BIOCHEMISTRY OF GRANULE CELL MIGRATION

这项研究的总体目标是使用体外模型 该系统是我们实验室开发的，用于研究分子 发育过程中神经胶质引导的神经元迁移机制 大脑。这项拟议的研究将提纯和刻画一部小说 糖蛋白、天冬氨酸、抗哪种抗体 微培养系统中神经元-神经胶质细胞的相互作用。单克隆 和选定的单特异性多克隆抗Astroactin抗体 通过体外功能分析，将用于纯化Astroactin， 从小鼠lambda 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。 然后高分辨率视频显微镜将与 计算机辅助图像处理，以可视化的效果 单特异性Aspotactin抗体的Fab片段 迁移神经元及其前导过程与神经细胞的相互作用 神经胶质纤维。 Astroactin及其蛋白水解酶的生物化学研究 产品将被用来分析尺寸、形状和功能 分子的结构域。此信息将与 Astroactin多肽融合的体外实验研究 用于定义区域的多肽和单抗 调节神经元-神经胶质细胞相互作用的Astroactin。 然后我们将使用免疫细胞化学和分子生物学 方法研究Astroactin在胚胎发育过程中的表达 C57BL/6J小鼠脑和织布鼠突变小鼠， 因为我们发现阿司匹林缺失或有缺陷 织女小脑的内侧部分。最后，我们会 制备抗Asproactin抗体的抗独特型抗体 并使用亲和层析试图纯化一种受体 从神经胶质细胞中提取Astroactin。

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.