39 Fluorescently Tagged Proteins to Monitor NADPH Oxidase Assembly During Phagocytosis

Abstract

The ability to examine the spatial and temporal localization of proteins in living cells through the use of fluorescent tags is a powerful tool for investigating the organization and dynamics of many cellular processes. We are applying this technology to studies on the NADPH oxidase expressed in neutrophils and monocyte/macrophages. This enzyme catalyzes the first step leading to the generation of potent microbicidal oxidants, which are critical for the killing of many bacterial and fungal pathogens. The importance of the NADPH oxidase to host defense is attested to by the impact of genetic defects in this enzyme, which result in Chronic Granulomatous Disease. In order to better understand the assembly and molecular regulation of this multi-subunit enzyme during phagocytosis, we are analyzing and testing fluorescently tagged derivatives of the NADPH oxidase subunits. The assembly of cytosolic factors p47^phox and p67^phox with flavocytochrome b₅₅₈ (gp91^phox and p22^phox) at the membrane is a crucial step in enzyme activation. The p47^phox and p67^phox subunits are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) in an SH3 domain, respectively, in their C-termini. This interaction is critical for p47^phox dependent p67^phox membrane translocation. To investigate molecular rearrangements of p47^phox and p67^phox during oxidase activation, a fragment derived from the p47^phox PRR and tagged with YFP (p47/YFP) was generated and shown to bind to recombinant p67^phox in vitro. When expressed in PLB-985 granulocytes, this fragment was recruited to phagosomes. This suggests that the C-terminal SH3 domain of p67^phox becomes accessible following oxidase assembly. Additional studies are examining the role of p40^phox, an additional regulatory subunit of the NADPH oxidase that has a phosphoinositide binding site critical for oxidase activity on phagosomes (Suh, et al, J Exp Med, In Press). Whether p40^phox also promotes recruitment of p67^phox to the membrane or acts as an allosteric regulator is being examined in similar studies using fluorescently tagged derivatives while imaging living granulocytes undergoing phagocytosis.

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