title: The recruitment of p47(phox) and Rac2G12V at the phagosome is transient and phosphatidylserine dependent.
abstract: BACKGROUND
  During phagocytosis, neutrophils internalise pathogens in a phagosome and produce reactive oxygen species (ROS) by the NADPH oxidase to kill the pathogen. The cytosolic NADPH oxidase subunits p40(phox), p47(phox), p67(phox) and Rac2 translocate to the phagosomal membrane to participate in enzyme activation. The kinetics of this recruitment and the underlying signalling pathways are only partially understood. Anionic phospholipids, phosphatidylserine (PS) and phosphoinositides (PPI) provide an important attachment site for numerous proteins, including several oxidase subunits.
  
  
  RESULTS
  We investigated the kinetics of p47(phox) and Rac2 phagosomal membrane recruitment. Both subunits are known to interact with anionic phospholipids; we therefore addressed the role of PS in this recruitment. Phagosomal accumulation of p47(phox) and Rac2 tagged with fluorescent proteins was analysed by videomicroscopy. We used the C2 domain of lactadherin (lactC2) that interacts strongly and specifically with PS to monitor intracellular PS localisation and to decrease PS accessibility. During phagocytosis of opsonised zymosan, p47(phox) and constitutively active Rac2G12V briefly translocated to the phagosomal membrane, whereas ROS production continued for a longer period. However, in the presence of lactC2, Rac2G12V recruitment was inhibited and the kinetics of p47(phox) recruitment and detachment were delayed. A reduced phagosomal ROS production was also observed during the first 7 min following the phagosome closure.
  
  
  CONCLUSIONS
  These results suggest that p47(phox) and Rac2 accumulate only transiently at the phagosome at the onset of NADPH activity and detach from the phagosome before the end of ROS production. Furthermore, lactC2, by masking PS, interfered with the phagosomal recruitment of p47(phox) and Rac2 and disturbed NADPH oxidase activity. Thus, PS appears as a modulator of NADPH oxidase activation.
authors.affiliation: Univ. Paris-Sud, Orsay, F-91405, France; INSERM UMRS757, Orsay, F-91405, France; Life Sciences Research Unit, University of Luxembourg, Luxembourg, L-1511, Luxembourg.
authors.forename: Marie Cécile
authors.initials: MC
authors.lastname: Faure
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Sulpice
authors.forename: Maud
authors.initials: M
authors.lastname: Delattre
authors.forename: Marc
authors.initials: M
authors.lastname: Lavielle
authors.forename: Magali
authors.initials: M
authors.lastname: Prigent
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Cuif
authors.forename: Chantal
authors.initials: C
authors.lastname: Melchior
authors.forename: Eric
authors.initials: E
authors.lastname: Tschirhart
authors.forename: Oliver
authors.initials: O
authors.lastname: Nüße
authors.forename: Sophie
authors.initials: S
authors.lastname: Dupré-Crochet
mesh_terms: D019943:Amino Acid Substitution; D000954:Antigens, Surface / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008894:Milk Proteins / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D009895:Opsonin Proteins / Q000378:metabolism; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015054:Zymosan / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D000097766:RAC2 GTP-Binding Protein
chemical_list: D000954:Antigens, Surface; D008894:Milk Proteins; D050505:Mutant Proteins; D019255:NADPH Oxidases; D009895:Opsonin Proteins; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D015054:Zymosan; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C098942:MFGE8 protein, human; C060505:neutrophil cytosolic factor 1
keywords: Anionic phospholipids; Live imaging; NADPH oxidase; Phagocytosis; ROS production