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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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Model of p40/p47/p67phox membrane translocation and potential roles for the actin cytoskeleton and actin-regulatory proteins in NADPH oxidase assembly and function. At rest the p40/p47/p67phox complex is tethered to F-actin either directly or indirectly through for example moesin (bottom). Following cell activation p40phox and p47phox are activated by phosphorylation to acquire phosphoinositide and cyt b558-binding properties (green curved arrows) and loss of tight binding to F-actin. In addition, phosphorylation may lead to binding of actin-associated proteins or adaptors like TRAF4 to the phox protein complex. This membrane recruitment part of the cycle is supported by actin depolymerization (F- to G-actin conversion). At the membrane p47phox bind cortical actin, phosphoinositides in the membrane and cyt b558 to position p67phox for catalysis of electron transfer to cyt b558. The specific role of actin-associated proteins in the recruitment, retention, or dissociation of p40/p47/p67phox at the membrane is unclear but WAVE1, moesin, and cortactin are likely candidates as recruiter proteins. Once the NADPH oxidase holo-enzyme has been assembled the magnitude and duration of NADPH oxidase activity is augmented by polymerization of actin (G- to F-actin conversion). Note, that cyt b558 itself is tightly associated with cortical actin and can be redistributed through the action of IQGAP. Evidence for the role of actin in disassembly of NADPH oxidase and deactivation of p40/p47/p67phox is unavailable, but likely p40/p47/p67phox returns to a tethered state with F-actin as end-point following dephosphorylation of p47phox.
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Figure 8: Model of p40/p47/p67phox membrane translocation and potential roles for the actin cytoskeleton and actin-regulatory proteins in NADPH oxidase assembly and function. At rest the p40/p47/p67phox complex is tethered to F-actin either directly or indirectly through for example moesin (bottom). Following cell activation p40phox and p47phox are activated by phosphorylation to acquire phosphoinositide and cyt b558-binding properties (green curved arrows) and loss of tight binding to F-actin. In addition, phosphorylation may lead to binding of actin-associated proteins or adaptors like TRAF4 to the phox protein complex. This membrane recruitment part of the cycle is supported by actin depolymerization (F- to G-actin conversion). At the membrane p47phox bind cortical actin, phosphoinositides in the membrane and cyt b558 to position p67phox for catalysis of electron transfer to cyt b558. The specific role of actin-associated proteins in the recruitment, retention, or dissociation of p40/p47/p67phox at the membrane is unclear but WAVE1, moesin, and cortactin are likely candidates as recruiter proteins. Once the NADPH oxidase holo-enzyme has been assembled the magnitude and duration of NADPH oxidase activity is augmented by polymerization of actin (G- to F-actin conversion). Note, that cyt b558 itself is tightly associated with cortical actin and can be redistributed through the action of IQGAP. Evidence for the role of actin in disassembly of NADPH oxidase and deactivation of p40/p47/p67phox is unavailable, but likely p40/p47/p67phox returns to a tethered state with F-actin as end-point following dephosphorylation of p47phox.

Mentions: Therefore we conclude that at least in microglia actin polymerization and depolymerization can both enhance NADPH oxidase activity. This apparently contradictory result is probably reconciled by the cyclic nature of NADPH oxidase assembly during superoxide production: cytosolic phox proteins and Rac1/2 bound to cyt b558 are constantly exchanged with new subunits [10,11], which require release from F-actin (mobilization), translocation to membrane (actin or actin-associated protein facilitated), and tethering to membrane (initial contacts with cortical F-actin or associated proteins?) as illustrated in Figure 8. During cell activation these processes may be aided preferentially by induced F-actin depolymerization. Superimposed on this subunit cycling is the mechanic influence of F-actin on the assembled and functional NADPH oxidase whose activity is increased and prolonged by active actin polymerization [41]. Therefore, increasing the concentration of an actin polymerizing or depolymerizing factor may increase NADPH oxidase activity, but only to a point, where after one or more steps in the continuous assembly/disassembly cycle of cytosolic phox proteins with cyt b558, facilitated by the opposite actin fate, will be inhibited to a degree where it negatively affects superoxide release. Our results also imply that NADPH oxidase function in stimulated phagocytes always will be in an enhanced state of activity regardless of the very different alterations of the actin cytoskeleton required to effect different immune cell tasks such as chemotaxis, invasion, phagocytosis, redox signaling, or immunological synapse formation. Such a capacity of the respiratory burst to accommodate the plasticity of the actin cytoskeleton may be particularly relevant for microglia, which undergoe dramatic changes in morphology following cell activation going from a highly elongated and ramified morphology to a rounded, amoeboid shape.


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)

Model of p40/p47/p67phox membrane translocation and potential roles for the actin cytoskeleton and actin-regulatory proteins in NADPH oxidase assembly and function. At rest the p40/p47/p67phox complex is tethered to F-actin either directly or indirectly through for example moesin (bottom). Following cell activation p40phox and p47phox are activated by phosphorylation to acquire phosphoinositide and cyt b558-binding properties (green curved arrows) and loss of tight binding to F-actin. In addition, phosphorylation may lead to binding of actin-associated proteins or adaptors like TRAF4 to the phox protein complex. This membrane recruitment part of the cycle is supported by actin depolymerization (F- to G-actin conversion). At the membrane p47phox bind cortical actin, phosphoinositides in the membrane and cyt b558 to position p67phox for catalysis of electron transfer to cyt b558. The specific role of actin-associated proteins in the recruitment, retention, or dissociation of p40/p47/p67phox at the membrane is unclear but WAVE1, moesin, and cortactin are likely candidates as recruiter proteins. Once the NADPH oxidase holo-enzyme has been assembled the magnitude and duration of NADPH oxidase activity is augmented by polymerization of actin (G- to F-actin conversion). Note, that cyt b558 itself is tightly associated with cortical actin and can be redistributed through the action of IQGAP. Evidence for the role of actin in disassembly of NADPH oxidase and deactivation of p40/p47/p67phox is unavailable, but likely p40/p47/p67phox returns to a tethered state with F-actin as end-point following dephosphorylation of p47phox.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Model of p40/p47/p67phox membrane translocation and potential roles for the actin cytoskeleton and actin-regulatory proteins in NADPH oxidase assembly and function. At rest the p40/p47/p67phox complex is tethered to F-actin either directly or indirectly through for example moesin (bottom). Following cell activation p40phox and p47phox are activated by phosphorylation to acquire phosphoinositide and cyt b558-binding properties (green curved arrows) and loss of tight binding to F-actin. In addition, phosphorylation may lead to binding of actin-associated proteins or adaptors like TRAF4 to the phox protein complex. This membrane recruitment part of the cycle is supported by actin depolymerization (F- to G-actin conversion). At the membrane p47phox bind cortical actin, phosphoinositides in the membrane and cyt b558 to position p67phox for catalysis of electron transfer to cyt b558. The specific role of actin-associated proteins in the recruitment, retention, or dissociation of p40/p47/p67phox at the membrane is unclear but WAVE1, moesin, and cortactin are likely candidates as recruiter proteins. Once the NADPH oxidase holo-enzyme has been assembled the magnitude and duration of NADPH oxidase activity is augmented by polymerization of actin (G- to F-actin conversion). Note, that cyt b558 itself is tightly associated with cortical actin and can be redistributed through the action of IQGAP. Evidence for the role of actin in disassembly of NADPH oxidase and deactivation of p40/p47/p67phox is unavailable, but likely p40/p47/p67phox returns to a tethered state with F-actin as end-point following dephosphorylation of p47phox.
Mentions: Therefore we conclude that at least in microglia actin polymerization and depolymerization can both enhance NADPH oxidase activity. This apparently contradictory result is probably reconciled by the cyclic nature of NADPH oxidase assembly during superoxide production: cytosolic phox proteins and Rac1/2 bound to cyt b558 are constantly exchanged with new subunits [10,11], which require release from F-actin (mobilization), translocation to membrane (actin or actin-associated protein facilitated), and tethering to membrane (initial contacts with cortical F-actin or associated proteins?) as illustrated in Figure 8. During cell activation these processes may be aided preferentially by induced F-actin depolymerization. Superimposed on this subunit cycling is the mechanic influence of F-actin on the assembled and functional NADPH oxidase whose activity is increased and prolonged by active actin polymerization [41]. Therefore, increasing the concentration of an actin polymerizing or depolymerizing factor may increase NADPH oxidase activity, but only to a point, where after one or more steps in the continuous assembly/disassembly cycle of cytosolic phox proteins with cyt b558, facilitated by the opposite actin fate, will be inhibited to a degree where it negatively affects superoxide release. Our results also imply that NADPH oxidase function in stimulated phagocytes always will be in an enhanced state of activity regardless of the very different alterations of the actin cytoskeleton required to effect different immune cell tasks such as chemotaxis, invasion, phagocytosis, redox signaling, or immunological synapse formation. Such a capacity of the respiratory burst to accommodate the plasticity of the actin cytoskeleton may be particularly relevant for microglia, which undergoe dramatic changes in morphology following cell activation going from a highly elongated and ramified morphology to a rounded, amoeboid shape.

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