BIOCHEMISTRY OF GRANULE CELL MIGRATION

本研究的总体目标是使用体外模型 系统，在我们的实验室开发，研究分子 发育中胶质细胞引导神经元迁移的机制 个脑袋 这项拟议中的研究将净化和表征一部小说， 糖蛋白，ASTROTACTIN，阻断抗体 微培养系统中神经元-神经胶质细胞的相互作用。 单克隆 和单特异性多克隆抗astrotactin抗体， 通过体外功能测定，将用于纯化astrotactin， 为了从小鼠λ gtll表达中选择cDNA克隆， 文库并从免疫阳性克隆中分离融合蛋白。 高分辨率视频显微镜将与 计算机辅助图像处理，以可视化 单特异性astrotactin抗体的Fab片段在 迁移神经元及其引导过程与 神经胶质纤维 astrotactin及其蛋白酶解的生化研究 产品将被用来分析的大小，形状和功能 分子的结构域。 这些信息将与 体外测定融合的astrotactin肽的作用 肽和单克隆抗体来定义 调节神经元-神经胶质相互作用的astrotactin。 然后我们将使用免疫细胞化学和分子生物学 方法研究astrotactin在发育中 C57 BL/6 J小鼠脑和韦弗小鼠突变小鼠中， 因为我们已经发现，阿司他汀是缺失或缺陷， 织布者小脑的内侧 最后我们将 针对抗astrotactin抗体产生抗独特型抗体 用亲和层析法来纯化受体 从神经胶质细胞中提取astrotactin。

The overall goal of this research is to use an in vitro model system, developed in our laboratory, to study the molecular mechanism of glial-guided neuronal migration in the developing brain. The proposed research will purify and characterize a novel glycoprotein, ASTROTACTIN, antibodies against which block neuron-glial interactions in the microculture system. Monoclonal and mono-specific polyclonal anti-astrotactin antibodies, selected with in vitro functional assays, will be used to purify astrotactin, to select cDNA clones from a mouse lambda gtll expression library and to isolate fusion proteins from immunopositive clones. High resolution video microscopy will then be combined with computer-assisted image processing to visualize the effects of Fab fragments of mono-specific astrotactin antibodies on the interaction of the migrating neuron and its leading process with the glial fiber. Biochemical studies on astrotactin and its proteolytic cleavage products will be used to analyze the size, shape and functional domains of the molecule. This information will be combined with in vitro assays of the effects of astrotactin peptides, fusion peptides and monoclonal antibodies to define the regions of astrotactin that regulate neuron-glial interactions. We will then use immunocytochemical and molecular biological methods to study the expression of astrotactin in the developing C57BL/6J mouse brain and in the weaver mouse mutant mouse, because we have found that astrotactin is missing or defective in the medial aspect of the weaver cerebellum. Finally, we will raise anti-idiotypic antibodies against anti-astrotactin antibodies and use affinity chromatography to attempt to purify a receptor for astrotactin from glial cells.