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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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Over-expression of p47phox alleviates the effects of latrunculin and jasplakinolide. Superoxide production of Ra2 cells over-expressing p47phox wild type (A,B) or the p47phox (S303D/S304D/S328D) mutant (C,D) and treated with increasing doses of latrunculin (A,C) or jasplakinolide (B,D) before stimulation with PMA. The ordinate in arbitrary units shows luminol E-CL signal normalized to cells receiving no toxin (no add.), and results are presented as mean of at least three independent experiments. Arrows indicate time point of agonist injection.
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Figure 2: Over-expression of p47phox alleviates the effects of latrunculin and jasplakinolide. Superoxide production of Ra2 cells over-expressing p47phox wild type (A,B) or the p47phox (S303D/S304D/S328D) mutant (C,D) and treated with increasing doses of latrunculin (A,C) or jasplakinolide (B,D) before stimulation with PMA. The ordinate in arbitrary units shows luminol E-CL signal normalized to cells receiving no toxin (no add.), and results are presented as mean of at least three independent experiments. Arrows indicate time point of agonist injection.

Mentions: It has been proposed that cytosolic phox proteins have to be released from an actin-sequestered state for NADPH oxidase assembly [8,16]. We therefore tested if the effects of latrunculin and jasplakinolide on NADPH oxidase activation could be overruled by over-expression of p47phox through titration of actin-related binding sites. Ra2 cells over-expressing p47phox produce five-fold more superoxide than control cells (not shown in Figure 2; see [25]). Figure 2A and 2C shows that latrunculin at concentrations, which caused PMA-stimulated Ra2 cells to produce ca. two-fold more superoxide, only had a modest enhancing effect in Ra2 cells over-expressing wild type p47phox. No further enhancement could be gained by expressing the p47phox(S303D/S304D/S320D) mutant, which mimics phosphorylation and partial activation of p47phox, and is known to induce phosphoinositide and p22phox binding [29], and a basal superoxide production in un-stimulated Ra2 cells [25]. The p47phox over-expressing cells were also partially insensitive to the effects of jasplakinolide (Figure 2B and 2D) although concentrations of more than 0.5 μM caused a delayed peak response of superoxide production.


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)

Over-expression of p47phox alleviates the effects of latrunculin and jasplakinolide. Superoxide production of Ra2 cells over-expressing p47phox wild type (A,B) or the p47phox (S303D/S304D/S328D) mutant (C,D) and treated with increasing doses of latrunculin (A,C) or jasplakinolide (B,D) before stimulation with PMA. The ordinate in arbitrary units shows luminol E-CL signal normalized to cells receiving no toxin (no add.), and results are presented as mean of at least three independent experiments. Arrows indicate time point of agonist injection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Over-expression of p47phox alleviates the effects of latrunculin and jasplakinolide. Superoxide production of Ra2 cells over-expressing p47phox wild type (A,B) or the p47phox (S303D/S304D/S328D) mutant (C,D) and treated with increasing doses of latrunculin (A,C) or jasplakinolide (B,D) before stimulation with PMA. The ordinate in arbitrary units shows luminol E-CL signal normalized to cells receiving no toxin (no add.), and results are presented as mean of at least three independent experiments. Arrows indicate time point of agonist injection.
Mentions: It has been proposed that cytosolic phox proteins have to be released from an actin-sequestered state for NADPH oxidase assembly [8,16]. We therefore tested if the effects of latrunculin and jasplakinolide on NADPH oxidase activation could be overruled by over-expression of p47phox through titration of actin-related binding sites. Ra2 cells over-expressing p47phox produce five-fold more superoxide than control cells (not shown in Figure 2; see [25]). Figure 2A and 2C shows that latrunculin at concentrations, which caused PMA-stimulated Ra2 cells to produce ca. two-fold more superoxide, only had a modest enhancing effect in Ra2 cells over-expressing wild type p47phox. No further enhancement could be gained by expressing the p47phox(S303D/S304D/S320D) mutant, which mimics phosphorylation and partial activation of p47phox, and is known to induce phosphoinositide and p22phox binding [29], and a basal superoxide production in un-stimulated Ra2 cells [25]. The p47phox over-expressing cells were also partially insensitive to the effects of jasplakinolide (Figure 2B and 2D) although concentrations of more than 0.5 μM caused a delayed peak response of superoxide production.

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