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The recruitment of p47phox and Rac2G12V at the phagosome is transient and phosphatidylserine-dependent

Abstract : Background information: During phagocytosis, neutrophils internalize pathogens in a phagosome and produce reactive oxygen species (ROS) by the NADPH oxidase to kill the pathogen. The cytosolic NADPH oxidase subunits p40phox, p47phox, p67phox 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 important attachment site for numerous proteins, including several oxidase subunits. Results: We investigated the kinetics of p47phox 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 p47phox and Rac2 tagged with fluorescent proteins was analyzed by videomicroscopy. We used the C2 domain of lactadherin (lactC2) that interacts strongly and specifically with PS to monitor intracellular PS localization and to decrease PS accessibility. During phagocytosis of opsonized zymosan, p47phox and constitutively active Rac2G12V briefly translocated to the phagosomal membrane while ROS production continued for a longer period. However, in the presence of lactC2, Rac2G12V recruitment was inhibited and the kinetics of p47phox recruitment and detachment were delayed. A reduced phagosomal ROS production was also observed during the first seven minutes following the phagosome closure. Conclusions: These results suggest that p47phox 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 p47phox and Rac2 and disturbed NADPH oxidase activity. Thus, PS appears as a modulator of NADPH oxidase activation.
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Submitted on : Tuesday, December 10, 2013 - 5:50:40 PM
Last modification on : Friday, October 23, 2020 - 4:41:39 PM
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Marie-Cécile Faure, Jean-Claude Sulpice, Maud Delattre, Marc Lavielle, Magali Prigent, et al.. The recruitment of p47phox and Rac2G12V at the phagosome is transient and phosphatidylserine-dependent. Biology of the Cell, Wiley, 2013, 105, pp.1--18. ⟨10.1111/boc.201300010⟩. ⟨hal-01555264v2⟩

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