REGULATION OF NEURONAL MOTILITY

神经元运动的调节

• 批准号: 3415262
• 负责人: PAUL FORSCHER
• 金额: $ 17.31万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3415262
• 关键词: Aplysia; actins; adhesions; alternatives to animals in research; biological signal transduction; chimeric proteins; cytoskeleton; digital imaging; extracellular; fluorescence; growth cones; intermolecular interaction; lasers; membrane proteins; membrane structure; micromanipulator; molecular cloning; molecular dynamics; neural cell adhesion molecules; neuronal guidance; neuronal transport; neutralizing antibody; nucleic acid probes; photoactivation; protein transport; tissue /cell culture

During the pathfinding phase of neuronal development, the growth cone functions as a specialized sensor, capable of guiding extending axons toward distant target sites. Growth cones display a high level of actin- based motility which, in an as yet unspecified manner, supports this guidance process. Specific receptors on the growth cone surface also appear to be involved: some of these receptors interact with extracellular matrix components that may serve as spatial cues, still others recognize cell adhesion molecules (CAMs) on other cell surfaces. Although there has been intense interest in characterizing molecules involved in neuronal guidance, very little is known about the actual signal transduction processes involved; for example, how receptor occupation leads to alterations of cytoskeletal structure and motility likely to underlie pathfinding decisions. The proposed research attempts to fill this gap in our knowledge: (1) by characterizing signal transduction mechanisms involved in regulation of growth cone motility and structure and (2) by investigating the molecular dynamics underlying the motility process itself. The results of this work have direct implications for clinical interpretation of developmental brain disorders involving aberrant neuronal pathway formation and will extend our understanding of the process of nerve regeneration. Diagnostic probes for developmental and regenerative neuronal disorders could also result from the proposed research. This project relies on the use of high spatial and temporal resolution digital imaging techniques to investigate the behavior of membrane proteins involved in growth cone guidance and synaptogenesis. To achieve these ends, a system utilizing pseudosubstrate probes has been developed that allows tracking of membrane proteins in growth cones. These probes (typically 100-200 nm beads derivatized with ligands of interest) are being used to investigate alterations of growth cone cytoskeletal structure and motility that occur both in response to the pseudosubstrates themselves and during growth cone target interactions. To facilitate the pseudosubstrate experiments, a single beam gradient optical trap (laser tweezers) will be constructed. The laser tweezers is a non invasive method for micropositioning of small objects (like microbeads) which can also be used to measure forces associated with the growth cone guidance and recognition processes. To compliment these studies, intracellular actin dynamics will be characterized using fluorescence photoactivation techniques. This will allow us to compare and contrast intracellular f-actin and cell surface protein movements involved in growth cone target recognition or substrate adhesion. Finally, a reverse genetic approach will be used to generate specific molecular probes for proteins likely to be involved in regulation of growth cone motility. Specifically, fusion proteins immunogens expressed subsequent to gene cloning from an Aplysia cDNA library will be used to generate antibodies to CAM and actin binding proteins of interest.

在神经元发育的寻路阶段，生长锥 作为一种特殊的传感器，能够引导延伸的轴突 远距离的目标地点。 生长锥显示出高水平的肌动蛋白- 基于运动性，以尚未指明的方式，支持这一点， 指导过程。 生长锥表面的特异性受体也 似乎参与其中：这些受体中的一些与 细胞外基质成分仍然可以作为空间线索， 其它的识别其它细胞表面上的细胞粘附分子（CAM）。 尽管人们对表征分子有着浓厚的兴趣， 涉及神经元的指导，很少有人知道的实际 参与的信号转导过程;例如，受体如何 占据导致细胞骨架结构和运动性的改变 很可能成为寻路决策的基础。 建议的研究尝试 为了填补这一空白，我们的知识：（1）通过表征信号 参与生长锥运动调节的转导机制 （2）通过研究分子动力学基础 运动过程本身。 这项工作的结果有直接的 脑发育障碍的临床解释 涉及异常神经元通路的形成，并将延长我们的 了解神经再生的过程。 诊断探针 发育和再生神经元紊乱也可能导致 从拟议的研究。 该项目依赖于使用高 空间和时间分辨率的数字成像技术， 参与生长锥引导的膜蛋白的行为， 突触发生 为了实现这些目标，一种系统利用 已经开发了伪基底探针， 生长锥中的膜蛋白。 这些探针（通常为100-200 nm 用感兴趣的配体衍生化的珠）被用于研究 生长锥细胞骨架结构和运动性的改变， 无论是对假基质本身还是在生长过程中 视锥靶相互作用 为了便于假基质实验， 将构造单光束梯度光学阱（激光镊子）。 激光镊子是一种非侵入性的微小组织微定位方法， 也可用于测量力的物体（如微珠） 与生长锥引导和识别过程相关。 到 补充这些研究，细胞内肌动蛋白动力学将是 使用荧光光活化技术表征。 这将 使我们能够比较和对比细胞内F-肌动蛋白和细胞表面 参与生长锥目标识别或底物的蛋白质运动 粘连 最后，反向遗传方法将用于生成 针对可能参与的蛋白质的特异性分子探针 生长锥运动的调节。 具体而言，融合蛋白 免疫原表达后，基因克隆从一个Aaplasia cDNA 文库将用于产生CAM和肌动蛋白结合的抗体 感兴趣的蛋白质