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
FRAP experiments reveal widely different actin recovery half-times in transduced Ra2 cells. Ra2 cell populations as indicated were superinfected with lentivectors expressing β-actin-YFP and used for FRAP. A) Panels (top, LIMK1-WT80 cells; two lower panels, LIMK1-DN80 cells) show time-lapse series of live Ra2 cells where the ROI bleached by the laser beam is indicated by arrows (the frame before and after bleach). In the top and middle row (LIMK1-WT80 and LIMK1-DN80 cells, respectively) the ROI covers podosomal structures, while the lower panel (LIMK1-DN80 cells) shows a ROI covering the opposing plasma membranes of two cells. B) Mean pixel intensity of β-actin-YFP fluorescence is depicted over time before and after bleach (bleach is at time point three) for LIMK1-WT80 cells, together with whole cell and background intensity (white symbols), and for LIMK1-DN80 cells (black). C) Mean pixel intensity of β-actin-YFP fluorescence was fitted to a curve for FRAP recovery to obtain the mean half-time of recovery for either plasma membrane or podosome ROI's for the indicated cell lines or drug-treated cells. The ordinate shows the recovery time in seconds and mean and SEM is based on FRAP curves obtained from at least ten different cell profiles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: FRAP experiments reveal widely different actin recovery half-times in transduced Ra2 cells. Ra2 cell populations as indicated were superinfected with lentivectors expressing β-actin-YFP and used for FRAP. A) Panels (top, LIMK1-WT80 cells; two lower panels, LIMK1-DN80 cells) show time-lapse series of live Ra2 cells where the ROI bleached by the laser beam is indicated by arrows (the frame before and after bleach). In the top and middle row (LIMK1-WT80 and LIMK1-DN80 cells, respectively) the ROI covers podosomal structures, while the lower panel (LIMK1-DN80 cells) shows a ROI covering the opposing plasma membranes of two cells. B) Mean pixel intensity of β-actin-YFP fluorescence is depicted over time before and after bleach (bleach is at time point three) for LIMK1-WT80 cells, together with whole cell and background intensity (white symbols), and for LIMK1-DN80 cells (black). C) Mean pixel intensity of β-actin-YFP fluorescence was fitted to a curve for FRAP recovery to obtain the mean half-time of recovery for either plasma membrane or podosome ROI's for the indicated cell lines or drug-treated cells. The ordinate shows the recovery time in seconds and mean and SEM is based on FRAP curves obtained from at least ten different cell profiles.

Mentions: F/G-actin ratios derived by end point measurements of F- and G-actin says little or nothing about the dynamics of the actin cytoskeleton, and we therefore considered that turn-over rates could be a better correlate of NADPH oxidase activity than F/G- actin ratios (compare Figure 3F with 4C, F; Figure 3G with 1B, D, F, H). We therefore performed FRAP analysis of β-actin-YFP-expressing Ra2 cells to obtain the actin recovery half time (recovery t1/2), which is a measure of actin off- and on-rates combined (Figure 6). We chose two different types of regions of interest (ROI) to work with: podosomal F-actin and cortical, plasma membrane-associated F-actin (see representative examples in Figure 6A and Additional Files 7, 8, 9). FRAP measurements were performed on Ra2 045 control cells, Ra2 LIMK1-WT80/200 and -DN80/200 cells, Ra2 cofilin shRNA-1 cells, and Ra2 cells treated with concentrations of latrunculin and jasplakinolide resulting in maximal positive or negative effect on superoxide production as determined previously (see Figure 1). As seen in Figure 6C, recovery t1/2 of actin-YFP fluorescence was depressed slightly by latrunculin or expression of LIMK1-DN at low levels (LIMK1-DN80 cells), which both increase the respiratory burst. Conversely, jasplakinolide at 1 μM, which increases the PMA-stimulated superoxide release to 150% of control levels, caused a modest increase in recovery t1/2 in plasma membrane ROI's, while a higher concentration of 8 μM (50% reduction in superoxide release) increased recovery t1/2 more than fourfold relative to controls. Expression of LIMK1-WT in WT80 cells distinguished itself from other conditions of enhanced superoxide production (latrunculin 100 ng/ml, LIMK1-DN80, and 1 μM jasplakinolide) by a greatly increased half-time of recovery (62 ± 18 seconds and < 20 seconds, respectively). Of note, cofilin shRNA-1 expression and jasplakinolide treatment both antagonized the formation of podosomes and caused distribution of F-actin to the dorsal plasma membrane.


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)

FRAP experiments reveal widely different actin recovery half-times in transduced Ra2 cells. Ra2 cell populations as indicated were superinfected with lentivectors expressing β-actin-YFP and used for FRAP. A) Panels (top, LIMK1-WT80 cells; two lower panels, LIMK1-DN80 cells) show time-lapse series of live Ra2 cells where the ROI bleached by the laser beam is indicated by arrows (the frame before and after bleach). In the top and middle row (LIMK1-WT80 and LIMK1-DN80 cells, respectively) the ROI covers podosomal structures, while the lower panel (LIMK1-DN80 cells) shows a ROI covering the opposing plasma membranes of two cells. B) Mean pixel intensity of β-actin-YFP fluorescence is depicted over time before and after bleach (bleach is at time point three) for LIMK1-WT80 cells, together with whole cell and background intensity (white symbols), and for LIMK1-DN80 cells (black). C) Mean pixel intensity of β-actin-YFP fluorescence was fitted to a curve for FRAP recovery to obtain the mean half-time of recovery for either plasma membrane or podosome ROI's for the indicated cell lines or drug-treated cells. The ordinate shows the recovery time in seconds and mean and SEM is based on FRAP curves obtained from at least ten different cell profiles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: FRAP experiments reveal widely different actin recovery half-times in transduced Ra2 cells. Ra2 cell populations as indicated were superinfected with lentivectors expressing β-actin-YFP and used for FRAP. A) Panels (top, LIMK1-WT80 cells; two lower panels, LIMK1-DN80 cells) show time-lapse series of live Ra2 cells where the ROI bleached by the laser beam is indicated by arrows (the frame before and after bleach). In the top and middle row (LIMK1-WT80 and LIMK1-DN80 cells, respectively) the ROI covers podosomal structures, while the lower panel (LIMK1-DN80 cells) shows a ROI covering the opposing plasma membranes of two cells. B) Mean pixel intensity of β-actin-YFP fluorescence is depicted over time before and after bleach (bleach is at time point three) for LIMK1-WT80 cells, together with whole cell and background intensity (white symbols), and for LIMK1-DN80 cells (black). C) Mean pixel intensity of β-actin-YFP fluorescence was fitted to a curve for FRAP recovery to obtain the mean half-time of recovery for either plasma membrane or podosome ROI's for the indicated cell lines or drug-treated cells. The ordinate shows the recovery time in seconds and mean and SEM is based on FRAP curves obtained from at least ten different cell profiles.
Mentions: F/G-actin ratios derived by end point measurements of F- and G-actin says little or nothing about the dynamics of the actin cytoskeleton, and we therefore considered that turn-over rates could be a better correlate of NADPH oxidase activity than F/G- actin ratios (compare Figure 3F with 4C, F; Figure 3G with 1B, D, F, H). We therefore performed FRAP analysis of β-actin-YFP-expressing Ra2 cells to obtain the actin recovery half time (recovery t1/2), which is a measure of actin off- and on-rates combined (Figure 6). We chose two different types of regions of interest (ROI) to work with: podosomal F-actin and cortical, plasma membrane-associated F-actin (see representative examples in Figure 6A and Additional Files 7, 8, 9). FRAP measurements were performed on Ra2 045 control cells, Ra2 LIMK1-WT80/200 and -DN80/200 cells, Ra2 cofilin shRNA-1 cells, and Ra2 cells treated with concentrations of latrunculin and jasplakinolide resulting in maximal positive or negative effect on superoxide production as determined previously (see Figure 1). As seen in Figure 6C, recovery t1/2 of actin-YFP fluorescence was depressed slightly by latrunculin or expression of LIMK1-DN at low levels (LIMK1-DN80 cells), which both increase the respiratory burst. Conversely, jasplakinolide at 1 μM, which increases the PMA-stimulated superoxide release to 150% of control levels, caused a modest increase in recovery t1/2 in plasma membrane ROI's, while a higher concentration of 8 μM (50% reduction in superoxide release) increased recovery t1/2 more than fourfold relative to controls. Expression of LIMK1-WT in WT80 cells distinguished itself from other conditions of enhanced superoxide production (latrunculin 100 ng/ml, LIMK1-DN80, and 1 μM jasplakinolide) by a greatly increased half-time of recovery (62 ± 18 seconds and < 20 seconds, respectively). Of note, cofilin shRNA-1 expression and jasplakinolide treatment both antagonized the formation of podosomes and caused distribution of F-actin to the dorsal plasma membrane.

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