Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia.
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.


CONCLUSIONS
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.
Rasmussen
Izabela
I
Dept of Cellular and Molecular Medicine, The Panum Institute, Copenhagen University, 2200N Copenhagen, Denmark.
Pedersen
Line H
LH
Byg
Luise
L
Suzuki
Kazuhiro
K
Sumimoto
Hideki
H
Vilhardt
Frederik
F
D051339:Actin Depolymerizing Factors / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D006801:Humans; D054461:Lim Kinases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D017628:Microglia / Q000378:metabolism* / Q000473:pathology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D058105:Polymerization / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D019076:Transgenes / Q000235:genetics
D051339:Actin Depolymerizing Factors; D000199:Actins; D034741:RNA, Small Interfering; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D054461:Lim Kinases
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