title: Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia.
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.
  
  
  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.
authors.affiliation: Dept of Cellular and Molecular Medicine, The Panum Institute, Copenhagen University, 2200N Copenhagen, Denmark.
authors.forename: Izabela
authors.initials: I
authors.lastname: Rasmussen
authors.forename: Line H
authors.initials: LH
authors.lastname: Pedersen
authors.forename: Luise
authors.initials: L
authors.lastname: Byg
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Suzuki
authors.forename: Hideki
authors.initials: H
authors.lastname: Sumimoto
authors.forename: Frederik
authors.initials: F
authors.lastname: Vilhardt
mesh_terms: 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
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D034741:RNA, Small Interfering; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D054461:Lim Kinases
references.citation: Mol Cell Biol. 2005 Mar;25(6):2320-30
references.pmid: 15743827
references.citation: Arch Biochem Biophys. 2006 Dec 15;456(2):183-93
references.pmid: 16854367
references.citation: J Biol Chem. 2008 Mar 21;283(12):7983-93
references.pmid: 18160398
references.citation: J Biol Chem. 2003 Sep 19;278(38):36830-40
references.pmid: 12855698
references.citation: J Biol Chem. 1994 Sep 2;269(35):22405-11
references.pmid: 8071369
references.citation: Biochem Biophys Res Commun. 2000 Oct 5;276(3):1186-90
references.pmid: 11027608
references.citation: J Biol Chem. 2002 Nov 1;277(44):42136-43
references.pmid: 12207012
references.citation: J Biol Chem. 1995 Aug 18;270(33):19551-6
references.pmid: 7642640
references.citation: Glia. 1999 Dec;28(3):265-71
references.pmid: 10559785
references.citation: Cell Biol Int. 2006 Feb;30(2):154-63
references.pmid: 16448823
references.citation: J Biol Chem. 2007 Aug 10;282(32):23284-95
references.pmid: 17562703
references.citation: Blood. 1999 May 15;93(10):3521-30
references.pmid: 10233905
references.citation: Histochem Cell Biol. 2004 Oct;122(4):277-91
references.pmid: 15293055
references.citation: J Cell Biochem. 2004 Jul 1;92(4):795-809
references.pmid: 15211576
references.citation: Mol Biol Cell. 2007 Feb;18(2):441-54
references.pmid: 17122360
references.citation: J Biol Chem. 2007 Oct 12;282(41):30273-84
references.pmid: 17698849
references.citation: J Biol Chem. 2002 May 31;277(22):19952-60
references.pmid: 11893732
references.citation: Biochem J. 2005 Apr 15;387(Pt 2):531-40
references.pmid: 15535802
references.citation: J Cell Sci. 1997 Dec;110 ( Pt 24):3071-81
references.pmid: 9365277
references.citation: J Biol Chem. 2002 Nov 22;277(47):45566-71
references.pmid: 12297504
references.citation: Biochem J. 1993 Feb 15;290 ( Pt 1):173-8
references.pmid: 8439286
references.citation: J Leukoc Biol. 2006 Jan;79(1):223-34
references.pmid: 16275890
references.citation: J Clin Invest. 1990 Mar;85(3):714-21
references.pmid: 2155923
references.citation: Arterioscler Thromb Vasc Biol. 2005 Nov;25(11):2295-300
references.pmid: 16179592
references.citation: J Leukoc Biol. 1995 Jul;58(1):108-13
references.pmid: 7616102
references.citation: Nat Methods. 2006 Feb;3(2):109-16
references.pmid: 16432520
references.citation: Arterioscler Thromb Vasc Biol. 2005 Mar;25(3):512-8
references.pmid: 15618548
references.citation: J Cell Biol. 2005 Dec 5;171(5):893-904
references.pmid: 16330715
references.citation: Biophys J. 1982 Oct;40(1):69-75
references.pmid: 7139035
references.citation: Biochem J. 2000 Jul 1;349(Pt 1):369-75
references.pmid: 10861249
references.citation: Nat Cell Biol. 1999 Sep;1(5):253-9
references.pmid: 10559936
references.citation: J Biol Chem. 2002 Jan 4;277(1):544-9
references.pmid: 11694543
references.citation: J Biol Chem. 2004 Mar 5;279(10):9097-102
references.pmid: 14623873
references.citation: J Clin Invest. 1991 Jan;87(1):352-6
references.pmid: 1985107
references.citation: J Biol Chem. 1999 Nov 19;274(47):33644-53
references.pmid: 10559253
references.citation: J Biol Chem. 2008 Jan 25;283(4):2108-19
references.pmid: 18029359
references.citation: FASEB J. 2007 Apr;21(4):1244-55
references.pmid: 17227953
references.citation: J Exp Med. 2006 Aug 7;203(8):1915-25
references.pmid: 16880255
references.citation: EMBO J. 2002 Dec 2;21(23):6312-20
references.pmid: 12456638
references.citation: J Biol Chem. 2002 Feb 8;277(6):4512-8
references.pmid: 11729195
references.citation: Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4474-9
references.pmid: 12672956
references.citation: J Cell Sci. 1998 Aug;111 ( Pt 16):2433-43
references.pmid: 9683637
references.citation: Biochem Biophys Res Commun. 2001 Nov 30;289(2):382-8
references.pmid: 11716484
references.citation: Breast Cancer Res Treat. 2006 Sep;99(2):143-53
references.pmid: 16541306