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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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Cofilin knock-down increases F/G-actin ratio and inhibits superoxide production. A and B) Levels of cofilin protein after tetracycline induction of two different cofilin shRNAs alone or in combination was determined by western blotting. Control cells refer to Ra2 cells transduced with empty pLVTHM dsRed vector. The bar graphs in B shows mean optical density and SEM of western blot bands derived from three independent experiments. C) Ratio of F- to G-actin in control and cofilin-1 shRNA-expressing Ra2 cells. Data represent mean ± SEM of two independent experiments, each performed in triplicate. D-G) Superoxide production in control or cofilin shRNA-transduced Ra2 cells before and after stimulation with FMLP (D,E) or PMA (F,G) was measured by luminol E-CL. The ordinate of the trace graphs show luminol E-CL normalized to control cells in arbitrary units. Injection of agonist is indicated by arrow. Bar graphs E and G) show normalized luminol E-CL mean and SEM of three independent experiments as performed in A or C, respectively.
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Figure 5: Cofilin knock-down increases F/G-actin ratio and inhibits superoxide production. A and B) Levels of cofilin protein after tetracycline induction of two different cofilin shRNAs alone or in combination was determined by western blotting. Control cells refer to Ra2 cells transduced with empty pLVTHM dsRed vector. The bar graphs in B shows mean optical density and SEM of western blot bands derived from three independent experiments. C) Ratio of F- to G-actin in control and cofilin-1 shRNA-expressing Ra2 cells. Data represent mean ± SEM of two independent experiments, each performed in triplicate. D-G) Superoxide production in control or cofilin shRNA-transduced Ra2 cells before and after stimulation with FMLP (D,E) or PMA (F,G) was measured by luminol E-CL. The ordinate of the trace graphs show luminol E-CL normalized to control cells in arbitrary units. Injection of agonist is indicated by arrow. Bar graphs E and G) show normalized luminol E-CL mean and SEM of three independent experiments as performed in A or C, respectively.

Mentions: We also targeted cofilin directly using shRNA knock-down as described in materials and methods, which decreased the level of cofilin protein in Ra2 cells expressing cofilin-shRNA-1 and shRNA-2 with 75% and 55% relative to control (pLVTHM vector) transduced cells, respectively (Figure 5A and 5B). A knock-down of cofilin would be expected to increase actin polymerization, which was confirmed (Figure 5C). The F/G-actin ratio achieved in Ra2 cofilin-shRNA-1 cells was even higher than that of LIMK1-WT200 cells (10 ± 1 and 6.9 ± 0.9, respectively). The degree of knockdown of cofilin obtained with either cofilin shRNA correlated with a similarly decreased superoxide production (Figure 5, D-G). Cofilin shRNA-1 reduced the respiratory burst following FMLP stimulation with roughly 80% and following PMA stimulation with 70%.


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)

Cofilin knock-down increases F/G-actin ratio and inhibits superoxide production. A and B) Levels of cofilin protein after tetracycline induction of two different cofilin shRNAs alone or in combination was determined by western blotting. Control cells refer to Ra2 cells transduced with empty pLVTHM dsRed vector. The bar graphs in B shows mean optical density and SEM of western blot bands derived from three independent experiments. C) Ratio of F- to G-actin in control and cofilin-1 shRNA-expressing Ra2 cells. Data represent mean ± SEM of two independent experiments, each performed in triplicate. D-G) Superoxide production in control or cofilin shRNA-transduced Ra2 cells before and after stimulation with FMLP (D,E) or PMA (F,G) was measured by luminol E-CL. The ordinate of the trace graphs show luminol E-CL normalized to control cells in arbitrary units. Injection of agonist is indicated by arrow. Bar graphs E and G) show normalized luminol E-CL mean and SEM of three independent experiments as performed in A or C, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Cofilin knock-down increases F/G-actin ratio and inhibits superoxide production. A and B) Levels of cofilin protein after tetracycline induction of two different cofilin shRNAs alone or in combination was determined by western blotting. Control cells refer to Ra2 cells transduced with empty pLVTHM dsRed vector. The bar graphs in B shows mean optical density and SEM of western blot bands derived from three independent experiments. C) Ratio of F- to G-actin in control and cofilin-1 shRNA-expressing Ra2 cells. Data represent mean ± SEM of two independent experiments, each performed in triplicate. D-G) Superoxide production in control or cofilin shRNA-transduced Ra2 cells before and after stimulation with FMLP (D,E) or PMA (F,G) was measured by luminol E-CL. The ordinate of the trace graphs show luminol E-CL normalized to control cells in arbitrary units. Injection of agonist is indicated by arrow. Bar graphs E and G) show normalized luminol E-CL mean and SEM of three independent experiments as performed in A or C, respectively.
Mentions: We also targeted cofilin directly using shRNA knock-down as described in materials and methods, which decreased the level of cofilin protein in Ra2 cells expressing cofilin-shRNA-1 and shRNA-2 with 75% and 55% relative to control (pLVTHM vector) transduced cells, respectively (Figure 5A and 5B). A knock-down of cofilin would be expected to increase actin polymerization, which was confirmed (Figure 5C). The F/G-actin ratio achieved in Ra2 cofilin-shRNA-1 cells was even higher than that of LIMK1-WT200 cells (10 ± 1 and 6.9 ± 0.9, respectively). The degree of knockdown of cofilin obtained with either cofilin shRNA correlated with a similarly decreased superoxide production (Figure 5, D-G). Cofilin shRNA-1 reduced the respiratory burst following FMLP stimulation with roughly 80% and following PMA stimulation with 70%.

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