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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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Changes in morphology and relative changes in superoxide production, F/G-actin ratio, and actin recovery half-time following different experimental perturbations of the actin cytoskeleton. A) Ra2 LIMK1-WT80, Ra2 LIMK1-DN80, Ra2 cofilin-shRNA1, and Ra2 wild type cells treated for five minutes with 100 ng/ml latrunculin or 8 μM jasplakinolide were paraformaldehyde fixed and stained with Alexa 568-conjugated phalloidin, and then Z-stacks (20-40 planes, ca. 0.25-0.75 μm section width) were acquired. In the case of 8 μM jasplakinolide treatment z-stacks were generated using β-actin-YFP fluorescence as jasplakinolide inhibits phalloidin binding to F-actin. Laser intensity and gain was adjusted to show detail and therefore does not reflect the levels of polymerized actin between cell lines. The images shown represent a XY-view of the most ventral z-plane of the cell (center) while a XZ projection is shown at the top, and a ZY projection on the side. B) For each parameter (superoxide production, F/G-actin ratio, and actin recovery half-time) absolute values derived from experiments above with PMA-stimulated cells were normalized to that of Ra2 045 control cells and depicted on the Y-axis in arbitrary units.
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Figure 7: Changes in morphology and relative changes in superoxide production, F/G-actin ratio, and actin recovery half-time following different experimental perturbations of the actin cytoskeleton. A) Ra2 LIMK1-WT80, Ra2 LIMK1-DN80, Ra2 cofilin-shRNA1, and Ra2 wild type cells treated for five minutes with 100 ng/ml latrunculin or 8 μM jasplakinolide were paraformaldehyde fixed and stained with Alexa 568-conjugated phalloidin, and then Z-stacks (20-40 planes, ca. 0.25-0.75 μm section width) were acquired. In the case of 8 μM jasplakinolide treatment z-stacks were generated using β-actin-YFP fluorescence as jasplakinolide inhibits phalloidin binding to F-actin. Laser intensity and gain was adjusted to show detail and therefore does not reflect the levels of polymerized actin between cell lines. The images shown represent a XY-view of the most ventral z-plane of the cell (center) while a XZ projection is shown at the top, and a ZY projection on the side. B) For each parameter (superoxide production, F/G-actin ratio, and actin recovery half-time) absolute values derived from experiments above with PMA-stimulated cells were normalized to that of Ra2 045 control cells and depicted on the Y-axis in arbitrary units.

Mentions: Observations made during FRAP trials prompted us to perform detailed analysis of F-actin distribution by Z-sectioning using fluorophore-conjugated phalloidin to visualize F-actin. Figure 7A shows for each condition a main panel with a XY-view of the most ventral plane of collected Z-stacks, and on the sides of this panel is shown XZ (top) or YZ (side) projections of the reconstructed Z-stack. This analysis confirmed that LIMK1-WT strongly induces podosome-like structures (see movies in Additional Files 3 and 4) and that 8 μM jasplakinolide and cofilin shRNA-1, which most effectively inhibited NADPH oxidase activity, both caused a marked redistribution of F-actin associated with the ventral aspect of the cell membrane to lateral and dorsal membrane (see Additional Files 5 and 6). To allow easier comparison of the effects of different perturbations of the actin cytoskeleton on superoxide production, Figure 7B depicts the normalized values for F/G-actin ratios, actin recovery half-times and superoxide production derived from PMA-stimulated cells overlaid in the same graph (absolute values derived from experiments shown above).


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)

Changes in morphology and relative changes in superoxide production, F/G-actin ratio, and actin recovery half-time following different experimental perturbations of the actin cytoskeleton. A) Ra2 LIMK1-WT80, Ra2 LIMK1-DN80, Ra2 cofilin-shRNA1, and Ra2 wild type cells treated for five minutes with 100 ng/ml latrunculin or 8 μM jasplakinolide were paraformaldehyde fixed and stained with Alexa 568-conjugated phalloidin, and then Z-stacks (20-40 planes, ca. 0.25-0.75 μm section width) were acquired. In the case of 8 μM jasplakinolide treatment z-stacks were generated using β-actin-YFP fluorescence as jasplakinolide inhibits phalloidin binding to F-actin. Laser intensity and gain was adjusted to show detail and therefore does not reflect the levels of polymerized actin between cell lines. The images shown represent a XY-view of the most ventral z-plane of the cell (center) while a XZ projection is shown at the top, and a ZY projection on the side. B) For each parameter (superoxide production, F/G-actin ratio, and actin recovery half-time) absolute values derived from experiments above with PMA-stimulated cells were normalized to that of Ra2 045 control cells and depicted on the Y-axis in arbitrary units.
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Related In: Results  -  Collection

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Figure 7: Changes in morphology and relative changes in superoxide production, F/G-actin ratio, and actin recovery half-time following different experimental perturbations of the actin cytoskeleton. A) Ra2 LIMK1-WT80, Ra2 LIMK1-DN80, Ra2 cofilin-shRNA1, and Ra2 wild type cells treated for five minutes with 100 ng/ml latrunculin or 8 μM jasplakinolide were paraformaldehyde fixed and stained with Alexa 568-conjugated phalloidin, and then Z-stacks (20-40 planes, ca. 0.25-0.75 μm section width) were acquired. In the case of 8 μM jasplakinolide treatment z-stacks were generated using β-actin-YFP fluorescence as jasplakinolide inhibits phalloidin binding to F-actin. Laser intensity and gain was adjusted to show detail and therefore does not reflect the levels of polymerized actin between cell lines. The images shown represent a XY-view of the most ventral z-plane of the cell (center) while a XZ projection is shown at the top, and a ZY projection on the side. B) For each parameter (superoxide production, F/G-actin ratio, and actin recovery half-time) absolute values derived from experiments above with PMA-stimulated cells were normalized to that of Ra2 045 control cells and depicted on the Y-axis in arbitrary units.
Mentions: Observations made during FRAP trials prompted us to perform detailed analysis of F-actin distribution by Z-sectioning using fluorophore-conjugated phalloidin to visualize F-actin. Figure 7A shows for each condition a main panel with a XY-view of the most ventral plane of collected Z-stacks, and on the sides of this panel is shown XZ (top) or YZ (side) projections of the reconstructed Z-stack. This analysis confirmed that LIMK1-WT strongly induces podosome-like structures (see movies in Additional Files 3 and 4) and that 8 μM jasplakinolide and cofilin shRNA-1, which most effectively inhibited NADPH oxidase activity, both caused a marked redistribution of F-actin associated with the ventral aspect of the cell membrane to lateral and dorsal membrane (see Additional Files 5 and 6). To allow easier comparison of the effects of different perturbations of the actin cytoskeleton on superoxide production, Figure 7B depicts the normalized values for F/G-actin ratios, actin recovery half-times and superoxide production derived from PMA-stimulated cells overlaid in the same graph (absolute values derived from experiments shown above).

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