Okadaic acid‐induced actin assembly in neutrophils: Role of protein phosphatases

Abstract

Activation of neutrophils results in morphological and functional alterations including changes in cell shape and initiation of motile bchavior that depend on assembly and reorganization ot the actin cytoskeleton. Phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal alterations and whereas much attention has been directed at the role of protein kinases, relatively little information is available on the importance of phosphatases. To elucidate the role of protein phosphatases, we studied thc effects of the phosphatase inhibitors okadaic acid and calyculin A on the actin cytoskeleton of human neutrophils. Exposure of cells to okadaic acid resulted in assembly and spatial redistribution of actin, which peaked at 25 min and returned to baseline levels by 45 min, as assessed by flow cytornetric analysis of NBD-phallacidin stained cells and confocal fluorescence microscopy, rcspec-tively. These effects correlated with an increase in protein phosphorylation, determined by incorporation of "P into cellular proteins using SDS-PAGE arid autoradiography. Similar but more rapid responses were observed in electropermeabilized cells treated with okadaic acid or calyculin A. The dose dependence of these effects was compatible with a role for phosphatase type 1 as the target enzyme. These findings also suggested the presence of constitutively active protein kinases capable of effecting actin polymerization. Phosphorylation of myosin light chain IMLC) has been postulated to promote actin assembly, but myosin light chain kinase (MLCK) appeared not to be involved because: (1) the effect of okadaic acid was not inhibited by the MLCK inhibitor KT5926 and ( 2 ) in permeabilized cells suspended in medium with free calcium [Ca"l < 10 n M (conditions under which MLCK is inactive), the effect of okadaic acid pcrsisted. The role of phosphatases in stimulus-induced actin assembly was assessed in cells preincubated with okadaic acid for 45 rnin, after F-actin levels had returned to baseline. Under these conditions, okadaic acid completely abrogated actin assembly induced by phorbol niyristate acetate, platelet activating factor, and leukotriene B,, whereas the effects of the chemotactic peptide fMLP and opsonized zymosan (OpZ) were unaffected. We conclude that swine and threonine phosphatases exert a tonic negative influence on actin assembly and organization. Furthermore, divergent pathways seem to mediate the response to lipidic stimuli, on one hand, and fMLP and OpZ, on the other, as evidenced by the difterential susceptibility to inhibition by okadaic acid. N e u t r o p h i l leukocytes are highly m o t i l e cells, a prope r t y w e l l suited t o t h e i r role as i t i n e r a n t phagocytes in i n f l a m m a t i o n . Locomotion, as w e l l as other functions crucial t o t h e process o f cell activation in response t o i n f l a m m a t o r y stimuli, i n c l u d i n g r e g u l a t i o n of cell shape, phagocytosis, and projection o f membrane ruffles and pseudopodia, are dependent o n t h e highl...

Shape, F‐actin, and surface morphology changes during chemotactic peptide‐induced polarity in human neutrophils

“…The signal transduction pathways responsible for these various shapes are unknown. Previous studies showed that exposure of neutrophils to PKC activators (e.g., diacylglycerols or phorbol esters) induced reorganization of F-actin and membrane ruffling (Roos et al, 1987;Zimmermann et al, 1988;Downey et al, 1992) similar to the changes described in the present study. However, these chemotactic peptide-induced effects cannot be prevented by PKC inhibitors, such as H7, staurosporine, or calphostine C; thus neither the reorganization of F-actin nor pseudopod extension induced by fMLP appears to depend on PKC activation.…”

Regulation of membrane trafficking and subcellular organization of endocytic compartments revealed with FM1-43 in resting and activated human T cells

“…Caly A induced the formation of a dense actin ring beneath the plasma membrane (Fig. 3D), consistent with effects described in other cell types [32][33][34][35][36]. Caly A-treated cells acquired a typical kidney-like shape and appeared to be curved around a single tightly condensed cytosolic actin bundle.…”

The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

“…In our study, structure and organization of cell morphology and the actin cytoskeleton of Clone 9 cells and primary cultured rat hepatocytes were affected by OA. Downey et al (1993), who described OA-induced actin assembly in neutrophils, observed similar alterations. In accordance with the previous work of Vilarino et al (2008) in neuroblastoma cells, an almost 10-fold higher concentration of MeOk than of OA was needed to induce a similar disruption in the actin cytoskeleton and morphology of Clone 9 hepatocytes.…”