechnaya et al.,
2000
). In our study we show that there is a high degree of
colocalization of MLCK with the dense networks of F-actin comprising the
dynamic T cell lamelli. It is tempting to speculate that the 166 kDa MLCK,
which we identify here, also contains extra actin-binding motifs,
strengthening binding to F-actin. Further characterization of T cell MLCK will
be needed to test this hypothesis.
ROCK, like MLCK, is a major serine/threonine kinase that phosphorylates
MLC, leading to activation of myosin ATPase activity
(Amano et al., 1996). The
negligible effect on T cell attachment assays of inhibiting ROCK and its
activator RhoA (supplementary figure 1, available at
jcs.biologists.org/supplemental)
suggested that they play no major role in the signaling events responsible for
T cell attachment to ICAM-1. However, video microscopy revealed a dramatic
effect of ROCK inhibition on the migratory ability of T cells. Blocking ROCK
resulted in the trailing edge of the T cell remaining firmly adhered to
ICAM-1, whereas the leading edge continued to progress forward. These two
opposing processes resulted in the generation of an artificially elongated
cell.
The failure of tail detachment when ROCK (and RhoA) activity is inhibited
could occur because of an increased number of LFA-1-mediated attachments
rather than an increase in stability of the pre-existing attachments. However,
as both cell attachment and cell spreading analyses provide no evidence for an
increase in attachments, the most likely explanation is that ROCK inhibition
increases the stability of pre-existing attachments, leading to the failure to
detach the trailing edge in coordination with the forward movement at the
front end of the cell. Alternatively, by causing MLC phosphorylation and
activating the myosin motor, ROCK may enhance the contractile force of the
trailing edge, thereby overcoming the binding strength of the LFA-1/ICAM-1
adhesions. ROCK also contributes to phosphorylated MLC stability via
inhibition of myosin phosphatase (Kimura
et al., 1996). Additionally, ROCK can phosphorylate other
substrates such as moesin, adducin, intermediate filament proteins, LIM kinase
and the NaH exchanger (Fukata et al.,
1998
; Fukata et al.,
1999
; Kosako et al.,
1997
; Sumi et al.,
2001
; Tominaga et al.,
1998
). ROCK may therefore promote de-adhesion by a pathway not
involving myosin. However preliminary evidence indicates that inhibition of
ROCK activity by Y-27632 decreases the phosphorylation of MLC on Ser 19, which
pinpoints MLC as the relevant downstream target of ROCK in migrating T cells
(data not shown). This same finding also makes it less likely that the effects
of Y-27632 are attributable to inhibition of the Rho/Rac binding kinase, PRK2,
as has recently been described (Davies et
al., 2000
).
The upstream activator of ROCK is RhoA in other cell types
(Ridley, 2001), and we show
here that the RhoA inhibitor C3 exoenzyme mimics the effects of ROCK
inhibition in causing tail elongation in migrating T cells. Other evidence
that RhoA is active in T cells comes from the analysis of RhoA activity using
an in vitro kinase pull-down assay with Rhotekin
(Ren et al., 1999
), which
showed a time-dependent increase in RhoA activity that was dependent upon
contact with ICAM-1 (data not shown). A similar role for Rho A and ROCK has
been described for de-adhesion of the tail end of migrating eosinophils
(Alblas et al., 2001
) and
monocytes (Worthylake et al.,
2001
). Thus ROCK appears to be required for detachment of the
trailing edge, allowing forward migration of several types of leukocytes.
Our data show that there is spatial regulation of MLCK and ROCK activity,
initiated by LFA-1 binding to ICAM-1, with MLCK operating at the leading edge
and ROCK found in highest concentration in the body and trailing edge of the
migrating T cell. The distribution of these two kinases suggests that there
are two compartments with distinct functions operating in the migrating T
cell. Separation of these two kinases has been observed previously in murine
3T3 (Totsukawa et al., 2000)
and human fibroblasts (Katoh et al.,
2001
). In these studies, ROCK activity was associated with stress
fibers in the center of cells, whereas MLCK was associated with peripheral
cortical microfilaments. In contrast, when neutrophil migration was examined,
blocking of both ROCK (Niggli,
1999
) and MLCK (Eddy et al.,
2000
) caused delayed retraction of the tail end of the cell,
suggesting they were both active in the same cellular compartment. Differences
in migratory machinery may exist between even closely related cell types that
are tailored to the role that migration performs in cellular function.
Separate control of MLCK and ROCK enables the front and rear of the cell to
contribute in different ways to T cell migration. There are several
speculations as to why these two kinases operate in different areas within a
migrating T cell. MLCK phosphorylates MLC in vitro with a sixfold higher Km
than ROCK (Amano et al., 1996).
It may be that the rapid lamellar activity, which is associated with MLCK,
requires a kinase with high Km, whereas the generalized contraction of the
tail end of the migrating cells may be better effected by a slow acting kinase
like ROCK. The dynamic lamellar movement at the leading edge may also benefit
from myosin phosphatase activity that would be enhanced in a compartment of
reduced ROCK activity. Coordination of the two compartments must lie farther
upstream from the immediate activation of MLCK and ROCK by CaM and RhoA,
respectively. In the BHK cell line there is a link between the GTPases Rac and
RhoA in control of MLC phosphorylation, with Rac causing MLCK inactivation
through PAK1-mediated phosphorylation, and RhoA causing ROCK activation
(Sanders et al., 1999
). How
these activities are coordinated and whether similar pathways exist in primary
T cells is not known.
Direct activation of LFA-1 followed by exposure to immobilized ICAM-1 is
sufficient to cause T cells to attach, polarize and randomly migrate within 3
minutes. This migration is dependent on MLCK and ROCK, which control the
activity of acto-myosin. Our in vitro analysis of T cell migration parallels a
recent two-photon microscopic study showing randomly moving T cells in the
lymph node with a speed of 11 µm/minute
(Miller et al., 2003). These
two techniques will provide a powerful combination to unravel the mechanisms
involved in T cell migration. ICAM-1 is not only expressed in lymphoid tissue
but is also rapidly induced in other tissues following inflammatory stimuli.
This suggests that T cell migration is relevant not only in the lymphoid
compartment but also in other tissues. LFA-1 has previously been associated
with the acto-myosin transport involved in delivering receptors to the contact
area or `synapse' by which leukocytes contact target cells, implying that
LFA-1 signaling also has a role in cell-cell contact events
(Wulfing and Davis, 1998
). In
summary, LFA-1 is not just an adhesion receptor but also a signaling molecule
that can direct acto-myosin-based T cell functions by its coordinated
activation of MLCK and ROCK.
![]() |
Acknowledgments |
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![]() |
Footnotes |
---|
Present address: Department Pharmacoepidemiology and Pharmacotherapy,
Utrecht Institute for Pharmaceutical Sciences, PO Box 80082, 3508 TB Utrecht,
The Netherlands
Present address: Sackler Institute for Muscular Skeletal Research,
Department of Medicine, University College London, 5 University Street, London
WC1E 6JJ, UK
¶ Present address: MRC Laboratory for Molecular Cell Biology, University
College London, Gordon Street, London WC1E 6BT, UK
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