Limits...
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.

Show MeSH
Effects of LIMK1-WT or LIMK1-DN expression on NADPH oxidase activity. Ra2 cells expressing LIMK1-WT (A,D) or LIMK1-DN (B,E) at increasing protein levels were stimulated with either FMLP (A-C) or PMA (D-F) and superoxide production measured by Luminol E-CL. The graphs represent traces from individual wells of one experiment, where the ordinate shows luminol E-CL normalized to control cells in arbitrary units; arrows indicate time point of agonist injection. The bar graphs shows peak luminol E-CL response for FMLP (C) or PMA (F) stimulated superoxide production, and represents mean and SD of at least three independent experiments. Differences statistically significant at the P < 0.05 (one sample t-test) level are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2944333&req=5

Figure 4: Effects of LIMK1-WT or LIMK1-DN expression on NADPH oxidase activity. Ra2 cells expressing LIMK1-WT (A,D) or LIMK1-DN (B,E) at increasing protein levels were stimulated with either FMLP (A-C) or PMA (D-F) and superoxide production measured by Luminol E-CL. The graphs represent traces from individual wells of one experiment, where the ordinate shows luminol E-CL normalized to control cells in arbitrary units; arrows indicate time point of agonist injection. The bar graphs shows peak luminol E-CL response for FMLP (C) or PMA (F) stimulated superoxide production, and represents mean and SD of at least three independent experiments. Differences statistically significant at the P < 0.05 (one sample t-test) level are indicated.

Mentions: Having confirmed that LIMK1 expression regulates cofilin and F-actin in Ra2 cells, we then examined the effect on FMLP or PMA-elicited superoxide production. Expression of LIMK1-DN dose-dependently increased the FMLP-induced respiratory burst but only expression of LIMK1-WT protein at low levels (Ra2 LIMK1-WT80 cells) significantly induced a two-fold increase in superoxide release (P < 0.05, one sample t-test; Figure 4A and 4C). After PMA stimulation, Ra2 LIMK1-DN80 cells had a significantly (P < 0.05) increased NADPH oxidase activity compared to control cells, and again expression of low levels of LIMK1-WT (WT80) caused an increased superoxide production, while higher levels in WT150 and WT200 Ra2 cells significantly (P < 0.05) decreased the superoxide output with up to 40% (Figure 4D-F). Therefore, despite opposing effects of transgene on F/G-actin ratio both LIMK1-WT80 and LIMK1-DN80 Ra2 cells increased superoxide production up to two-fold following FMLP or PMA stimulation. PAK1 increases FMLP and PMA-stimulated superoxide generation in Ra2 microglia through phosphorylation (partial activation) of the cytosolic p47phox subunit [25], but we also considered that PAK1 through activation of LIMK1 [30] might additionally contribute to NADPH oxidase activity by modulating actin dynamics. However, analysis of superoxide production in a series of Ra2 cell lines co-expressing PAK1 and LIMK1 mutants indicates a parallel rather than sequential organization of these two kinases with respect to NADPH oxidase activity (see Additional File 2).


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)

Effects of LIMK1-WT or LIMK1-DN expression on NADPH oxidase activity. Ra2 cells expressing LIMK1-WT (A,D) or LIMK1-DN (B,E) at increasing protein levels were stimulated with either FMLP (A-C) or PMA (D-F) and superoxide production measured by Luminol E-CL. The graphs represent traces from individual wells of one experiment, where the ordinate shows luminol E-CL normalized to control cells in arbitrary units; arrows indicate time point of agonist injection. The bar graphs shows peak luminol E-CL response for FMLP (C) or PMA (F) stimulated superoxide production, and represents mean and SD of at least three independent experiments. Differences statistically significant at the P < 0.05 (one sample t-test) level are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effects of LIMK1-WT or LIMK1-DN expression on NADPH oxidase activity. Ra2 cells expressing LIMK1-WT (A,D) or LIMK1-DN (B,E) at increasing protein levels were stimulated with either FMLP (A-C) or PMA (D-F) and superoxide production measured by Luminol E-CL. The graphs represent traces from individual wells of one experiment, where the ordinate shows luminol E-CL normalized to control cells in arbitrary units; arrows indicate time point of agonist injection. The bar graphs shows peak luminol E-CL response for FMLP (C) or PMA (F) stimulated superoxide production, and represents mean and SD of at least three independent experiments. Differences statistically significant at the P < 0.05 (one sample t-test) level are indicated.
Mentions: Having confirmed that LIMK1 expression regulates cofilin and F-actin in Ra2 cells, we then examined the effect on FMLP or PMA-elicited superoxide production. Expression of LIMK1-DN dose-dependently increased the FMLP-induced respiratory burst but only expression of LIMK1-WT protein at low levels (Ra2 LIMK1-WT80 cells) significantly induced a two-fold increase in superoxide release (P < 0.05, one sample t-test; Figure 4A and 4C). After PMA stimulation, Ra2 LIMK1-DN80 cells had a significantly (P < 0.05) increased NADPH oxidase activity compared to control cells, and again expression of low levels of LIMK1-WT (WT80) caused an increased superoxide production, while higher levels in WT150 and WT200 Ra2 cells significantly (P < 0.05) decreased the superoxide output with up to 40% (Figure 4D-F). Therefore, despite opposing effects of transgene on F/G-actin ratio both LIMK1-WT80 and LIMK1-DN80 Ra2 cells increased superoxide production up to two-fold following FMLP or PMA stimulation. PAK1 increases FMLP and PMA-stimulated superoxide generation in Ra2 microglia through phosphorylation (partial activation) of the cytosolic p47phox subunit [25], but we also considered that PAK1 through activation of LIMK1 [30] might additionally contribute to NADPH oxidase activity by modulating actin dynamics. However, analysis of superoxide production in a series of Ra2 cell lines co-expressing PAK1 and LIMK1 mutants indicates a parallel rather than sequential organization of these two kinases with respect to NADPH oxidase activity (see Additional File 2).

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