Department of Pathology, University of Bern, Murtenstr. 31, 3010 Bern, Switzerland
(e-mail: niggli{at}patho.unibe.ch)
Accepted 4 December 2002
![]() |
Summary |
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Key words: Neutrophil, Microtubule, Migration, Rho, Rho-kinase, Phosphatidylinositol 3-kinase
![]() |
Introduction |
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Microtubule disruption thus may activate Rho, at least in 3T3 cells
(Ren et al., 1999). Another
small GTP-binding protein, Rac1, in contrast, is activated in those cells by
microtubule growth, resulting in increased lamellipodial protrusions in
fibroblasts (Waterman-Storer et al.,
1999
). Microtubule growth at the leading edge and shortening at
the rear could thus locally activate Rho or Rac and result in local
contraction or ruffling in migrating fibroblasts
(Wittmann and Waterman-Storer,
2001
). In contrast to the data obtained in neutrophils,
microtubule disruption reduces polarization, ruffling and the speed of
migration in fibroblasts (Wittmann and
Waterman-Storer, 2001
), which suggests that different mechanisms
operate in the different cell types.
We describe here the signalling from microtubule disruption to migration in human neutrophils. We provide evidence for the activation in this process of Rho-kinase but not of heterotrimeric Gi proteins, the PI 3-kinase pathway or the p42/44 mitogen-activated protein kinase (MAPK). This differs from chemotactic peptide-induced migration where Gi proteins are activated as well as PI 3-kinase and the MAPK cascade in addition to Rho family proteins and their downstream targets.
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Materials and Methods |
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Antibodies
A monoclonal antibody specific for RhoA was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA) and a monoclonal antibody directed against
Rok was obtained from Transduction Laboratories (Lexington, KY). A
polyclonal antibody specific for p42/44 MAPK phosphorylated on threonine and
tyrosine was obtained from New England Biolabs, Beverly, MA. A polyclonal
antibody specific for Akt phosphorylated on serine 473 and a polyclonal
antibody reacting with Akt independent of its phosphorylation state were
obtained from Cell Signaling Technology Inc. (Beverly, MA). A polyclonal
antibody produced in rabbits against a peptide corresponding to myosin light
chain (MLC) phosphorylated on Thr-16 and Ser-19 (PPMLC)
(Ratcliffe et al., 1999
) was
kindly provided by J. M. Staddon (Eisai London Research Laboratories, London,
UK). Goat anti-mouse and goat anti-rabbit antibodies conjugated to alkaline
phosphatase were obtained from Bio-Rad (Richmond, CA).
Isolation of human neutrophils
Neutrophils were isolated from heparinized human blood (10 units/ml). In a
first step, red blood cells were removed with a solution containing 12% (w/v)
OptiPrep and 16.6 g/l methocel in 130 mM NaCl
(Böyum, 1968).
Subsequently, mononuclear cells were removed using NIM, which contains Hypaque
and Ficoll (Ferrante and Thong,
1980
). Approximately 95% of the leukocytes were neutrophils.
Analysis of shape changes
Neutrophils (3x106 cells/ml) were incubated in Gey's
medium containing 0.1% HSA but lacking divalent cations in a reciprocating
waterbath at 37°C without or with inhibitors, colchicine, taxol or
fNLPNTL, as indicated in Results. The reaction was stopped by fixing the cells
in 1% glutaraldehyde (final concentration) for 30 minutes. The cells were
washed and resuspended in 0.9% NaCl containing NaN3 (1 mg/ml). Cell
shape was determined using differential interference contrast microscopy
(Nomarski optics) using a Zeiss IM 35 microscope with a 100x objective
(NA 1.25). The shapes of neutrophils were classified into the following
categories as previously described (Keller
and Niggli, 1993): spherical (for examples, see
Fig. 1, control), spherical
with unifocal projections, polarized (for examples, see
Fig. 1, fNLPNTL, 10 minutes)
and non-polar cells with surface projections (for examples, see
Fig. 1, fNLPNTL, 1 minute).
|
Treatment with pertussis toxin
Neutrophils (3x106/ml) were preincubated in Iscove's
medium with 10% fetal calf serum without or with pertussis toxin (400 ng/ml)
for 2 hours at 37°C in a humidified atmosphere. After 2 hours, the cells
were centrifuged and the cells resuspended in Gey's medium with divalent
cations, 0.5% HSA with or without pertussis toxin (450 ng/ml). Cell shape was
then determined after incubation in the presence or absence of fNLPNTL or
colchicine.
Chemokinesis
Neutrophils (4x106/ml, 0.45 ml per assay) were incubated
in Gey's medium containing divalent cations and 2% (experiments with
colchicine) or 10% (experiments with fNLPNTL) HSA with inhibitors and/or
stimuli at 37°C in a reciprocating waterbath as indicated in Results. The
cells were centrifuged at 300 g for 5 minutes and resuspended
in 50 µl of the supernatant. Neutrophils (5 µl containing
0.18x106 cells) were placed between a slide and a round
coverslip (25 mm diameter). The slide-coverslip preparation was sealed with
paraffin and placed on the heated stage (37°C) of a Zeiss IM 35 microscope
with a 63x objective. The locomotor behavior of the cells (7-33 cells
for each condition) was recorded for 10 minutes using video microscopy. The
outline of the cell at the initial and final position and the path traveled
during the 10 minutes was drawn on a transparency. Cells remaining totally or
partially within the outline of the initial position are defined as
stationary; cells found outside of the outline after 10 minutes are defined as
locomoting. The length of the path of individual cells was measured using a
KS-300 analysis system (Kontron, Eching, Germany).
Neutrophil fractionation and detection of RhoA and Rok
After treatment with DFP to block endogenous proteases
(Amrein and Stossel, 1980),
neutrophils (12x106 cells/ml) were resuspended in Gey's
medium without divalent cations and HSA. Aliquots (450 µl) of this
suspension were incubated with inhibitors and/or stimuli as described in
Results. The reaction was stopped by centrifugation (300 g, 5
minutes, 4°C). Cells were then resuspended in 500 µl relaxation buffer
(Dusi et al., 1996
) containing
100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1 mM EGTA, 10 mM Pipes, pH
7.3, 1 mM EDTA, 10 mM NaF. This suspension was incubated for 5 minutes on ice
followed by sonication (Branson tip sonifier B-10, output 3.5, 1x10
seconds). After addition of 4 mM DFP to the cell lysate and a low-speed
centrifugation for 5 minutes at 700 g and 4°C to remove
unbroken cells and nuclei, the supernatants were centrifuged at 100,000
g for 30 minutes at 4°C in a Beckmann table top
ultracentrifuge. The resulting pellets (corresponding to the membrane
fraction) were dissolved in 100 µl of Laemmli sample buffer
(Laemmli, 1970
) by incubation
at 95°C with vortexing for 10 minutes. The solubilized pellets, together
with aliquots of the high-speed supernatants (soluble cytosolic fractions),
were subjected to electrophoresis on 5-10% (for Rok
) or 5-20% (for
RhoA) SDS-polyacrylamide gradient gels and transblotted to nitrocellulose
using a mini-genie blotter from Idea Scientific (Minneapolis, MN) for 70
minutes, 24 Volts. Blots were exposed to antibodies to RhoA (diluted 1:1000)
or to Rok
(diluted 1:500). For visualization of bound antibody an
anti-mouse antibody conjugated to alkaline phosphatase, diluted 1:7000,
followed by ECL detection of the second antibody, was used. The bands were
scanned with a Camag TLC scanner at 590 nm and the area of the peaks was taken
as a relative measure for the amount of protein present in the bands.
Analysis of phosphorylation of p42/44 MAPK, Akt or MLC in
neutrophils
Neutrophils in 0.45 ml Gey's medium lacking divalent cations and HSA
(0.5x106 cells for analysis of p42/44 MAPK,
1.5x106 cells for analysis of Akt and 2x106
cells for analysis of PPMLC) were exposed to stimuli as described in Results.
The reaction was stopped by addition of 0.5 ml of a solution containing 20%
(w/v) trichloroacetic acid (TCA), 40 mM NaF and 10 mM
Na2HPO4. After a 20 minute incubation on ice, the
precipitates were collected by centrifugation and washed once with 0.5 ml of
5% TCA and once with 0.5% TCA. The pellets were solubilized in sample buffer
(see above), followed by separation of proteins on a 5-10% SDS-polyacrylamide
gradient gel for analysis of p42/44 MAPK, a 10% gel for Akt and a 15% gel for
analysis of PPMLC. Phosphorylated proteins were detected using a polyclonal
antibody specific for p42/44 MAPK phosphorylated on threonine and tyrosine,
diluted 1:1000, or a polyclonal antibody specific for Akt phosphorylated on
serine 473, diluted 1:1000, or a polyclonal antibody specifically reacting
with PPMLC, diluted 1:500. For visualization of PPMLC, blots were blocked in
PBS containing 2% BSA and 0.05% Tween, followed by incubation with the primary
antibody overnight at 4°C and a 1 hour incubation with the goat
anti-rabbit IgG antibody prior to ELC detection. Detection of phosphorylated
Akt was carried out as recommended by the producer.
Statistical analysis of data
Differences between data were analyzed with the Student's t-test
for paired data with a P value of <0.05 considered
significant. Data correspond to the mean±s.e.m.
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Results |
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|
Colchicine-induced motile responses in neutrophils are suppressed by
inhibition of Rho-kinase but are insensitive to pertussis toxin and the PI
3-kinase inhibitor wortmannin
On the basis of the data obtained in fibroblasts where microtubule
disruption activates Rho (Ren et al.,
1999), we investigated a possible role for the Rho-effector
Rho-kinase in colchicine-induced development of polarity and migration in
human neutrophils. Preincubation of cells with the Rho-kinase inhibitor
Y-27632 suppressed development of polarity in a concentration-dependent manner
(Figs 1 and
3), which is comparable to
effects on chemotactic-peptide-stimulated cells
(Niggli, 1999
). Maximal
inhibition was obtained at 10 µM of the inhibitor (95±3% inhibition,
n=5). Half-maximal values were obtained at 2.6±0.8 µM
(n=5), which is comparable to the concentration necessary for
inhibition of the purified enzyme (Uehata
et al., 1997
). As shown in Fig.
1, most of the cells preincubated with 10 µM Y-27632 before
addition of colchicine were spherical, although a few cells still showed small
projections. This is different from chemotactic-peptide-stimulated cells
pretreated with Y-27632, which still exhibit large F-actin-rich ruffles
(Niggli, 1999
). We also
assessed the effects of Y-27632 on colchicine-induced chemokinesis and
observed a highly significant (P<0.0025) reduction of 76±9%
of the fraction of migrating cells (Table
2). The speed of migration of the remaining motile cells was also
significantly (P<0.005) decreased by 66-86%. These findings
suggest that microtubule disassembly activates the Rho/Rho-kinase cascade in
neutrophils and that this process is required for colchicine-induced
neutrophil migration.
|
|
We assessed the effect of inhibiting activity of heterotrimeric Gi proteins with pertussis toxin on chemotactic-peptide- and colchicine-induced development of polarity. As shown in Fig. 4, preincubation with 400 ng pertussis toxin/ml completely abolished chemotactic peptide-induced polarity. The cells assumed the spherical morphology typical of resting cells. By contrast, colchicine-induced polarity was not affected, suggesting that microtubule disassembly does not activate motile processes in neutrophils via Gi.
|
PI 3-kinase plays an important role in chemotactic-factor-induced
neutrophil migration (Hirsch et al.,
2000; Stephens et al.,
2002
). As shown in Fig.
5A and in our previous work
(Niggli and Keller, 1997
), the
PI 3-kinase inhibitor wortmannin markedly reduces chemotactic-peptide-induced
development of polarity and migration in human neutrophils, correlating with
inhibition of phosphatidylinositol (3,4,5)-trisphosphate
[PtdIns(3,4,5)P3] production. Half-maximal effects of
wortmannin on cell polarization were obtained in the range of 14-32 nM of the
drug. Development of polarity was inhibited by 58±8% (n=5) by
100 nM wortmannin (Fig. 5A)
(Niggli and Keller, 1997
). We
assessed whether the incomplete inhibition of chemotactic-peptide-induced cell
polarization by wortmannin is due to incomplete inhibition of PI 3-kinase by
monitoring the effect of wortmannin on activation of a major target of PI
3-kinase, the serine-threonine protein kinase Akt (or protein kinase B; PKB).
Hirsch et al. showed that chemotactic-peptide-induced increased
phosphorylation of Akt is completely abolished in neutrophils of mice lacking
PI 3-kinase
, whereas chemotaxis of neutrophils from these mice is only
partially impaired (Hirsch et al.,
2000
). We now show that wortmannin (100 nM) also completely
suppressed fNLPNTL-induced Akt phosphorylation in human neutrophils
(Fig. 5B). By contrast, this
concentration of wortmannin did not affect colchicine-induced development of
polarity (Fig. 5A). Moreover we
could not detect any Akt phosphorylation in cells treated with colchicine for
5 to 30 minutes, whereas exposure of cells to 1 nM fNLPNTL for 5 minutes
induced a marked signal (Fig.
5B).
|
Previous data suggested that microtubule disruption activates p42/44 MAPK
rapidly in human pro-monocytic cells
(Schmid-Alliana et al., 1998).
However, we could not observe a significant increase in phosphorylation of
p42/44 MAPK in neutrophils exposed to colchicine for 5-30 minutes, whereas
activation with fNLPNTL for 5 minutes had a very marked effect
(Fig. 6).
|
Rho and Rho-kinase are translocated to the membrane in neutrophils
exposed to colchicine or chemotactic peptide
Translocation of Rho-kinase to the plasma membrane may correlate with its
activation (Taggart et al.,
1999). Therefore we studied the effect of colchicine on membrane
association of Rok
(Rho-kinase II) in human neutrophils and compared it
with the effects of chemotactic peptide. In resting cells, 12±7%
(n=3) of total enzyme was recovered in the membrane-containing
fraction. Incubation of cells with colchicine (5 minutes) induced a marked,
99±20% increase (n=7, P<0.0025) in
membrane-associated Rok
, which is comparable with that induced by
chemotactic peptide (171±50% increase, n=3), as shown in
Fig. 7A,B. Membrane association
of Rok
was maximal 5 minutes after addition of colchicine and decreased
to a lower level, which was still significantly higher than control levels 10
minutes after addition of the drug (data not shown). This response is thus
more transient than the effect of colchicine on cell polarity where 93% of the
maximal polarization response was observed 5 minutes after addition of
colchicine and 100% 10 minutes after addition of the drug. Similar to effects
on cell shape and migration (Fig.
2, Table 1),
preincubation with taxol also completely prevented colchicine-induced membrane
association of Rok
(Fig.
7A,B), whereas that induced by chemotactic peptide was only
partially affected (63±16% inhibition, n=6,
Fig. 7C,D). In the latter case
the extent of inhibition was quite variable in different experiments.
Lumicolchicine, which is inactive on microtubules, did not induce membrane
association of Rok
(Fig.
7A,B).
|
Activation of neutrophils with chemotactic peptide induces recruitment of a
fraction of Rho and Rac from the cytosol to the membrane
(Dusi et al., 1996;
Marcil et al., 1999
).
Incubation of cells with 10 µM colchicine for 5 minutes also induced a
significant shift of RhoA to the membrane (50-230% increase). This increase
was comparable to that induced by a 30 minutes incubation with 1 nM fNLPNTL
(102±47% increase) (data not shown).
Our data strongly suggest that microtubule disassembly in neutrophils
activates RhoA and its effector Rok.
Microtubule disassembly induces phosphorylation of myosin light
chain
We wanted to obtain information on downstream targets of Rho-kinase
activated in cells by colchicine. As shown previously, exposure to chemotactic
peptide induces a sustained increase in myosin light chain phosphorylation in
human neutrophils that can be abolished by the Rho-kinase inhibitor Y-27632
(Niggli, 1999). As assessed
with an antibody that specifically reacts with myosin light chain
phosphorylated on Thr-16 and Ser-19 (PPMLC)
(Ratcliffe et al., 1999
),
treatment of neutrophils with 10 µM colchicine induced a time-dependent
increase in the amount of PPMLC; this increase was maximal 20-30 minutes after
addition of the drug (a 223±65% increase, n=5 at 20 minutes,
P<0.025; Fig. 8).
34 to 57% of the response was attained 5 minutes after addition of the drug to
the cells. As shown in Fig. 8,
the maximal increase obtained 20 minutes after addition of colchicine was even
higher than that induced by a 5 minute incubation with chemotactic peptide and
could be completely abolished by preincubation of the cells with 10 µM
Y-27632. Preincubation with 10 µM taxol also completely abolished the
colchicine-induced increase in PPMLC (data not shown).
|
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Discussion |
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Differential signalling pathways are involved in
microtubule-disruption- and in chemotactic-peptide-induced neutrophil polarity
and migration
We demonstrate here that neutrophil motility induced by microtubule
disruption is probably not due to direct effects on actin organization;
instead it is probably due to activation of specific signalling pathways. We
studied four different signalling pathways that could conceivably be affected
by microtubule disruption: heterotrimeric G-proteins, the Rho-Rho-kinase
pathway, the PI 3-kinase pathway and activation of p42/44 MAPK. Concerning
heterotrimeric G-proteins, membrane-bound tubulin has been reported to
directly activate Gs, G
1 and G
q
(Popova et al., 1997
).
However, Gi inhibition in neutrophils by pertussis toxin did not suppress
colchicine-induced cell polarization in our system, which excludes
Gi proteins from having a role in this response.
Chemotactic-peptide-induced shape changes in contrast were completely
prevented by pertussis toxin.
We have shown previously that inhibition of Rho-kinase activity results in
almost complete suppression of chemotactic-peptide-induced polarization and
migration of human neutrophils (Niggli,
1999). We now show that Rho-kinase inhibition also almost
completely suppresses colchicine-induced development of polarity and markedly
inhibits colchicine-induced chemokinesis in neutrophils. This finding
correlates with reports showing that microtubule disassembly activates the
Rho-Rho-kinase pathway in other cell types (Witman and Waterman-Storer, 2001).
Indeed we could show that colchicine treatment results in increased membrane
association of Rho-kinase, which can be prevented by taxol-based stabilization
of microtubules (Fig. 6). Previous evidence obtained in smooth muscle cells suggests that membrane
association of Rho-kinase correlates with its activation. There, carbachol
stimulates association of Rho-kinase with the plasma membrane as demonstrated
using immunofluorescence staining (Taggart
et al., 1999
). We now show that this also occurs in stimulated
neutrophils exposed to chemotactic peptide or colchicine. We also provide
evidence that Rho-kinase, activated by microtubule disassembly, induces a
marked sustained increase in myosin light chain phosphorylation, comparable to
that induced by exposure of cells to chemotactic peptide, which then leads to
tail contraction and is crucial for colchicine-induced migration.
Interestingly, the time course of colchicine-induced membrane association of
Rho-kinase is somewhat faster than that of myosin light chain phosphorylation
and development of polarity. Moreover the first event is transient, whereas
the two other events are sustained for up to at least 30 minutes after
addition of the stimulus. Transient activation of Rho-kinase thus results in
sustained myosin light chain phosphorylation and polarization. Rho-kinase is
thought to act by phosphorylating the myosin-binding subunit of myosin light
chain phosphatase, thereby inhibiting this enzyme
(Somlyo and Somlyo, 2000
). A
transient activation of Rho-kinase may be sufficient to inactivate the
phosphatase for longer. This suggests that phosphatases acting on the myosin
light chain phosphatase are inactive in the migrating cells. Interestingly,
the phosphorylated myosin-binding subunit of type 1 protein phosphatase is
highly resistant to dephosphorylation
(Takizawa et al., 2002
).
Alternatively, activation of Rho-kinase may be maintained even after its
dissociation from the membrane.
Regulatory subunits of PI 3-kinase interact with tubulin
(Inukai et al., 2000).
Activity of this enzyme could thus be affected by microtubule disassembly.
However, colchicine-induced polarization was not sensitive to treatment with
concentrations of the PI 3-kinase inhibitor wortmannin, which completely
suppress chemotactic-peptide-induced phosphorylation of a major target of PI
3-kinase, Akt (Fig. 5B), and
which markedly inhibit neutrophil migration induced by exposure to chemotactic
peptide or to membrane-permeable PtdIns(3,4,5)P3
(Niggli and Keller, 1997
;
Niggli, 2000
). This strongly
suggests that colchicine does not act via PI 3-kinase. In line with this
notion, stimulation of cells with colchicine did not induce any detectable Akt
phosphorylation (Fig. 5B).
A substantial fraction of p42/44 MAPK is associated with microtubules, and
microtubule disruption activates these enzymes within minutes of addition of
colchicine in human pro-monocytic cells
(Schmid-Alliana et al., 1998).
However, this was not the case in human neutrophils, suggesting differential
regulation of different signalling systems depending on the cell type.
Our data strongly suggest that microtubule disassembly induces a selective
activation of the Rho-Rho-kinase pathway but not of PI 3-kinase or of p42/44
MAPK. This activation of Rho-kinase, resulting in increased phosphorylation of
myosin light chain on Thr-18 and Ser-19, is required for the
colchicine-induced development of polarity and the subsequent migratory
response in neutrophils. Concerning the speed of migration and the fraction of
migrating cells, this response compares well with that induced by chemotactic
peptides. However, in colchicine-induced cells, less ruffling occurs at the
leading edge (Fig. 1)
(Keller et al., 1984). This
may be due to the lack of activation of PI 3-kinase and lack of local
accumulation of PtdIns(3,4,5)P3, which is thought to be
crucial for chemotactic-peptide-induced polarity in neutrophils
(Knall et al., 1997
;
Niggli and Keller, 1997
;
Niggli, 2000
;
Hirsch et al., 2000
;
Weiner, 2002
;
Stephens et al., 2002
).
Migration is thus not obligatorily coupled to localized production of
PtdIns(3,4,5)P3.
Concerning the mechanism of microtubule-disassembly-induced Rho activation,
evidence has recently been provided for a direct regulation of the nucleotide
exchange factor GEF-H1 by interaction with microtubules. This factor appears
to be inactive when bound to microtubules. It is activated by microtubule
disassembly and selectively activates Rho but not Rac1 or Cdc42
(Krendel et al., 2002).
Our data thus show that different stimuli activate migration in human neutrophils involving differential sets of signalling proteins depending on the initial stimulus.
Mechanisms of microtubule-disruption-induced cytoskeletal
reorganization
Microtubule disassembly in neutrophils induces development of a polarized
shape. The front of these polarized cells, as assessed by videomicroscopy, is
defined as that part of the cell where protrusions are extended in the
direction of migration, whereas pseudopod protrusion is suppressed in the rear
of the cell, which is characterized by a narrow, contracted tail
(Fig. 1)
(Keller et al., 1984).
Interestingly, polarization of cells induced by microtubule disassembly is
accompanied by accumulation of F-actin and
-actinin in the front and
myosin II in the tail; these results are similar to findings in
chemotactic-peptide-stimulated cells. Also, a small increase in the fraction
of stable actin resistant to Triton X-100 could be detected in
colchicine-treated cells (Keller and
Niggli, 1993
), which is similar to previous findings by another
group (Tsai et al., 1998
).
Relatively small changes in the F-actin:G-actin ratio are thus sufficient to
sustain rapid migration. The mechanism of this cytoskeletal reorganization
remains to be clarified. We observed previously that Rho-kinase inhibition
does not affect actin reorganization in human neutrophils
(Niggli, 1999
). Moreover, Rho
inhibition in neutrophils did not suppress chemotactic-peptide-induced actin
polymerization (Ehrengruber et al.,
1995
). However, we observed that in neutrophils pretreated with
Y-27632 prior to addition of colchicine, F-actin was diffusely distributed in
the cytosol, which is similar to results for untreated controls, suggesting a
role for Rho-kinase in regulating the polarized accumulation of F-actin in
colchicine-treated cells (data not shown).
Mechanisms of microtubule-disruption-induced development of polarity
in neutrophils
Localized activation of signalling proteins and generation of diffusable
inhibitors may be prerequisites for development of polarity and chemotaxis
(Weiner, 2002;
Stephens et al., 2002
).
Neutrophils develop polarity not only in gradients of chemoattractants but
also in uniform concentrations of stimuli. In the latter case, stochastic
differences in concentration of stimuli are thought to induce polarity. It is
not clear how global depolymerization of microtubules induced by colchicine
can result in localized activation of Rho/Rho-kinase and localized myosin
activation presumably required for development of polarity. One would have to
assume a fortuitous local higher activation of the Rho/Rho-kinase system,
which would result in generation of a diffusable inhibitor and localized
positive feedback. However such factors regulating the Rho/Rho-kinase system
have not yet been identified in neutrophils
(Stephens et al., 2002
).
The role of microtubule disassembly in migration of
chemotactic-peptide-stimulated cells
As outlined by Wittmann and Waterman-Storer, cells such as fibroblasts or
macrophages require an intact microtubule network for development of polarity
and migration whereas other cells such as neutrophils do not
(Wittmann and Waterman-Storer,
2001). The exact role of microtubules in maintaining polarity in
specific cell types is still under debate. Microtubules may be required for
transport of membranes to the leading edge and/or for selectively stabilizing
the leading edge and/or for regulating focal adhesion dynamics ensuring
disassembly of focal contacts in the rear of the cell
(Wittmann and Waterman-Storer,
2001
). Neutrophils polarize not only when adhering to a substrate
but also in suspension. Possibly the need for intact microtubules for
fibroblast polarization is related to the role of cell substrate contacts in
this process. Adhesion-independent polarization of neutrophils does not
require intact microtubules. As microtubule disassembly on its own can induce
a migratory response in neutrophils, the question arises whether localized
microtubule disassembly induced by chemotactic peptide also plays a role in
receptor-linked chemotactic responses. The microtubule-stabilizing drug taxol
does not affect chemotactic-peptide-induced cell polarization in suspension
but partially inhibits chemotactic-peptide-induced membrane association of
Rho-kinase and markedly inhibits chemotactic-peptide-induced chemokinesis
(Fig. 7C,D;
Table 1B). The third
observation is in agreement with earlier data showing abolishment of
fMLP-induced chemotaxis in neutrophils by taxol
(Roberts et al., 1982
).
According to these authors, taxol reduces spreading of neutrophils. Localized
microtubulin depolymerization occurring during cell migration, which in turn
results in localized Rho/Rho-kinase activation, may contribute to regulation
of cell adhesion. The partial inhibition of Rho-kinase activation by taxol
observed in chemotactic-peptide-stimulated cells may not be sufficient to
abolish development of polarity in suspension but may significantly impair
cell migration.
In summary, we provide novel data showing that microtubule disassembly in human neutrophils selectively activates the Rho/Rho-kinase pathway but not PI 3-kinase or p42/44 MAPK. Motility ensuing from microtubule disruption is independent of PI 3-kinase but requires Rho-kinase activity. This is in contrast to development of polarity and migration induced by chemotactic peptides or membrane-permeable PtdIns(3,4,5)P3 where the PI 3-kinase pathway plays a major role. Differential sets of signalling pathways are thus required for migration depending on the initial stimulus.
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Acknowledgments |
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