REGULATION OF NEURONAL MOTILITY

在神经元发育的寻径阶段，生长锥 作为专门的传感器，能够引导延伸的轴突 朝向遥远的目标地点。生长锥体显示出高水平的肌动蛋白- 以尚未指明的方式支持这一点的基于运动性的 指导过程。生长锥体表面的特定受体也 似乎参与了：其中一些受体与 可能作为空间线索的细胞外基质成分，仍然 另一些则识别其他细胞表面的细胞黏附分子(CAM)。 尽管人们对分子的特性有着浓厚的兴趣 参与神经引导，对实际情况知之甚少 涉及的信号转导过程；例如，如何受体 职业导致细胞骨架结构和运动能力的改变 很可能成为寻路决定的基础。拟议的研究尝试 为了填补我们知识中的这一空白：(1)通过表征信号 参与生长激素运动调节的信号转导机制 和结构以及(2)通过研究潜在的分子动力学 运动过程本身。这项工作的结果直接 对发育性脑疾病临床解释的启示 与异常的神经通路形成有关，并将延长我们的 了解神经再生的过程。诊断探头 也可能导致发育和再生神经元障碍 来自这项拟议的研究。该项目依赖于使用高 空间和时间分辨率数字成像技术研究 膜蛋白在生长锥体导向和调控中的作用 突触发生。为了实现这些目的，一个利用 已经开发出伪底物探针，其允许跟踪 生长锥体中的膜蛋白。这些探针(通常为100-200 nm 用感兴趣的配体衍生的珠子)正被用来研究 发生的生长锥细胞骨架结构和运动性的改变 既是对假底物本身的反应，也是在生长期间 锥形目标相互作用。为了便于进行伪底物实验， 将构建一个单光束梯度光学陷阱(激光镊子)。 激光镊子是一种非侵入性的微定位方法。 也可以用来测量力的物体(如微珠) 与生长锥体指导和识别过程相关。至 补充这些研究，细胞内肌动蛋白的动力学将是 用荧光光活化技术进行了表征。这将是 使我们能够比较和对比细胞内的f-肌动蛋白和细胞表面 生长激素靶标识别或底物中的蛋白质运动 粘附力。最后，将使用反向遗传方法来生成 可能参与的蛋白质的特定分子探针 生长锥运动的调节。具体来说，融合蛋白 海兔基因克隆后的免疫原性表达 文库将用于产生抗CAM和肌动蛋白结合的抗体 感兴趣的蛋白质。

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.