Fcy receptor (FcR)-mediated phagocytosis in macrophages internalizes IgG-coated particles by complex movements of membranes and the actin cytoskeleton. Phagocytosis requires the G proteins Cdc42 and Rac, and phosphoinositide 3'- kinase (PI3K), which generates 3' phosphoinositides (3'Pls) in the membrane of the forming phagosome. Inhibitors of PI3K affect phagocytosis of large particles more than that of small particles, which indicates particle size-dependent contributions of 3'Pls to phagocytosis. We hypothesize that FcR engagement initiates two kinds of signals: 3'PI-independent signals (class I), which scale directly with particle size; and 3'PI-dependent signals (class II), which appear during phagocytosis of larger particles. Class I signals include activation of Cdc42 and the recruitment of proteins into FcR complexes, whereas class II signals include activation of Rac and the recruitment or activation of proteins with 3'PI-binding domains. This hypothesis will be tested by biochemical, immunofluorescence, ratiometric fluorescence (RF) and fluorescence resonance energy transfer (FRET)-based microscopic methods for measuring protein recruitment and the activation of Rac and Cdc42 during individual phagocytic events. IgG-opsonized particles of various sizes will be prepared and calibrated, and the magnitudes of signaling responses will be measured as a function of particle size. Biochemistry and immunofluorescence microscopy will be used to measure protein recruitment to phagosomes. RF microscopy will be used to analyze phagocytosis in cells expressing cyan fluorescent protein (CFP) plus yellow fluorescent protein (YFP) chimeras of actin, PI3K, the lipid phosphatase SHIP-1, Rac1, Cdc42, the p21- binding domain of Pak1, and 3'PI-binding domains from signaling proteins. Rac and Cdc42 activation during phagocytosis will be measured by FRET stoichiometry, a method for quantifying protein-protein interactions in microscopic images. Finally, methods for manipulating 3'PI levels will be used to determine the 3'PI-dependence of protein recruitment and Rac or Cdc42 activation during phagocytosis. PI3K inhibitors and overexpressed SHIP-1 or PH domains will be used to manipulate 3'Pl signaling during phagocytosis. The attendant effects on actin dynamics, protein recruitment to phagosomes, and Cdc42 and Rac activation will be measured. Because these studies will measure signal amplitudes as a function of particle size, they should identify size thresholds for FcR signaling and determine the relative contribution of 3'Pls to those thresholds.
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