Premium
The recruitment of p47 phox and Rac2G12V at the phagosome is transient and phosphatidylserine dependent
Author(s) -
Faure Marie Cécile,
Sulpice JeanClaude,
Delattre Maud,
Lavielle Marc,
Prigent Magali,
Cuif MarieHélène,
Melchior Chantal,
Tschirhart Eric,
Nüße Oliver,
DupréCrochet Sophie
Publication year - 2013
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1111/boc.201300010
Subject(s) - phagosome , nadph oxidase , phagocytosis , microbiology and biotechnology , biology , cytosol , intracellular , reactive oxygen species , phosphatidylserine , zymosan , p22phox , biochemistry , membrane , enzyme , in vitro , phospholipid
Background information 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.