Regulation of Actin Filament Formation in Phagocytes

描述(由申请人提供)：中性粒细胞和巨噬细胞中肌动蛋白细丝动态的调节允许细胞爬行到感染部位并摄取入侵的病原体。目前的拨款集中在巨噬细胞中最丰富的肌动蛋白调节蛋白CAPG上。目的1将探讨敲除CAPG基因对小鼠的功能影响。CAPG基因缺失的小鼠在巨噬细胞、中性粒细胞和树突状细胞移动和吸收异物的能力方面存在严重缺陷。为了确定CAPG与细胞中其他蛋白质的功能关系，将通过二维凝胶电泳和微测序以及基因芯片分析来评估其他巨噬细胞蛋白质的代偿性变化。这些适应性蛋白质变化的功能意义将通过过度表达识别的蛋白质和通过RNA干扰降低它们的浓度来评估。它们与CAPG结合的能力将通过下拉试验和酵母双杂交试验进行评估。CAPG的缺失会增加对细胞内李斯特菌感染的敏感性，这表明细胞免疫功能存在缺陷。将测量炎症介质IL-2和干扰素-γ的水平。将检测CD4和CDS淋巴细胞反应，巨噬细胞和树突状细胞抗原处理和与CD4淋巴细胞的沟通，靶细胞的杀伤细胞裂解，以及B细胞抗体的产生。CAPG在吞噬细胞中的表达浓度远远高于调节肌动蛋白组装所需的浓度，这增加了CAPG可能发挥其他功能的可能性。过表达CAPG对不同细胞系存活的影响将被检测。CAPG缺失的中性粒细胞和巨噬细胞的凋亡将与野生型细胞进行比较。目的2将分析CAPG功能获得突变蛋白的结构-功能关系。肌动蛋白细丝的切断和封端是巨噬细胞运动的关键步骤。聚合酶链式反应诱变产生了一系列功能获得的CAPG切割蛋白，结晶学研究揭示了它们的结构。核仁肌动蛋白和分析离心法被用来确定这些重要过程的结构-功能关系。CAPG的研究有望为细胞运动性、先天免疫和细胞介导性免疫以及细胞存活的机制提供新的见解。这些研究可能为预防感染、控制自身免疫性疾病和调节细胞死亡的时间提供新的策略。

DESCRIPTION (provided by applicant): The regulation of actin filament dynamics in neutrophils and macrophages allows cells to crawl to sites of infection and ingest invading pathogens. The present grant focuses on the most abundant actin regulatory protein in macrophages, CapG. Aim 1 will explore the functional consequences of knocking out CapG in mice. CapG-null mice have profound defects in the ability of their macrophages, neutrophils and dendritic cells to move and take in foreign material. In order to determine how CapG functionally relates to other proteins in the cell, the compensatory changes in other macrophage proteins will be assessed by 2-D gel electrophoresis and microsequencing, as well as by gene microarray analysis. The functional significance of these adaptive protein changes will be assessed by over-expressing the identified proteins and by lowering their concentrations by RNA interference. Their ability to bind to CapG will be assessed by pull-down and yeast-two hybrid assays. Loss of CapG results in increased susceptibility to infection by the intracellular bacterium Listeria, indicating a defect in cell-mediated immunity. Levels of the inflammatory mediators IL-2 and interferon-gamma will be measured. CD4 and CDS lymphocyte responses, macrophage and dendritic cell antigen processing and communication with CD4 lymphocytes, killer cell lysis of target cells, and B cell antibody production will be examined. CapG is expressed in phagocytes at far higher concentrations than required to regulate actin assembly, raising the possibility that CapG may serve other functions. The effects of over-expressing CapG on the survival of different cell lines will be examined. Apoptosis of CapG-null neutrophils and macrophages will be compared to wild-type cells. Aim 2 will analyze structure-function relationships in CapG gain-of-function mutant proteins. Actin filament severing and capping are critical steps for macrophage movement. PCR mutagenesis has created a series of gain-of-function CapG severing proteins and crystallographic studies have revealed their structure. Pyrenyl actin and analytical centrifugation are being used to define the structure-function relationship of these vital processes. Investigations of CapG promise to provide new insights into cell motility, innate and cell-mediated immunity, and the mechanisms underlying cell survival. These studies may provide new strategies for defending against infections, controlling auto-immune diseases and regulating the timing of cell death.