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Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia.

Rasmussen I, Pedersen LH, Byg L, Suzuki K, Sumimoto H, Vilhardt F - BMC Immunol. (2010)

Bottom Line: 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.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. moderate 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept of Cellular and Molecular Medicine, The Panum Institute, Copenhagen University, 2200N Copenhagen, Denmark.

ABSTRACT

Background: 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.

Results: Here, 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.

Conclusion: moderate 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.

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Latrunculin and jasplakinolide modulate superoxide production in Ra2 microglia. Superoxide production in Ra2 cells pre-incubated with latrunculin or jasplakinolide as indicated before stimulation with A-D) FMLP or E-H) PMA. The traces presented in the left column show superoxide production in one individual well of an experiment, while the graph on the right show mean ± SEM of peak superoxide production from three independent experiments all in at least three separate runs with duplicate wells on the plate reader. The ordinates show superoxide production as measured by luminol E-CL normalized to control cells not receiving toxin (arbitrary units). Arrows indicate time point of PMA or FMLP injection.
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Figure 1: Latrunculin and jasplakinolide modulate superoxide production in Ra2 microglia. Superoxide production in Ra2 cells pre-incubated with latrunculin or jasplakinolide as indicated before stimulation with A-D) FMLP or E-H) PMA. The traces presented in the left column show superoxide production in one individual well of an experiment, while the graph on the right show mean ± SEM of peak superoxide production from three independent experiments all in at least three separate runs with duplicate wells on the plate reader. The ordinates show superoxide production as measured by luminol E-CL normalized to control cells not receiving toxin (arbitrary units). Arrows indicate time point of PMA or FMLP injection.

Mentions: Latrunculin causes F-actin depolymerization by sequestration of monomeric G-actin, and is known to increase the respiratory burst in neutrophils [8,27]. In Figure 1 we compared the effect of latrunculin with that of jasplakinolide, an actin filament-stabilizing toxin, on the FMLP and PMA-induced superoxide production of the murine microglia cell line Ra2 [22], which express all the subunits of the phagocyte NADPH oxidase [28]. We used FMLP because it is a physiological and commonly used phagocyte stimulant. However, any perturbation of the actin cytoskeleton may affect FMLP receptor signaling in addition to effects on the NADPH oxidase complex, for which reason we have also included stimulation with the strong agonist PMA, which works solely on a post-receptor level by PKC activation. Both single well traces (Figure 1A,C,E, and 1G) and mean peak chemiluminescence of at least three independent experiments (Figure 1B,C,F, and 1H) are shown. Latrunculin at a concentration of 40-50 ng/ml induced a 2-3 fold increase in superoxide release measured by luminol-enhanced chemiluminescence (luminol E-CL) for both FMLP and PMA-stimulated cells (Figure 1A,B,E, and 1F). Conversely, jasplakinolide dose-dependently blocked the FMLP-induced response (ca. 90% inhibition at 2 μM), while jasplakinolide in PMA-stimulated cells exerted an enhancing effect on superoxide production (maximum of 60% increase at 1 μM), in the concentration range from ca 0.25-4 μM before becoming inhibitory at concentrations above 4 μM.


Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia.

Rasmussen I, Pedersen LH, Byg L, Suzuki K, Sumimoto H, Vilhardt F - BMC Immunol. (2010)

Latrunculin and jasplakinolide modulate superoxide production in Ra2 microglia. Superoxide production in Ra2 cells pre-incubated with latrunculin or jasplakinolide as indicated before stimulation with A-D) FMLP or E-H) PMA. The traces presented in the left column show superoxide production in one individual well of an experiment, while the graph on the right show mean ± SEM of peak superoxide production from three independent experiments all in at least three separate runs with duplicate wells on the plate reader. The ordinates show superoxide production as measured by luminol E-CL normalized to control cells not receiving toxin (arbitrary units). Arrows indicate time point of PMA or FMLP injection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2944333&req=5

Figure 1: Latrunculin and jasplakinolide modulate superoxide production in Ra2 microglia. Superoxide production in Ra2 cells pre-incubated with latrunculin or jasplakinolide as indicated before stimulation with A-D) FMLP or E-H) PMA. The traces presented in the left column show superoxide production in one individual well of an experiment, while the graph on the right show mean ± SEM of peak superoxide production from three independent experiments all in at least three separate runs with duplicate wells on the plate reader. The ordinates show superoxide production as measured by luminol E-CL normalized to control cells not receiving toxin (arbitrary units). Arrows indicate time point of PMA or FMLP injection.
Mentions: Latrunculin causes F-actin depolymerization by sequestration of monomeric G-actin, and is known to increase the respiratory burst in neutrophils [8,27]. In Figure 1 we compared the effect of latrunculin with that of jasplakinolide, an actin filament-stabilizing toxin, on the FMLP and PMA-induced superoxide production of the murine microglia cell line Ra2 [22], which express all the subunits of the phagocyte NADPH oxidase [28]. We used FMLP because it is a physiological and commonly used phagocyte stimulant. However, any perturbation of the actin cytoskeleton may affect FMLP receptor signaling in addition to effects on the NADPH oxidase complex, for which reason we have also included stimulation with the strong agonist PMA, which works solely on a post-receptor level by PKC activation. Both single well traces (Figure 1A,C,E, and 1G) and mean peak chemiluminescence of at least three independent experiments (Figure 1B,C,F, and 1H) are shown. Latrunculin at a concentration of 40-50 ng/ml induced a 2-3 fold increase in superoxide release measured by luminol-enhanced chemiluminescence (luminol E-CL) for both FMLP and PMA-stimulated cells (Figure 1A,B,E, and 1F). Conversely, jasplakinolide dose-dependently blocked the FMLP-induced response (ca. 90% inhibition at 2 μM), while jasplakinolide in PMA-stimulated cells exerted an enhancing effect on superoxide production (maximum of 60% increase at 1 μM), in the concentration range from ca 0.25-4 μM before becoming inhibitory at concentrations above 4 μM.

Bottom Line: 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.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. moderate 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept of Cellular and Molecular Medicine, The Panum Institute, Copenhagen University, 2200N Copenhagen, Denmark.

ABSTRACT

Background: 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.

Results: Here, 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.

Conclusion: moderate 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.

Show MeSH
Related in: MedlinePlus