MOLECULAR MECHANISMS OF GRANULE CELL MIGRATION

颗粒细胞迁移的分子机制

• 批准号: 3396254
• 负责人: Mary Elizabeth Hatten
• 金额: $ 29.77万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3396254
• 关键词: PC12 cells; animal tissue; antibody; biological models; cell adhesion molecules; cell cell interaction; cell migration; cerebellum; chickens; chimeric proteins; complementary DNA; confocal scanning microscopy; developmental neurobiology; electron microscopy; enzyme linked immunosorbent assay; fibronectins; fluorescence microscopy; gene expression; genetic library; genetic manipulation; genetic regulation; glia; glycoproteins; granule cell; image processing; immunocytochemistry; immunologic techniques; immunoprecipitation; integrins; ion exchange chromatography; laboratory mouse; laminin; molecular cloning; mutant; neural cell adhesion molecules; neuronal guidance; neurons; nucleic acid sequence; oligonucleotides; polylysine; protein structure function; radiotracer; receptor; thymidine; tissue /cell culture; video recording system

The overall goal of this research is to understand the molecular mechanism of glial-guided neuronal migration and its genetic regulation in the developing mammalian brain. To analyze the molecular mechanism of migration, the proposed research will use an in vitro model system, developed in our laboratory, to define the role of cell adhesion ligands in the locomotion of the cerebellar granule neuron along the glial guide. We will assay the effects of antibodies gainst the neuron-glia ligand astrotactin, and compare these with effects of antibodies against the neuron-neuron ligands L1, N-CAM, N-cadherin and TAG-1, and of the receptor for fibronectin and laminin, integrin. Migrating cells will be imaged with video-enhanced differential interference contrast microscopy in microcultures to examine the frequency, rate of movement or cytology of migrating granule neurons. In companion studies, we will develop methods to image the dynamics of migration of dye-labeled granule neurons in tissue slices of cerebellum, and assess the effects of antibodies against cell adhesion ligands on migration in situ. To provide information on the predicted protein structure of astrotactin, including its overall structure, interaction with the membrane and homology with other cell adhesion ligands, affinity- purified anti-astrotactin antibodies will be used to clone cDNAs for the 100KD protein component of the astrotactin activity. We will use our biological assays to demonstrate that putative astrotactin clones encode functional epitopes of the astrotactin activity. Astrotactin clones will be analyzed at the molecular level and the function of astrotactin cDNAs will be xamined by expression of identified clones in PC12 neurons and weaver granule cells. To analyze the genetic regulation of neuronal migration, we will continue our studies on neurological mutant mice with defects in glial-guided migration, weaver and meander tail. Meander tail is an exciting new mutation with a severe disruption of cellular organization restricted restricted to the anterior lobe of the cerebellum. In vitro recombination experiments will be used to define the cellular site of action of the meander tail gene. The dynamics of granule cell migration in tissue slices of meander tail cerebellum will then be compared with results from weaver. To analyze whether cell-cell interactions with neurons or axons provide the "stop signals" for glial-guided neuronal migration, we will examine whether cerebellar granule neurons terminate migration upon encountering lanes of neurons or axons in and in vitro model system.

本研究的总体目标是了解分子 胶质引导的神经元迁移机制及其基因调控 在发育中的哺乳动物大脑中。 分析分子机制 迁移，拟议的研究将使用体外模型系统， 我们实验室开发的，用于定义细胞粘附配体的作用 小脑颗粒神经元沿神经胶质细胞的运动 指导。 我们将检测抗体对神经元胶质细胞的影响 配体 astrotactin，并将其与抗体的效果进行比较 神经元-神经元配体 L1、N-CAM、N-钙粘蛋白和 TAG-1，以及 纤连蛋白和层粘连蛋白、整合素的受体。 迁移细胞将 使用视频增强微分干涉显微镜成像 在微培养物中检查运动的频率、速率或细胞学 迁移的颗粒神经元。 在同伴研究中，我们将开发 染料标记颗粒迁移动态成像的方法 小脑组织切片中的神经元，并评估 原位迁移时针对细胞粘附配体的抗体。 提供有关预测的蛋白质结构的信息 astrotactin，包括其整体结构、与 膜和与其他细胞粘附配体的同源性，亲和力 纯化的抗 astroactin 抗体将用于克隆 cDNA 100KD 蛋白质成分的 astrotactin 活性。 我们将使用我们的 生物测定证明推定的 astrotactin 克隆编码 astrotactin 活性的功能表位。 Astrotactin克隆 将在分子水平上分析astrotactin的功能 将通过 PC12 神经元中已识别克隆的表达来检查 cDNA 和织布颗粒细胞。 为了分析神经元迁移的遗传调控，我们将 继续我们对具有缺陷的神经突变小鼠的研究 神经胶质引导的迁移、编织尾和蜿蜒尾。 曲折尾是一个 令人兴奋的新突变会严重破坏细胞组织 局限于小脑前叶。体外 重组实验将用于定义细胞位点 曲折尾基因的作用。 颗粒细胞迁移的动力学 然后将曲尾小脑的组织切片与 织工的结果。 分析细胞与细胞之间是否存在相互作用 神经元或轴突为神经胶质引导的神经元提供“停止信号” 迁移，我们将检查小脑颗粒神经元是否终止 在体外和体外遇到神经元或轴突泳道时的迁移 模型系统。