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First published online 15 January 2003
doi: 10.1242/jcs.00306
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Research Article |
Department of Pathology, University of Bern, Murtenstr. 31, 3010 Bern, Switzerland
(e-mail: niggli{at}patho.unibe.ch)
Accepted 4 December 2002
Neutrophil granulocytes rely on a functional actin network for directed
migration. Microtubule disassembly does not impair receptor-linked chemotaxis,
instead it induces development of polarity and chemokinesis in neutrophils
concomitant with polarized distribution of 
-actinin and F-actin. Cells
stimulated with colchicine, which disassembles microtubules, migrate with a
speed comparable to cells exposed to chemotactic peptide. We investigated
signalling pathways involved in colchicine-induced neutrophil polarization and
migration. Colchicine-induced development of polarity was insensitive to
treatment with pertussis toxin, in contrast to chemotactic-peptide-induced
shape changes, which were completely abolished by this treatment. Thus,
colchicine does not appear to act via activating heterotrimeric Gi
proteins. Colchicine does also not seem to act via phosphatidylinositol
3-kinase, as it failed to induce phosphorylation of its downstream target Akt
and the potent phosphatidylinositol 3-kinase inhibitor wortmannin failed to
inhibit colchicine-induced shape changes. By contrast, wortmannin
significantly reduced chemotactic-peptide-induced shape changes. However, the
Rho-kinase inhibitor Y-27632 (10 µM) inhibited colchicine-induced
development of polarity by 95±3% (n=5) and chemokinesis by
76±9% (n=3), which suggests that the Rho-Rho-kinase pathway
has a crucial role in polarity and migration. Indeed, treatment of cells with
colchicine induced a significant increase in membrane-bound Rho-kinase II,
which is indicative of activation of this protein. This membrane translocation
could be prevented by taxol, which stabilizes microtubules. Colchicine also
induced a marked increase in myosin light chain phosphorylation, which could
be suppressed by Y-27632 and by taxol. In summary, we provide evidence that
microtubule disassembly induces in neutrophils a selective activation of
Rho-kinase, bypassing activation of heterotrimeric Gi proteins and
phosphatidylinositol 3-kinase. This process is sufficient for induction of
chemokinesis and mediates increased phosphorylation of myosin light chain and
accumulation of F-actin and -actinin in the leading edge.
Key words: Neutrophil, Microtubule, Migration, Rho, Rho-kinase, Phosphatidylinositol 3-kinase
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