title: PtdIns3P and Rac direct the assembly of the NADPH oxidase on a novel, pre-phagosomal compartment during FcR-mediated phagocytosis in primary mouse neutrophils.
abstract: The generation of reactive oxygen species (ROS) by the nicotinamide adenine dinucleotide phosphate oxidase is an important mechanism by which neutrophils kill pathogens. The oxidase is composed of a membrane-bound cytochrome and 4 soluble proteins (p67(phox), p40(phox), p47(phox), and GTP-Rac). These components form an active complex at the correct time and subcellular location through a series of incompletely understood mutual interactions, regulated, in part, by GTP/GDP exchange on Rac, protein phosphorylation, and binding to lipid messengers. We have used a variety of assays to follow the spatiotemporal assembly of the oxidase in genetically engineered primary mouse neutrophils, during phagocytosis of both serum- and immunoglobulin G-opsonized targets. The oxidase assembles directly on serum-Staphylococcus aureus-containing phagosomes within seconds of phagosome formation; this process is only partially dependent (∼ 30%) on PtdIns3P binding to p40(phox), but totally dependent on Rac1/2 binding to p67(phox). In contrast, in response to immunoglobulin G-targets, the oxidase first assembles on a tubulovesicular compartment that develops at sites of granule fusion to the base of the emerging phagosome; oxidase assembly and activation is highly dependent on both PtdIns3P-p40(phox) and Rac2-p67(phox) interactions and delivery to the phagosome is regulated by Rab27a. These results define a novel pathway for oxidase assembly downstream of FcR-activation.
authors.affiliation: Inositide Laboratory, Babraham Institute, Babraham Research Campus, Cambridge, UK.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Anderson
authors.forename: Tamara A M
authors.initials: TA
authors.lastname: Chessa
authors.forename: Keith
authors.initials: K
authors.lastname: Davidson
authors.forename: Robert B
authors.initials: RB
authors.lastname: Henderson
authors.forename: Simon
authors.initials: S
authors.lastname: Walker
authors.forename: Tanya
authors.initials: T
authors.lastname: Tolmachova
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Grys
authors.forename: Oliver
authors.initials: O
authors.lastname: Rausch
authors.forename: Miguel C
authors.initials: MC
authors.lastname: Seabra
authors.forename: Victor L J
authors.initials: VL
authors.lastname: Tybulewicz
authors.forename: Len R
authors.initials: LR
authors.lastname: Stephens
authors.forename: Phillip T
authors.initials: PT
authors.lastname: Hawkins
mesh_terms: D000818:Animals; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology* / Q000276:immunology; D010587:Phagocytosis / Q000502:physiology*; D018129:Phosphatidylinositol Phosphates / Q000276:immunology / Q000378:metabolism*; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism; D011961:Receptors, Fc / Q000276:immunology; D020744:rac GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*
chemical_list: D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D011961:Receptors, Fc; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; C055525:phosphatidylinositol 3-phosphate; D019255:NADPH Oxidases; D020744:rac GTP-Binding Proteins
references.citation: J Leukoc Biol. 2005 Nov;78(5):1025-42
references.pmid: 16204621
references.citation: Mol Cell. 2000 Oct;6(4):899-907
references.pmid: 11090627
references.citation: J Immunol. 2006 Nov 1;177(9):6388-97
references.pmid: 17056570
references.citation: Nat Cell Biol. 2001 Jul;3(7):675-8
references.pmid: 11433300
references.citation: Antioxid Redox Signal. 2002 Feb;4(1):49-60
references.pmid: 11970843
references.citation: J Cell Sci. 1998 Jan;111 ( Pt 1):81-91
references.pmid: 9394014
references.citation: J Immunol Methods. 1999 Dec 17;232(1-2):3-14
references.pmid: 10618505
references.citation: Blood. 2009 Oct 8;114(15):3309-15
references.pmid: 19692703
references.citation: Blood. 2005 Aug 15;106(4):1432-40
references.pmid: 15878979
references.citation: Nat Rev Immunol. 2006 Mar;6(3):173-82
references.pmid: 16498448
references.citation: J Leukoc Biol. 2004 Dec;76(6):1093-103
references.pmid: 15466916
references.citation: Methods Enzymol. 2008;438:77-93
references.pmid: 18413242
references.citation: Biochem J. 2007 Mar 1;402(2):229-39
references.pmid: 17090228
references.citation: Biochem J. 1994 Mar 15;298 Pt 3:585-91
references.pmid: 8141770
references.citation: J Leukoc Biol. 1999 May;65(5):629-34
references.pmid: 10331491
references.citation: Nat Rev Immunol. 2006 Jul;6(7):541-50
references.pmid: 16799473
references.citation: J Immunol. 2003 Jun 1;170(11):5652-7
references.pmid: 12759446
references.citation: Blood. 2008 Nov 1;112(9):3867-77
references.pmid: 18711001
references.citation: Blood. 2008 Dec 1;112(12):4712-22
references.pmid: 18682599
references.citation: Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):7933-8
references.pmid: 10859366
references.citation: Nat Rev Immunol. 2008 Jan;8(1):34-47
references.pmid: 18064051
references.citation: Annu Rev Immunol. 2002;20:825-52
references.pmid: 11861619
references.citation: Biochem Biophys Res Commun. 1996 Jan 5;218(1):54-60
references.pmid: 8573175
references.citation: J Cell Biol. 2001 Oct 1;155(1):19-25
references.pmid: 11581283
references.citation: Immunity. 1999 Feb;10(2):183-96
references.pmid: 10072071
references.citation: Biochem J. 2005 Mar 15;386(Pt 3):401-16
references.pmid: 15588255
references.citation: Genes Dev. 2002 Jul 1;16(13):1587-609
references.pmid: 12101119
references.citation: J Clin Invest. 1987 Dec;80(6):1550-60
references.pmid: 2445780
references.citation: Mol Cell. 2003 Jan;11(1):35-47
references.pmid: 12535519
references.citation: Nat Immunol. 2007 Jan;8(1):31-8
references.pmid: 17159984
references.citation: Blood. 1998 Nov 1;92(9):3007-17
references.pmid: 9787133
references.citation: J Immunol Methods. 1985 Sep 3;82(1):161-7
references.pmid: 3928762
references.citation: Blood. 1990 Nov 15;76(10):2105-16
references.pmid: 2173636
references.citation: EMBO J. 2006 Oct 4;25(19):4468-78
references.pmid: 16990793
references.citation: Nat Cell Biol. 2007 Apr;9(4):367-78
references.pmid: 17351642
references.citation: J Cell Sci. 2006 Jul 1;119(Pt 13):2749-57
references.pmid: 16772332
references.citation: Biochim Biophys Acta. 2004 Jun 28;1657(1):1-22
references.pmid: 15238208
references.citation: Nat Cell Biol. 2001 Jul;3(7):679-82
references.pmid: 11433301
references.citation: J Immunol. 2001 Jan 15;166(2):1223-32
references.pmid: 11145705
references.citation: Blood. 2008 Dec 15;112(13):5202-11
references.pmid: 18755982
references.citation: J Leukoc Biol. 2004 Oct;76(4):760-81
references.pmid: 15240752
references.citation: Nat Immunol. 2001 Mar;2(3):211-5
references.pmid: 11224519
references.citation: Hematology Am Soc Hematol Educ Program. 2005;:89-95
references.pmid: 16304364
references.citation: J Biol Chem. 2004 Mar 5;279(10):9097-102
references.pmid: 14623873
references.citation: Curr Biol. 1998 Jun 4;8(12):684-91
references.pmid: 9637919
references.citation: Infect Immun. 2008 Nov;76(11):5373-80
references.pmid: 18779337
references.citation: J Exp Med. 2006 Aug 7;203(8):1927-37
references.pmid: 16880254
references.citation: Mol Cell. 2001 Oct;8(4):829-39
references.pmid: 11684018
references.citation: J Leukoc Biol. 2010 Apr;87(4):545-55
references.pmid: 19955208
references.citation: Curr Biol. 2001 Oct 16;11(20):1631-5
references.pmid: 11676926