Coordination of signaling during phagocytosis

描述(由申请人提供)：FCY受体(FCR)介导的巨噬细胞吞噬作用通过膜和肌动蛋白细胞骨架的复杂运动将包被免疫球蛋白的颗粒内化。吞噬需要G蛋白CDC42和Rac，以及磷脂酰肌醇3‘-激酶(PI3K)，从而在形成的吞噬体膜上产生3’-磷脂酰肌醇(3‘Pls)。PI3K抑制剂对大颗粒吞噬作用的影响大于对小颗粒的影响，这表明3‘Pls对吞噬功能的贡献依赖于颗粒大小。我们假设FCR参与启动了两种信号：3‘PI无关信号(I类)，它与颗粒大小直接相关；3’PI依赖信号(II类)，它出现在较大颗粒的吞噬过程中。I类信号包括CDC42的激活和FCR复合体中蛋白质的募集，而II类信号包括RAC的激活和带有3‘PI结合区的蛋白质的募集或激活。这一假设将通过基于生化、免疫荧光、比率荧光(RF)和荧光共振能量转移(FRET)的微观方法来验证，这些方法用于测量RAC和CDC42在个别吞噬事件中的蛋白质募集和激活。不同大小的免疫球蛋白调理颗粒将被制备和校准，信号响应的大小将作为颗粒大小的函数进行测量。生物化学和免疫荧光显微镜将被用来测量蛋白质向吞噬小体的募集。射频显微镜将被用来分析细胞中的吞噬作用，这些细胞表达肌动蛋白、PI3K、脂磷酸酶Ship-1、rac1、CDc42的青色荧光蛋白(CFP)和黄色荧光蛋白(YFP)嵌合体，Ak1的p21结合域，以及来自信号蛋白的3‘PI结合域。RAC和CDC42在吞噬过程中的激活将通过FRET化学计量学来测量，FRET化学计量学是一种在显微图像中量化蛋白质-蛋白质相互作用的方法。最后，将使用控制3‘PI水平的方法来确定吞噬过程中蛋白质募集和RAC或CDC42激活的3’PI依赖关系。PI3K抑制剂和过表达的SHIP-1或PH结构域将被用来操纵吞噬过程中的3‘Pl信号。随之而来的对肌动蛋白动力学的影响，吞噬小体的蛋白质募集，以及Cdc42和Rac的激活将被测量。由于这些研究将测量信号幅度作为颗粒大小的函数，他们应该确定FCR信号的大小阈值，并确定3‘P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.