Activation of RhoA and ROCK Are Essential for Detachment of Migrating Leukocytes

Abstract

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 GSTRhotekin 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.

Type Iγ PIP Kinase Is a Novel Uropod Component that Regulates Rear Retraction during Neutrophil Chemotaxis

“…Interestingly, the rear retraction defect noted upon overexpression of kinase-dead GFP-PIPKI␥661 phenocopies previous results observed upon inhibition of RhoA (Alblas et al, 2001;Worthylake et al, 2001), ROCK (Somlyo et al, 2000;Alblas et al, 2001;Worthylake et al, 2001), and myosin IIa (Eddy et al, 2000). Previous work has also shown that PIPKI␥ interacts with RhoA (Chong et al, 1994;Ren et al, 1996) and that this interaction increases PtdIns(4,5)P 2 production by PIPKI family members (Chong et al, 1994;Weernink et al, 2004).…”

“…Primary neutrophils expressing GFP-PIPKI␥661 exhibited a normal polarized morphology and targeting of GFP-PIPKI␥ 661 to the uropod (Figure 6, Supplementary Video S7). However, treatment of primary neutrophils expressing GFP-PIPKI␥ 661 with the ROCK inhibitor Y-27632 induced an abnormal elongated morphology with defects in rear release during chemotaxis, in agreement with previous reports (Somlyo et al, 2000;Alblas et al, 2001;Worthylake et al, 2001). However, targeting of GFP-PIPKI␥661 to the uropod was not affected by ROCK inhibition ( Figure 6, Supplementary Video S8), suggesting that the localization of GFP-PIPKI␥661 to the uropod is independent of ROCK activity.…”

Infection with Replication-deficient Adenovirus Induces Changes in the Dynamic Instability of Host Cell Microtubules

“…Inhibition of RhoA was achieved by pretreatment of cells with Clostridium botulinum exoenzyme C3 (Cytoskeleton) at a concentration of 10 g/ml (Aikawa et al, 1999;Alblas et al, 2001). Rac1 was inhibited by pretreating HFF cells with NSC23766 (EMD Biosciences, San Diego, CA) at 100 M for 2 h. NSC23766 inhibited Rac1 activity by blocking the interaction between Rac1 and its guanine nucleotide exchange factors TrioN and Tiam1 (Gao et al, 2004).…”

Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration

“…In T cells and other leukocytes, expression of dominant-negative Rho GTPases or treatment with chemical inhibitors or bacterial toxins has implicated several Rho GTPases in leukocyte migration (Tybulewicz and Henderson, 2009). For example, inhibitors of the closely related Rho isoforms RhoA, RhoB, and RhoC and/or their downstream targets, ROCKs, have been reported to reduce leukocyte adhesion by inhibiting activation of the integrin LFA-1 (Giagulli et al, 2004) and to reduce migration and chemotaxis by inhibiting contraction of the uropod at the rear (Alblas et al, 2001;Vicente-Manzanares et al, 2002;Smith et al, 2003;Worthylake and Burridge, 2003). In knockout mice, Cdc42, Rac1, and Rac2 contribute to the recruitment of leukocytes to inflamed sites (Roberts et al, 1999;Yamauchi et al, 2004;Szczur et al, 2006;Filippi et al, 2007), although their exact role in the individual steps of TEM is not yet defined.…”