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First published online 15 January 2003
doi: 10.1242/jcs.00306

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Microtubule-disruption-induced and chemotactic-peptide-induced migration of human neutrophils: implications for differential sets of signalling pathways

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 G_i 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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