Coordination of signaling during phagocytosis

描述（由申请人提供）：巨噬细胞中Fcy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Cdc42和Rac和磷酸肌苷3′-激酶（PI3K）， PI3K在形成吞噬体的膜上产生3′磷酸肌苷（3′pls）。PI3K抑制剂对大颗粒吞噬的影响大于对小颗粒吞噬的影响，这表明3'Pls对吞噬的贡献依赖于颗粒大小。我们假设FcR的参与引发了两种信号：与3' pi无关的信号（I类），它与颗粒大小直接成比例；3' pi依赖信号（II类），它们出现在大颗粒的吞噬过程中。I类信号包括Cdc42的激活和蛋白募集到FcR复合体中，而II类信号包括Rac的激活和3' pi结合域蛋白的募集或激活。这一假设将通过生化、免疫荧光、比率荧光（RF）和荧光共振能量转移（FRET）为基础的显微方法进行检验，以测量单个吞噬事件中蛋白质的募集和Rac和Cdc42的激活。将制备和校准各种大小的igg调理颗粒，并测量信号响应的大小作为颗粒大小的函数。生物化学和免疫荧光显微镜将用于测量蛋白质向吞噬体的募集。RF显微镜将用于分析细胞中表达青色荧光蛋白（CFP）和黄色荧光蛋白（YFP）嵌合体的细胞的吞噬情况，这些嵌合体包括肌动蛋白、PI3K、脂质磷酸酶SHIP-1、Rac1、Cdc42、Pak1的p21结合域和信号蛋白的3' pi结合域。在吞噬过程中，Rac和Cdc42的活化将通过FRET化学计量学来测量，这是一种在显微镜图像中定量蛋白质-蛋白质相互作用的方法。最后，将使用操纵3'PI水平的方法来确定吞噬过程中蛋白质募集和Rac或Cdc42激活对3'PI的依赖性。PI3K抑制剂和过表达的SHIP-1或PH结构域将用于在吞噬过程中操纵3'Pl信号。将测量其对肌动蛋白动力学、吞噬体蛋白募集以及Cdc42和Rac活化的影响。因为这些研究将测量信号振幅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'Pls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.