MOLECULAR MECHANISMS OF GRANULE CELL MIGRATION

颗粒细胞迁移的分子机制

• 批准号: 2379572
• 负责人: Mary Elizabeth Hatten
• 金额: $ 35.11万
• 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 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2379572
• 关键词: PC12 cells; animal tissue; antibody; biological models; cadherins; 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; 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.

这项研究的总体目标是了解分子 胶质细胞引导神经元迁移的机制及其遗传调控 在哺乳动物大脑发育过程中。 分析其分子机制 迁移，拟议的研究将使用体外模型系统， 在我们的实验室开发，以确定细胞粘附配体的作用， 在小脑颗粒神经元沿着胶质细胞的运动中 为着力 我们将检测抗体对神经胶质细胞的影响， 配体astrotactin，并比较这些抗体的影响， 神经元-神经元配体L1、N-CAM、N-cadherin和TAG-1，以及 纤维连接蛋白和层粘连蛋白的受体，整合素。 迁移细胞将是 用视频增强微分干涉对比显微镜成像 在微培养中检查频率、运动速率或细胞学 迁移的颗粒神经元。 在伴随研究中，我们将开发 染料标记颗粒迁移动力学成像方法 小脑组织切片中的神经元，并评估 抗细胞粘附配体的抗体对原位迁移的影响。 为了提供关于预测的蛋白质结构的信息， astrotactin，包括它的整体结构，相互作用， 膜和同源性与其他细胞粘附配体，亲和力- 纯化的抗astrotactin抗体将用于克隆用于 100 KD蛋白组分的astrotactin活性。 我们将用我们 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 通过在PC 12神经元中表达鉴定的克隆来检查cDNA 和织纹颗粒细胞。 为了分析神经元迁移的遗传调节，我们将 继续我们对神经系统突变小鼠的研究， 神经胶质引导的迁移，编织和蜿蜒的尾巴。 曲流尾巴是一种 令人兴奋的新突变严重破坏了细胞组织 局限于小脑的前叶。体外 重组实验将被用来定义细胞位点的 曲流尾基因的作用。 颗粒细胞迁移的动力学 在曲折尾小脑的组织切片中，然后将与 结果来自Weaver。 为了分析细胞间的相互作用是否与 神经元或轴突提供“停止信号”， 迁移，我们将检查小脑颗粒神经元是否终止 在体内和体外遇到神经元或轴突时的迁移 模型系统