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Vol. 12, Issue 7, 2137-2145, July 2001
and§
Detachment of the rear of the cell from its substratum is an
important aspect of locomotion. The signaling routes involved in this
adhesive release are largely unknown. One of the few candidate proteins
to play a role is RhoA, because activation of RhoA in many cell types
leads to contraction, a mechanism probably involved in detachment. To
study the role of RhoA in detachment regulation, we analyzed several
subsets of expert migratory leukocytes by video microscopy. In contrast
to fast-migrating neutrophils, eosinophils do not detach the rear of
the cell unless stimulated with serum. When measuring the amount of
active RhoA, with the use of a GST-Rhotekin pulldown assay, we
found that serum is an excellent activator of RhoA in granulocytes.
Inhibition of RhoA or one of Rho's target proteins, the kinase ROCK,
in neutrophils leads to the phenotype seen in eosinophils: the rear of
the cell is firmly attached to the substratum, whereas the cell body is
highly motile. ROCK-inhibition leads to impaired migration of
granulocytes in filters, on glass, and through endothelial monolayers.
Also, the ROCK signaling pathway is involved in changes of
integrin-mediated adhesion. Eosinophil transduction by a
tat-fusion construct containing active RhoA resulted in detachment
stimulation in the presence of chemoattractant. From these results we
conclude that activation of the RhoA-ROCK pathway is essential for
detachment of migratory leukocytes.
UMC Utrecht, Department of Pulmonary Diseases, 3584 CX Utrecht, The Netherlands; and CLB, Department of
Experimental Immunohematology, 1066 CX Amsterdam, The Netherlands
Online version of this article contains video material
for some figures. Online version is available at www.molbiolcell.org.
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