Coordination of signaling during phagocytosis

描述（由申请人提供）：巨噬细胞中Fc γ受体（FcR）介导的吞噬作用通过膜和肌动蛋白细胞骨架的复杂运动内化IgG包被颗粒。吞噬作用需要G蛋白Cdc 42和Rac以及磷酸肌醇3 '-激酶（PI 3 K），其在形成吞噬体的膜中产生3'磷酸肌醇（3 'Pls）。PI 3 K的抑制剂对大颗粒的吞噬作用的影响大于小颗粒的吞噬作用，这表明3 'PI对吞噬作用的颗粒尺寸依赖性贡献。我们假设FcR参与启动两种信号：3 'PI独立信号（I类），其直接与颗粒大小成比例;和3' PI依赖信号（II类），其出现在较大颗粒的吞噬过程中。I类信号包括Cdc 42的激活和蛋白质向FcR复合物中的募集，而II类信号包括Rac的激活和具有3 'PI结合结构域的蛋白质的募集或激活。这一假设将测试生化，免疫荧光，比率荧光（RF）和荧光共振能量转移（FRET）为基础的显微镜方法，用于测量蛋白质的招聘和激活的Rac和Cdc 42在个别吞噬事件。将制备并校准各种尺寸的IgG调理颗粒，并将测量信号传导响应的幅度作为颗粒尺寸的函数。将使用生物化学和免疫荧光显微镜来测量蛋白质向吞噬体的募集。RF显微镜将用于分析表达肌动蛋白、PI 3 K、脂质磷酸酶SHIP-1、Rac 1、Cdc 42、Pak 1的p21结合结构域和来自信号蛋白的3 'PI结合结构域的青色荧光蛋白（CFP）加黄色荧光蛋白（YFP）嵌合体的细胞中的吞噬作用。吞噬作用期间的Rac和Cdc 42活化将通过FRET化学计量法测量，FRET化学计量法是一种用于定量显微图像中蛋白质-蛋白质相互作用的方法。最后，操纵3 'PI水平的方法将用于确定吞噬作用期间蛋白质募集和Rac或Cdc 42活化的3' PI依赖性。PI 3 K抑制剂和过表达的SHIP-1或PH结构域将用于在吞噬作用期间操纵3 'P1信号传导。随之而来的肌动蛋白动力学，蛋白质招募吞噬体，Cdc 42和Rac激活的影响将被测量。因为这些研究将测量作为颗粒尺寸的函数的信号幅度，所以它们应该确定FcR信号传导的尺寸阈值并确定3 'Pl对这些阈值的相对贡献。

DESCRIPTION (provided by applicant): 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.