Abstract
BackgroundMost in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton.ResultsHere, we in addition to toxins use conditional expression of the major actin regulatory protein LIM kinase-1 (LIMK1), and shRNA knock-down of cofilin to modulate the cellular F/G-actin ratio in the Ra2 microglia cell line, and we use Fluorescence Recovery after Photobleaching (FRAP) in β-actin-YFP-transduced cells to obtain a dynamic measure of actin recovery rates (actin turn-over rates) in different F/G-actin states of the actin cytoskeleton. Our data demonstrate that stimulated NADPH oxidase function was severely impaired only at extreme actin recovery rates and F/G-actin ratios, and surprisingly, that any moderate changes of these parameters of the actin cytoskeleton invariably resulted in an increased NADPH oxidase activity.Conclusionmoderate actin polymerization and depolymerization both increase the FMLP and PMA-stimulated NADPH oxidase activity of microglia, which is directly correlated with neither actin recovery rate nor F/G- actin ratio. Our results indicate that NADPH oxidase functions in an enhanced state of activity in stimulated phagocytes despite widely different states of the actin cytoskeleton.
Highlights
Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton
Any perturbation of the actin cytoskeleton may affect N-Formylmethionine leucyl-phenylalanine (FMLP) receptor signaling in addition to effects on the NADPH oxidase complex, for which reason we have included stimulation with the strong agonist Phorbol myristate acetate (PMA), which works solely on a post-receptor level by PKC activation
Latrunculin at a concentration of 40-50 ng/ml induced a 2-3 fold increase in superoxide release measured by luminol-enhanced chemiluminescence for both FMLP and PMA-stimulated cells (Figure 1A,B,E, and 1F)
Summary
Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton. The superoxide-producing NADPH oxidase is expressed at high levels in professional phagocyte cells It is composed of membrane subunits gp91phox (NOX-2) and p22phox, which form a heterodimeric flavocytochrome b558 complex (cyt b558), and cytosolic subunits p40phox, p47phox, p67phox, and the small GTPase Rac or 2 [1]. Rac by GDP/GTP exchange factors, and phosphorylation of critical serine residues in p40phox and p47phox by serine/threonine kinases including PKCδ, AKT, and PAK1 (see [2] for references) This phosphorylation unmasks latent binding sites in p47phox for cyt b558 and exposes the PHOX domains of p40phox and p47phox, which bind to phosphoinositol lipids in the membrane [3,4,5]. Translocation of Rac and the cytosolic phox proteins complex takes place simultaneously but independently of each other [2,9], and continuous exchange of cytosolic phox subunits and Rac1/2 are necessary to sustain production of superoxide during the respiratory burst [10,11]
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