MOLECULAR/FUNCTIONAL ANALYSIS OF AXON FASCICLE PROTEINS

描述（来自申请人摘要） 在早期阶段正确的神经通路的形成 胚胎发生涉及许多导航策略。 突出 其中之一是生长锥沿着特定轴突的延伸 大片，其中的选择很可能是我调解的层次结构 分子引导线索的能力这一过程可能在以下方面发挥关键作用： 神经系统的正确布线本提案的目的 是通过识别和 剖析了一个定义明确的 水蛭外周感觉神经元的数量， 具体路径选择。在这个系统中这样做的承诺是 几种新的分子--它们之间的相互作用很可能 参与这一进程-已经确定， 这些分子的轴突束 分布描述。因此，我们将进行分子和 这些蛋白质的功能分析，这些蛋白质是由小 在水蛭中形成特定束的轴突子集具有特殊的 重点是克隆Lan 3 -2/4-2抗原。此外，我们将 分子特征1）一种新发现蛋白质， 血小板反应蛋白同源物，L（tsp），可能与lan 3 -2相互作用 抗原，和2）200 kD蛋白，L（p200），其与 calsensin，一种小EF-手型钙结合蛋白，在一个亚群中表达 在单个轴突束中成束的外周神经元。在这些 实验中我们将特别检验LAN 3 -2 抗原可以既作为促生长嗜同性的 粘附分子，它可能参与嗜异性 与L（tsp）的相互作用，这是由CNS效应表达，在某种程度上， 帮助引导外周感觉神经元轴突到达中枢神经系统。 我们将通过用抗体的体内功能阻断测定来实现这一点， 重组蛋白片段和通过CNS消融和易位 胚胎发育中的实验。 从所提出的实验中，我们将对 这些分子的结构和功能意义，它们可能的 等级组织及其发展规律 表情正如我们之前的结果所证明的，相对简单的 水蛭胚胎的可接近系统提供了一个独特的机会 用于鉴定介导轴突通路形成的分子， 将它们的结构与它们在体内的功能相关联。既然已经 确定了许多重要的结构蛋白质序列基序 在整个进化过程中，这些研究在功能上是保守的， 应该可以增强我们对神经元识别的基本理解， 选择性肌束震颤，并提供新的见解， 神经连接异常和大脑发育异常的原因。

DESCRIPTION (from applicant's abstract) The formation of correct neuronal pathways during the early stages of embryogenesis involves a number of navigational strategies. Prominent among them is the extension of growth cones along particular axon tracts, the selection of which is likely to me mediated by a hierarchy of molecular guidance cues. This process may play a crucial role in the correct wiring of the nervous system. The object of the present proposal is to increase our knowledge of such molecules by identifying and dissecting the molecular guidance mechanisms of a well defined population of peripheral sensory neurons in leech which make highly specific pathway choices. The promise of doing this in this system is that several novel molecules - the interactions between which are likely to be involved in this process - have already been identified and that these molecules have some of the most restricted axonal tract distributions yet described. Thus, we will perform a molecular and functional analysis of such proteins which are expressed by small subsets of axons forming specific fascicles in the leech with special emphasis on the cloning of lan3-2/4-2 antigens. In addition, we will molecularly characterize 1) a newly discovered protein with partial thrombospondin homology, L(tsp), which may interact with the lan3-2 antigen, and 2) a 200 kD protein, L(p200) which interacts with calsensin, a small EF-hand calcium-binding protein expressed in a subset of peripheral neurons fasciculating in a single axon tract. In these experiments we will in particular test the hypothesis that the lan3-2 antigen may be functioning both as a growth promoting homophilic adhesion molecule and that it may participate in heterophilic interactions with L(tsp), which is expressed by CNS effects, in a way that helps guide that axons of peripheral sensory neurons to the CNS. We will do this by in vivo function blocking assays with antibody and recombinant protein fragments and by CNS ablation and translocation experiments in the developing embryo. From the proposed experiments we will gain a basic understanding of the structure and functional significance of these molecules, their possible hierarchial organization, and their developmental regulation of expression. As our previous results demonstrate, the relatively simple and accessible system of the leech embryo provides a unique opportunity for identifying molecules mediating axonal pathway formation and to correlate their structure with their function in vivo. Since it has been established that many important structural protein sequence motifs have been functionally conserved throughout evolution, these investigations should enhance our basic understanding of neuronal recognition and selective fasciculation and provide new insights into the underlying causes of aberrant neural connections and abnormal brain development.