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1
*
Department of Clinical Medicine, University of Dublin, Trinity College, Dublin, Ireland; and
Royal College of Surgeons, Dublin, Ireland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, are targeted to the cytoskeleton with specific
localization corresponding to the microtubule-organizing center (MTOC)
and microtubules, as detected by immunocytochemistry and
immunoblotting. Clustering of LFA-1 associated with its signaling
function also occurs at the membrane sites adjacent to the MTOC. We
further show that cells of a PKC-ß-deficient clone derived from
parental PKC-ß-expressing T cell line can neither crawl nor develop a
polarized microtubule array upon integrin cross-linking. However, their
adhesion and formation of actin-based pseudopodia remain unaffected.
Our data demonstrate the critical importance of the microtubule
cytoskeleton in T cell locomotion and suggest a novel
microtubule-directed intracellular signaling pathway mediated by
integrins and involving two distinctive PKC
isoforms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and protein kinase C
(PKC)3 (1). However, the
exact sequence of integrin-mediated signaling events resulting in
cytoskeletal rearrangements and cell locomotion is not well
defined.
Cross-linking of cell surface adhesion receptors by mAbs
mimicking to a certain extent multivalent interactions with natural
ligands (2) has been successfully used as a model to study
intracellular signaling processes mediated by integrins and CD44 (3, 4). Ab-induced effects in this case are often judged by homotypic
aggregation (4), clustering of cytoskeletal and signaling proteins (2),
or cell motile characteristics (3). To investigate LFA-1-mediated
signaling in T cells, we gave preference to the reporter system based
on the induction of cell locomotion, as potentially more closely
related to physiological phenomena taking place, for instance, at the
stage of cell extravasation. In the present study, we used the model
described earlier (5) in which cells of the human T lymphoma line
HUT-78 or activated human peripheral blood T lymphocytes (PBTL)
were exposed to a triggering signal via LFA-1 by immobilized mAb
(mAb(i)) specific for its 
L-chain. In this system, T
cells adopted a locomotion-associated phenotype on anti-LFA-1 mAb.
Preactivation via TCR-CD3 complex or phorbol ester (PMA) treatment was
required for the development of motile phenotype in normal PBTL (5) and
represents an essential step in LFA-1-mediated lymphocyte adhesion (6).
PMA is a potent activator of the intracellular phosphorylation
enzymes of the PKC family. It includes the growing number of isoenzymes
grouped as follows: classical (Ca2+-dependent and activated
by diacylglycerol and PMA (, ß(I), ß(II), and )), novel
(Ca2+-independent (, 
, 
, 
, µ)), and atypical
(phospholipid- and Ca2+-independent (
, 
,
)). PKC-ß has been demonstrated to undergo translocation to
the plasma membrane from the cytosolic pool and cytoplasmic vesicles
containing ß2 integrins in response to phorbol ester
treatment (7). This isoform also has been reported to colocalize with
microtubule-associated proteins and to be physically linked with the
actin cytoskeleton (7, 8). Redistribution of novel PKC isoforms and
between cytosolic and cytoskeletal fractions can be modulated by
PKC agonists and specific inhibitors (9, 10). We herein demonstrate
that cross-linking of LFA-1 resulted in specific translocation of
PKC-ß(I) and isoforms to the cytoskeleton with a pattern
consistent with microtubule-organizing center (MTOC) and microtubules.
We analyzed this phenomenon in conjunction with the other LFA-1-induced
intracellular changes from the point of view of its impact on T cell
locomotory behavior.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal human PBTL, isolated as described (5), were preactivated by treatment with 25 ng/ml PMA (Sigma, St. Louis, MO) for 48–72 h at 37°C unless specifically indicated otherwise in the text and figure legends. Human T lymphoma cell line HUT-78 (American Type Culture Collection, Manassas, VA) were used nontreated or preactivated by PMA in the same concentration for 60 min in several experiments. RPMI 1640 culture medium on HEPES buffer (Life Technologies, Paisley, U.K.) supplemented with antibiotics and 10% FBS was used in all experiments, unless stated otherwise in the text.
Cell adhesion, motility, and transmigration
HUT-78 or activated PBTL (10–20 x 103/well)
were added to 8-well Permanox plastic chamber slides (Nunc, Naperville,
IL) coated with mAb to -chain of LFA-1, clone SPV-L7 (Sanbio, Uden,
The Netherlands) at 1.75 µg/ml as described (5). Control chambers
were treated similarly with isotype-matched murine IgG (Dako, Bucks,
U.K.). In several experiments, the chambers were coated with another
locomotion-inducing mAb to human -LFA-1 (clone YTH-81.5) at 5
µg/ml (Serotec, Oxford, U.K.). These mAbs both proved to be equally
potent in inducing cytoskeletal changes in T cells. Anti-LFA-1 mAb
MEM-83 were used for the same purposes at 2 µg/ml. Chimeric ICAM-1-Fc
fusion protein (kindly provided by A. Craig, Oxford, U.K.) was used in
motility studies at 10 µg/ml coating concentration, and cellular
fibronectin from human foreskin fibroblasts (Sigma) was used at 25
µg/ml. Cell motility on different integrin ligands was also
assessed using 3-µm membrane pore filters precoated with fibronectin,
laminin, and collagen type I and IV by the manufacturer (Becton
Dickinson Labware, Bedford, MA).
Broad spectrum kinase inhibitor staurosporine and selective PKC- and
-ß inhibitor Go6976 used in functional studies were purchased from
Calbiochem (Nottingham, U.K.). After 4 h of incubation in culture
medium or under specific experimental conditions as described in the
figure legends, unattached cells were removed by triple gentle washing
of wells with warmed culture medium. The fraction of adherent cells was
calculated as a percentage of cells remaining attached to substrate
from the initial cell count (before washing the wells). Motility was
assessed by estimating the ratio of cells undergoing cytoskeletal
rearrangements and formation of uropods (locomotion-associated
phenotype, Figs. 1
d and
2, a–f) of the total
number of adherent cells per microscopic field. At least five randomly
chosen fields at x400 magnification were analyzed for each
experimental condition. Transmigration experiments were performed in a
modified Boyden chamber assay using polyethylene terephtalate
track-etched membrane filters with 3 µm pore size (Becton Dickinson
Labware, Bedford, MA) in 24-well plates. Anti-LFA-1 mAb or control IgG
were immobilized on the upper side of the filters in the same way as
for chamber slides (5). The lower side of the filters was left
uncoated. Activated PBTL (500 µl of suspension at 106
cells/ml) were added to the filter chambers, while the lower
compartments were filled with culture medium alone or supplemented with
50 ng/ml human recombinant RANTES (Sigma) and incubated overnight at
37°C. After removing the filter chambers, transmigrated cells from
the bottom of the plate wells were collected by pipetting and counted
in a haemocytometer. Four grids corresponding to 0.1 mm3
suspension volume were averaged to estimate cell number in each well.
Mean values for each experimental condition were obtained from six
wells.
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Nikon Diaphot inverted microscope (Nikon Europe, Badhoevedozp, The Netherlands) with CCD video camera (Sony Corporation, Tokyo, Japan) was used for image acquisition, phase contrast observations, and microphotographs. Analysis of acquired video and photographic images was performed on a Macintosh computer using the National Institute of Health Image program (developed at the U.S. National Institutes of Health and available on the Internet).
Bead preparation
Polystyrene beads (0.8 µm) (Sigma) coated with anti-LFA-1 mAb as described for chamber slides (5) were added to the chambers (1000:1 bead:cell ratio) when the cells had established a locomotory phenotype (60 min after contact with mAb(i). Following 15 min incubation, unbound particles were removed by gentle washing and refilling of the chambers with warmed culture medium.
Immunofluorescence microscopy
The slides with attached cells were fixed/permeabilized in
acetone at -20°C. Ab to PKC isoforms , , , , , and
(Research & Diagnostic Abs, Berkeley, CA) and ß(I) and ß(II)
(Sigma) were used with FITC-labeled secondary Ab (Sigma). Ab blocked
with relevant peptide Ag were used for negative control staining. The
tetramethylrhodamine isothiocyanate (TRITC) conjugate of phalloidin
(Sigma) was used to stain filamentous actin (F-actin); mAb to LFA-1
(Sanbio), vimentin (Dako), and -tubulin were used with TRITC-labeled
secondary affinity-purified Ab (both from Sigma). Light and fluorescent
microscopy and microphotography were performed on a Leica
photomicroscope (Leica Microscopy Systems, Heerbrugg, Switzerland)
using Kodak Panther 1600 or Kodak Elite II-400 reversible films
(Eastman Kodak, Rochester, NY). Equal exposure times were used to
photograph cells with specific and negative control staining.
Western blot analysis
To analyze distribution of PKC isoforms between the subcellular
fractions, HUT-78 either kept in suspension or attached to plastic via
mAb(i) were lyzed on ice in buffer A (20 mM Tris-HCl, pH 7.5,
containing 0.25 M sucrose, 2 mM EGTA, 2 mM EDTA, 1 mM PMSF, and 10
mg/ml leupeptin (all reagents from Sigma)), sonicated for 5 s and
spun down at 600 x g to remove the nuclei and unlyzed
cells. After centrifugation at 100,000 x g for 10 min
the resulting supernatant was designated as the cytosolic fraction. The
pellet was resuspended in buffer B (20 mM Tris-HCl, pH 7.5, containing
1% (w/v) Nonidet P-40, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, and protease
inhibitors), and centrifuged at 15,000 x g for 30 min.
The supernatant was designated as the detergent-soluble membrane
fraction. The pellet representing the detergent-resistant cytoskeletal
fraction was dissolved in boiling buffer C (20 mM Tris-HCl, pH 7.5, 1%
SDS, 150 mM NaCl, 1 mM EGTA, and 1 mM EDTA). Equal amounts of proteins
were separated on a 10% SDS-polyacrylamide gel, electrotransferred
onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA),
probed with mAb to PKC isoforms ß and (Transduction Laboratories,
Lexington, KY) or -tubulin (Sigma), and visualized with
Phototope-horseradish peroxidase detection system (New England
Biolabs, Hertfordshire, U.K.). Densitometry of the blots was performed
using National Institute of Health Image software. In a number of
control experiments, similar results were reproduced using rabbit
polyclonal Ab to PKC-ß(I) (Santa Cruz Biotechnology, Santa Cruz, CA)
and sheep Ab to PKC-, which were a gift from J. Lord (Department of
Immunology, University of Birmingham, U.K.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HUT-78 and preactivated PBTL (both originally nonadherent, Fig. 1
a) exposed to a triggering signal from anti-LFA-1
mAb(i) started spreading and subsequently underwent dramatic
cytoskeletal changes resulting in a polarized phenotype with long
cytoplasmic projections (up to 50–60 µm) over 60 min after the
beginning of the incubation (Fig. 1d). Stimulation with PMA
alone was not sufficient to induce these changes in either of cell
types (Fig. 1b), but it enhanced homotypic aggregation of
PBTL. Pretreatment with phorbol ester was necessary for adhesion and
the development of cytoskeletal rearrangements upon LFA-1 cross-linking
in PBTL, but not in HUT-78 (Fig. 1, c and d). As
seen from the time-lapse video images (Fig. 2, a–f),
the acquisition of this phenotype was directly associated with active
cell body translocation, while cytoplasmic processes were represented
by extended trailing cell tails (or uropods). Characteristic
migratory phenotype was induced in T cells by anti-LFA-1 mAb(i)
clones SPV-L7 and YTH-81.5, but not by mAb(i) to a number of other
abundant cell surface proteins including CD3, CD43, and MHC class I
(data not shown) or mAb to LFA-1 clone MEM-83 (5). Specific properties
of various LFA-1-binding mAb in respect to cell locomotion might be
dependent on the differences in the functional significance of the
recognized epitopes (11) or their binding affinity (12). This question
has been further addressed in this study (see below).
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Locomotory T cells triggered via LFA-1 mAb(i) in the absence of
obvious chemotactic gradient produced movements in an apparently random
manner (Fig. 2, a–f and Fig. 3, a–c), often undergoing a
complete 180° change in the direction of migration. We next examined
whether LFA-1 cross-linking could affect T cell migration in response
to the chemokine RANTES. We used a modified Boyden chamber assay with
small, relative to PBTL, diameter pore size (3 µm), where mAb were
immobilized on the upper side of the filter and human recombinant
RANTES (50 ng/ml) was added to the culture medium in the lower chamber
compartment. A selected concentration of the chemokine was established
as optimal in preliminary experiments. As shown in the Fig. 2g, transmigration of PBTL triggered by mAb(i) to LFA-1 was
significantly enhanced in the presence of the chemotactic gradient of
RANTES in comparison to control IgG (with or without the
chemoattractant). A relatively high rate of activated PBTL
transmigration on mAb(i) without RANTES is evidently due to increased
background cell locomotory potential.
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Dynamic redistribution of adhesion receptors in locomotory cells
was demonstrated using polystyrene beads coupled to anti-LFA-1 mAb
(Fig. 3, a–c). The mAb-coated beads added to HUT-78
initially underwent centripetal migration away from the leading edge
toward the rear of the nucleus over the MTOC (Fig. 3a and
4c, lower panel). Here they formed a
characteristic "necklace" of aggregated particles (Fig. 3a) reflecting the site of integrin clustering and were
subsequently redistributing in accordance with cyclic cytoskeletal
changes. LFA-1 aggregation was not induced by the beads themselves, as
shown by immunostaining of HUT-78 in bead-free conditions (Fig. 3d) and represented a specific step in cell migration,
because it had not been registered using the beads coated with
isotype-matched IgG or mAb to CD3 (data not shown).
A typical sequence of events reflecting a "successful" cell
movement (resulting in a net translocation of the cell body to a new
position) involved formation of the leading lamella (Figs. 2d and e and 3a), translocation of the
nucleus termed as "nucleokinesis" (13) (Fig. 2, b–d),
and extension of the trailing tail (uropod) accompanied by rearward
movement of deaggregated integrins, as indicated by LFA-1-coupled beads
(Fig. 3b). A contraction of the uropod with rear release of
bound membrane integrins on the substrate (Fig. 3e) and
centripetal recondensation of integrin clusters (Fig. 3c)
preceded the new locomotion cycle. Rear release of LFA-1 on the
substrate may in fact represent membrane "ripping" as one of the
mechanisms of uropod detachment in the migrating cell determining
sometimes the overall locomotion rate (14). This process has been
previously described in detail for fibroblasts migrating on laminin
using ß1 integrins (15).
Cytoskeletal rearrangements in migrating T cells involve actin, microtubules, and intermediate filaments
At the intracellular level, induction of motility in HUT-78
affected the actin cytoskeleton and microtubules as well as vimentin
intermediate filaments (Fig. 4).
Actin-containing filopodia were present at the leading edge and
trailing tails (Fig. 4a). The cell body and axial
longitudinal cytoskeleton contained thick vimentin filaments (Fig. 4b). The characteristic array of microtubules displayed that
the MTOC was located at the side of the nucleus opposing the direction
of cell migration and from here long microtubules extended to the
uropods, reflecting the state of cell polarization (Fig. 4c).
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to the microtubule cytoskeleton
We analyzed the redistribution of PKC isoforms representing all
three groups of isoenzymes (classical ( and ß(I)), novel (,
, , and ), and atypical ()) expressed in HUT-78 and PBTL in
conjunction with major cytoskeletal components. Resting HUT-78 stained
for PKC- developed a diffuse granular cytoplasmic pattern with
clearly distinguishable spots at the centrosomes of mitotic cells (Fig. 5, a and b).
PKC- displayed a dramatic translocation with a loss of diffuse
cytoplasmic pattern and localized to a compact spot corresponding to
the MTOC in migrating HUT-78 (Fig. 5, c and d).
Classical PKC-ß(I) demonstrated granular cytoplasmic staining as well
as a translocation pattern consistent with the position of MTOC and
microtubules in the uropods of locomotory cells in comparison to
relatively diffuse distribution in resting HUT-78 (Fig. 5, e–h).
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Western blot analysis of fractionated HUT-78 lysates confirmed the
increased association of PKC-ß isoenzyme with the detergent-insoluble
cytoskeletal fraction in motile cells in response to stimulation
through PMA and anti-LFA-1 (Fig. 5j). Interestingly,
there were apparently three closely related species of PKC-ß
specifically distributed between cytosol, membrane, and cytoskeletal
fractions and originally almost indistinguishable in resting HUT-78.
Accumulation of 83-kDa species of PKC-ß in detergent-resistant
skeleton of motile cells occurred concomitantly with the losses in the
85-kDa cytosolic and 80-kDa membrane-associated bands. This phenomenon
could be possibly due to the existence of alternatively spliced PKC-ß
variant forms with selective substrate specificity or, on the other
hand, could be reflecting different degrees of phosphorylation of the
PKC-ß molecule, by analogy to the data reported for classical PKC-
(17). PKC- was detected predominantly in the cytoskeleton of the
HUT-78 migrating on mAb(i) to LFA-1 (Fig. 5i). This
translocation was significant and comparable to that induced by the
potent PKC activator, PMA. The overall reduction of detectable PKC-
in migrating HUT-78 induced by cross-linking of LFA-1 (either alone or
following pretreatment with PMA) does not appear to represent a
nonspecific proteolytic cleavage event as this was not induced by PMA
alone nor was it observed for PKC-ß isoform.
Redistribution of PKC isoenzymes to the cytoskeleton in motile cells
was concordant with the enrichment of the microtubule content in this
subcellular fraction (Fig. 5k). By contrast, resting HUT-78
lysates yielded a high amount of soluble (unpolymerized) tubulin in the
cytosol. This observation can be possibly explained as related to the
increased microtubule stability properties in LFA-1-triggered
locomotory T cells, potentially required, for instance, at the stage of
the extension of trailing uropods. On the other hand, the increased
skeleton:cytosol ratio of tubulin may be due to a higher overall number
of long polymerized microtubules present in the motile (vs resting)
cells. Consequently, a substantial pool of assembled tubulin polymers
is retained in the cytoskeletal fraction even under the cold extraction
conditions used in our experiments to minimize PKC degradation.
However, these data do not exclude a possibility that the
cytoskeleton-directed redistribution of PKC may be also mediated via
its isoform-specific interactions with certain microtubule-associated
proteins.
Taken together, these findings correlate well with MTOC- and
microtubule-attributed immunohistochemical staining patterns of
PKC-ß(I) and isoforms and suggest their involvement in
integrin-mediated cytoskeletal rearrangement processes likely depending
on phosphorylation (1).
Selective PKC-ß blocking affects T cell locomotory potential but not adhesion on immobilized mAb to LFA-1
We further elucidated regulatory factors of T cell motility (Fig. 6). Induction of T cell locomotion upon
cross-linking of LFA-1 proved to be energy- and temperature-dependent
and required the presence of divalent Ca2+ and
Mg2+ cations, consistent with their participation in
classical PKC activation and magnesium ion-regulated integrin
functioning (18). Neither a broad-spectrum protein kinase inhibitor
staurosporine nor selective PKC- and -ß blocker Go6976 (19)
significantly changed cell adhesiveness to substrate, while they both
abrogated HUT-78 locomotion. Therefore, we assume that acquisition of a
migratory phenotype in T cells in our model is primarily dependent on
LFA-mediated PKC activation, while adhesion modification via
high-affinity LFA-1 molecules induced by inside-out signaling pathways
(20) may develop as a secondary event.
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, ß(I), ß(II), and —only the first
two are expressed in HUT-78 cells; therefore, we used a
PKC-ß-deficient clone K-4 derived from parental HUT-78 line (21) to
determine the PKC isoform playing the leading role in T cell locomotion
by exclusion method. K-4 cells proved to be nonmotile on mAb(i)
to LFA-1, though their adhesion remained unaffected (Fig. 6a). These cells were capable of spreading and generating
multiple filopodia, thus reflecting that the function of actin-based
locomotory mechanisms was not abrogated (Fig. 6b). At the
same time, PKC-ß-deficient K-4 cells failed to develop a highly
polarized microtubular array (Fig. 6c) typically displayed
by parental HUT-78 upon LFA-1 cross-linking. This provides strong
supporting evidence for the role of PKC-ß in the regulation of the
tubulin-based cytoskeleton and hence cell motility. As the actin-based
cytoskeleton appeared to function normally in K-4 cells, it is entirely
possible that the defects in microtubule reorganization seen in these
cells could be due to aberrations in PKC-ß-mediated
actin-microtubules link-up. T cell locomotion patterns on different integrin-binding substrates are characterized by ligand and epitope specificity
This part of study was aimed to investigate the correlation of the
T cell locomotion-associated phenomena observed for anti-LFA-1 Abs
with the events induced by other integrin-specific ligands. As seen
from the Fig. 7a, PBTL
preactivated by PMA and exposed to immobilized chimeric ICAM-1-Fc
protein imitating a native LFA-1 ligand are indeed characterized by a
phenotype similar to those described above that are induced by
anti-LFA mAb (compare with Figs. 1 and 2). PBTL in this
system also produced active crawling movements with net cell body
translocation, as confirmed by video recording (data not shown). Of
note, staining of the fixed slides with anti-LFA-1 mAb (Fig. 7, b and c) demonstrated the frequent presence of
integrin-positive tracks left behind by the migrating cells, by analogy
to the ripping release of LFA-1 taking place when the crawling
substrate was provided by immobilized Abs (Fig. 3e). This
may potentially represent one of the mechanisms regulating T cell
migration enabling the uropod detachment from the supporting matrix and
described earlier for non-T cell types (14).
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a). Clearly developed but
less pronounced locomotory phenotype (with shorter uropods) was induced
by this ligand in constitutively activated HUT-78 (Fig. 8b).
HUT-78 also displayed motile behavior with similar phenotypic patterns
on laminin and collagen IV, but not on collagen I (data not shown).
Cells of the PKC-ß-deficient HUT-78-derived subclone K-4 were able to
attach and spread on fibronectin, but remained nonmotile (Fig. 8c). Therefore, cell motility could not be induced via the
ß1 or ß2 integrins studied (see Fig. 6b) in the absence of this particular PKC isoform.
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a) and did not acquire a
typical locomotory phenotype. At the same time, as shown on Fig. 9, b and c, respectively, neither PKC-ß(I) nor
PKC- underwent a dramatic redistribution demonstrated above for
motile cells (compare to Fig. 5, g and c). In
these experimental conditions, a substantial portion of PKC-attributed
staining remained in the cytosol in a pattern characteristic of resting
(static) cells (as on Fig. 5, a and e).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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with centrosomes demonstrated in the present study is in
agreement with its previously shown importance for mitotic division
(25) and can reflect the involvement of this enzyme in
MTOC-orchestrated cell functions. PKC recruitment in regulation of
microtubule-dependent motility systems deserves special attention in
view of their impact on polarization and direction of pseudopodial
activity in epithelial cells (26), neutrophils (27), and T cells (28).
Microtubule rearrangement can also generate forces contributing to
nucleokinesis in migrating neurons, for example (29). PKC engagement in
cytoskeletal assembly induced by phorbol esters has been shown earlier
in human T lymphocytes (30) and neutrophils (31). From this point of
view, the selective accumulation of PKC-ß and - in the
cytoskeletal fraction commencing upon LFA-1 cross-linking suggests
distinct cellular functions of individual PKC isoenzymes (32).
Taken together, our data demonstrate that cross-linking of LFA-1 in
HUT-78 and activated PBTL by mAb(i) induces dynamic redistribution of
PKC isoenzymes and cytoskeletal rearrangements and is accompanied by
cell locomotion. Redistribution of PKC isoforms ß(I) and may play
a critical role in the regulation of microtubule-driven events in T
cell motility. This can involve either direct phosphorylation of
microtubule proteins or closely associated molecules that regulate
their assembly. Potential specific functions of other PKC isoforms in
lymphoid cell locomotion still remain to be identified. Disclosure of
these functions will contribute to the dissection of the mechanisms of
cell motility and to the understanding of factors affecting both the
migration of normal cells and the metastatic potential of malignant
cells possibly leading to the development of new therapeutic
strategies.
The nature of LFA-1 interaction with its potential physiological ligands remains to be examined. Motility-inducing properties of purified ICAM-1 on reconstituted lipid bilayers have been shown earlier in large granular lymphocytes (33). It has been also reported that adhesion and cytoskeletal rearrangements in T cells can be triggered by recombinant soluble ICAM-1-Fc fusion protein (18, 34, 35). The phenotypic changes demonstrated for T lymphoblasts adhering to ICAM-1 (18, 36) are different from those described in this study. In our experiments, maximal length of trailing uropods was usually reached after 3–4 h incubation on immobilized Abs and 5–6 h following the T cell exposure to ICAM-1 or extracellular matrix components. Therefore, the short-term (30–40 min) incubations commonly used in adhesion assays may be not sufficient for developing the advanced picture of motility-associated phenomena. Of note, the phenotype closely resembling that registered in our model system was documented for the human Ag-specific T cell line CFTS 4:2.80 exposed to a number of extracellular matrix components 5 h after triggering with anti-ß1 and ß2 integrin Abs (3). The potential physiological significance of the observed phenomena is further supported by the finding that LFA-1 cross-linking augments RANTES-directed chemotaxis of PBTL. These data suggest the possibility that signaling through LFA-1 in activated T cell could potentially facilitate its migration through vascular endothelium along a chemotactic gradient.
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Acknowledgments |
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Abs, respectively. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Yuri Volkov, Department of Clinical Medicine, University of Dublin, Trinity College, The Trinity Centre for Health Sciences, James’s Street, Dublin 8, Ireland. E-mail address:
3 Abbreviations used in the paper: PKC, protein kinase C; HUT-78, T lymphoma cell line HUT-78; PBTL, peripheral blood T cells; mAb(i), immobilized mAbs; MTOC, microrubule-organizing center; F-actin, filamentous actin; TRITC, tetramethylrhodamine isothiocyanate.
Received for publication April 20, 1998. Accepted for publication August 17, 1998.
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 V. Denis and M. S. Cyert Molecular Analysis Reveals Localization of Saccharomyces cerevisiae Protein Kinase C to Sites of Polarized Growth and Pkc1p Targeting to the Nucleus and Mitotic Spindle Eukaryot. Cell, January 1, 2005; 4(1): 36 - 45. [Abstract] [Full Text] [PDF]
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 M. Conrad, J. DeNobile, I. Chaikhoutdinov, D. Escribano, K.-G. Lee, and W. D. Cohen Cytoskeletal Organization of Limulus Amebocytes Pre- and Post-Activation: Comparative Aspects Biol. Bull., August 1, 2004; 207(1): 56 - 66. [Abstract] [Full Text] [PDF]
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 A. Sundaresan, D. Risin, and N. R. Pellis Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes J Appl Physiol, June 1, 2004; 96(6): 2028 - 2033. [Abstract] [Full Text] [PDF]
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A. Vroon, C. J. Heijnen, M. S. Lombardi, P. M. Cobelens, F. Mayor Jr, M. G. Caron, and A. Kavelaars Reduced GRK2 level in T cells potentiates chemotaxis and signaling in response to CCL4 J. Leukoc. Biol., May 1, 2004; 75(5): 901 - 909. [Abstract] [Full Text] [PDF]
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D. Chen, A. Purohit, E. Halilovic, S. J. Doxsey, and A. C. Newton Centrosomal Anchoring of Protein Kinase C {beta}II by Pericentrin Controls Microtubule Organization, Spindle Function, and Cytokinesis J. Biol. Chem., February 6, 2004; 279(6): 4829 - 4839. [Abstract] [Full Text] [PDF]
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A. Sumoza-Toledo and L. Santos-Argumedo The spreading of B lymphocytes induced by CD44 cross-linking requires actin, tubulin, and vimentin rearrangements J. Leukoc. Biol., February 1, 2004; 75(2): 233 - 239. [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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K. A. Carnevale and M. K. Cathcart Protein Kinase C {beta} Is Required for Human Monocyte Chemotaxis to MCP-1 J. Biol. Chem., July 3, 2003; 278(28): 25317 - 25322. [Abstract] [Full Text] [PDF]
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A. Nakhost, N. Kabir, P. Forscher, and W. S. Sossin Protein Kinase C Isoforms Are Translocated to Microtubules in Neurons J. Biol. Chem., October 18, 2002; 277(43): 40633 - 40639. [Abstract] [Full Text] [PDF]
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M. Slevin, S. Kumar, and J. Gaffney Angiogenic Oligosaccharides of Hyaluronan Induce Multiple Signaling Pathways Affecting Vascular Endothelial Cell Mitogenic and Wound Healing Responses J. Biol. Chem., October 18, 2002; 277(43): 41046 - 41059. [Abstract] [Full Text] [PDF]
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J. L. Rodriguez-Fernandez, L. Sanchez-Martin, C. A. de Frutos, D. Sancho, M. Robinson, F. Sanchez-Madrid, and C. Cabanas LFA-1 integrin and the microtubular cytoskeleton are involved in the Ca2+-mediated regulation of the activity of the tyrosine kinase PYK2 in T cells J. Leukoc. Biol., March 1, 2002; 71(3): 520 - 530. [Abstract] [Full Text] [PDF]
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S. J. Hyduk and M. I. Cybulsky {alpha}4 Integrin Signaling Activates Phosphatidylinositol 3-Kinase and Stimulates T Cell Adhesion to Intercellular Adhesion Molecule-1 to a Similar Extent As CD3, but Induces a Distinct Rearrangement of the Actin Cytoskeleton J. Immunol., January 15, 2002; 168(2): 696 - 704. [Abstract] [Full Text] [PDF]
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A. Long, D. Kelleher, S. Lynch, and Y. Volkov Cutting Edge: Protein Kinase C{{beta}} Expression Is Critical for Export of IL-2 from T Cells J. Immunol., July 15, 2001; 167(2): 636 - 640. [Abstract] [Full Text] [PDF]
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C. Buensuceso, D Woodside, J. Huff, G. Plopper, and T. O'Toole The WD protein Rack1 mediates protein kinase C and integrin-dependent cell migration J. Cell Sci., January 5, 2001; 114(9): 1691 - 1698. [Abstract] [PDF]
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 J. Szalay, P. Bruno, R. Bhati, J. Adjodha, D. Schueler, V. Summerville, and R. Vazeos Associations of PKC Isoforms with the Cytoskeleton of B16F10 Melanoma Cells J. Histochem. Cytochem., January 1, 2001; 49(1): 49 - 66. [Abstract] [Full Text]
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L. Herreros, J. L. Rodriguez-Fernandez, M. C. Brown, J. L. Alonso-Lebrero, C. Cabanas, F. Sanchez-Madrid, N. Longo, C. E. Turner, and P. Sanchez-Mateos Paxillin Localizes to the Lymphocyte Microtubule Organizing Center and Associates with the Microtubule Cytoskeleton J. Biol. Chem., August 18, 2000; 275(34): 26436 - 26440. [Abstract] [Full Text] [PDF]
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