Abstract
Gelsolin is an actin filament-capping protein that has been shown to play a key role in cell migration. Here we have studied the involvement of phosphoinositide 3-kinase (PI 3-kinase) and GTP-binding proteins (G-proteins) in the regulation of gelsolin-actin interactions in neutrophils. Inhibition of PI 3-kinase activity in vivo by wortmannin did not affect the dissociation of actin-gelsolin (1:1) complexes induced by neutrophil stimulation with N-formyl-Met-Leu-Phe. Guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) indirectly promoted the dissociation of actin-gelsolin complexes in a cell-free system using neutrophil cytosol, and this effect was blocked by the GDP dissociation inhibitor for Rho (Rho-GDI). The GTPgammaS-loaded ialpha2 and the beta1gamma2 subunits of heterotrimeric G-proteins (Gialpha2 and Gbeta1gamma2) also triggered actin-gelsolin dissociation in a Rho-GDI-sensitive manner. GTP-loaded activated Rac, but not activated Rho, induced the dissociation of cytosolic actin-gelsolin complexes. The guanine nucleotide exchange on Rac was increased by addition of GTPgammaS-loaded Gialpha2 or Gbeta1gamma2 to neutrophil cytosol. These findings suggest that activation of Rac by G-protein-coupled receptors in neutrophils triggers uncapping of actin filaments, independently of PI 3-kinase.
Highlights
Complex [6, 7, 11], which can, in vitro, be dissociated by anionic phospholipids (PtdIns[4,5]P2 and PtdIns[4]P) [10]
Effect of PI 3-Kinase Inhibition and Ca2ϩ Depletion on Agonist-induced Actin-Gelsolin Dissociation and Actin Polymerization in Neutrophils—While in resting neutrophils a large fraction of gelsolin is associated with actin, and exposure of the cells to fMLP leads to the rapid release of actin from gelsolin [14]
When neutrophil cytosol was supplemented with 2.8 M recombinant Rho-GDI, the protein reversed the action of subsequently added Guanosine 5-[␥-thio]triphosphate (GTP␥S) but did not affect the level of actin-gelsolin complexes in untreated cytosol (Fig. 4A)
Summary
Complex [6, 7, 11], which can, in vitro, be dissociated by anionic phospholipids (PtdIns[4,5]P2 and PtdIns[4]P) [10]. When neutrophil cytosol was supplemented with 2.8 M recombinant Rho-GDI, the protein reversed the action of subsequently added GTP␥S but did not affect the level of actin-gelsolin complexes in untreated cytosol (Fig. 4A).
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