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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 wild type or dominant negative LIMK1 regulates cofilin phosphorylation and actin polymerization in Ra2 cells. Ra2 cells received increasing doses (80, 150, or 200 equivalents) of lentivector expressing LIMK1-WT or LIMK1-DN. A) Immunofluorescence of doxycycline-induced Ra2 cell populations using Alexa488-conjugated phalloidin (green) and anti-cofilin-ser3(P) rabbit antibodies (red). Detection gain was adjusted to include the signal for phalloidin or cofilin-ser3(P) from all Ra2 cell populations within the dynamic range and was then kept constant throughout. Bars, 10 μm. B) Expression of LIMK1 protein and effect on phosphorylation of cofilin was determined by western blotting with polyclonal rabbit anti-LIMK1 antibodies and anti-Ser3(P)-cofilin antibodies. Ra2 045 control cells express only the tetracycline-responsive rtTA-transactivator protein. Note that the LIMK1 antibody used only recognizes transgene human LIMK1 protein. C and D) Mean and SEM integrated optical density of western blot bands of LIMK protein (C) and Ser3(P)-cofilin (D) of three individual experiments performed as in B. E and F) The F/G-actin ratios of LIMK1-expressing cells (E) or toxin-treated control cells (F) represent mean and SEM of at least three independent experiments.
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Figure 3: Over-expression of wild type or dominant negative LIMK1 regulates cofilin phosphorylation and actin polymerization in Ra2 cells. Ra2 cells received increasing doses (80, 150, or 200 equivalents) of lentivector expressing LIMK1-WT or LIMK1-DN. A) Immunofluorescence of doxycycline-induced Ra2 cell populations using Alexa488-conjugated phalloidin (green) and anti-cofilin-ser3(P) rabbit antibodies (red). Detection gain was adjusted to include the signal for phalloidin or cofilin-ser3(P) from all Ra2 cell populations within the dynamic range and was then kept constant throughout. Bars, 10 μm. B) Expression of LIMK1 protein and effect on phosphorylation of cofilin was determined by western blotting with polyclonal rabbit anti-LIMK1 antibodies and anti-Ser3(P)-cofilin antibodies. Ra2 045 control cells express only the tetracycline-responsive rtTA-transactivator protein. Note that the LIMK1 antibody used only recognizes transgene human LIMK1 protein. C and D) Mean and SEM integrated optical density of western blot bands of LIMK protein (C) and Ser3(P)-cofilin (D) of three individual experiments performed as in B. E and F) The F/G-actin ratios of LIMK1-expressing cells (E) or toxin-treated control cells (F) represent mean and SEM of at least three independent experiments.

Mentions: We next analyzed the effects of over-expression of wild type and a dominant negative mutant of LIM kinase-1 (LIMK1) as an alternative means of modulating the actin cytoskeleton. Activated LIMK1 shapes the actin cytoskeleton by phosphorylating and thereby inactivating cofilin, which in its active form severs and depolymerizes actin filaments. We created Ra2 cell lines conditionally expressing wild type LIMK1 (LIMK1-WT) or the kinase-dead LIMK1-D406A mutant (LIMK1-DN) at different expression levels (80, 150, and 200 virus dose equivalents). Expression of LIMK1-WT dose-dependently increased phosphorylation of serine-3 in cofilin as expected (Figure 3A, B and 3C) and increased F/G-actin ratios in Ra2 cells (Figure 3E). Curiously, while LIMK1-DN decreased F/G-actin ratios, at all levels of expression it slightly increased the levels of phosphorylated cofilin above the levels of control cells (Figure 3B). We show in Additional File 1 that in fact introducing dominant negative mutants of any of the proteins in the important VAV1, Rac1, PAK1, LIMK1 signaling axis slightly increases p-cofilin levels above control cells, possibly because altered PAK1 or RhoA activation (which is negatively regulated by Rac1) changes the tonus of cofilin phosphatases. For comparison F/G-actin ratios in Ra2 045 control cells treated with select concentrations of latrunculin and jasplakinolide are shown in Figure 3F.


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 wild type or dominant negative LIMK1 regulates cofilin phosphorylation and actin polymerization in Ra2 cells. Ra2 cells received increasing doses (80, 150, or 200 equivalents) of lentivector expressing LIMK1-WT or LIMK1-DN. A) Immunofluorescence of doxycycline-induced Ra2 cell populations using Alexa488-conjugated phalloidin (green) and anti-cofilin-ser3(P) rabbit antibodies (red). Detection gain was adjusted to include the signal for phalloidin or cofilin-ser3(P) from all Ra2 cell populations within the dynamic range and was then kept constant throughout. Bars, 10 μm. B) Expression of LIMK1 protein and effect on phosphorylation of cofilin was determined by western blotting with polyclonal rabbit anti-LIMK1 antibodies and anti-Ser3(P)-cofilin antibodies. Ra2 045 control cells express only the tetracycline-responsive rtTA-transactivator protein. Note that the LIMK1 antibody used only recognizes transgene human LIMK1 protein. C and D) Mean and SEM integrated optical density of western blot bands of LIMK protein (C) and Ser3(P)-cofilin (D) of three individual experiments performed as in B. E and F) The F/G-actin ratios of LIMK1-expressing cells (E) or toxin-treated control cells (F) represent mean and SEM of at least three independent experiments.
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Figure 3: Over-expression of wild type or dominant negative LIMK1 regulates cofilin phosphorylation and actin polymerization in Ra2 cells. Ra2 cells received increasing doses (80, 150, or 200 equivalents) of lentivector expressing LIMK1-WT or LIMK1-DN. A) Immunofluorescence of doxycycline-induced Ra2 cell populations using Alexa488-conjugated phalloidin (green) and anti-cofilin-ser3(P) rabbit antibodies (red). Detection gain was adjusted to include the signal for phalloidin or cofilin-ser3(P) from all Ra2 cell populations within the dynamic range and was then kept constant throughout. Bars, 10 μm. B) Expression of LIMK1 protein and effect on phosphorylation of cofilin was determined by western blotting with polyclonal rabbit anti-LIMK1 antibodies and anti-Ser3(P)-cofilin antibodies. Ra2 045 control cells express only the tetracycline-responsive rtTA-transactivator protein. Note that the LIMK1 antibody used only recognizes transgene human LIMK1 protein. C and D) Mean and SEM integrated optical density of western blot bands of LIMK protein (C) and Ser3(P)-cofilin (D) of three individual experiments performed as in B. E and F) The F/G-actin ratios of LIMK1-expressing cells (E) or toxin-treated control cells (F) represent mean and SEM of at least three independent experiments.
Mentions: We next analyzed the effects of over-expression of wild type and a dominant negative mutant of LIM kinase-1 (LIMK1) as an alternative means of modulating the actin cytoskeleton. Activated LIMK1 shapes the actin cytoskeleton by phosphorylating and thereby inactivating cofilin, which in its active form severs and depolymerizes actin filaments. We created Ra2 cell lines conditionally expressing wild type LIMK1 (LIMK1-WT) or the kinase-dead LIMK1-D406A mutant (LIMK1-DN) at different expression levels (80, 150, and 200 virus dose equivalents). Expression of LIMK1-WT dose-dependently increased phosphorylation of serine-3 in cofilin as expected (Figure 3A, B and 3C) and increased F/G-actin ratios in Ra2 cells (Figure 3E). Curiously, while LIMK1-DN decreased F/G-actin ratios, at all levels of expression it slightly increased the levels of phosphorylated cofilin above the levels of control cells (Figure 3B). We show in Additional File 1 that in fact introducing dominant negative mutants of any of the proteins in the important VAV1, Rac1, PAK1, LIMK1 signaling axis slightly increases p-cofilin levels above control cells, possibly because altered PAK1 or RhoA activation (which is negatively regulated by Rac1) changes the tonus of cofilin phosphatases. For comparison F/G-actin ratios in Ra2 045 control cells treated with select concentrations of latrunculin and jasplakinolide are shown in Figure 3F.

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