GROWTH CONE GUIDANCE--INTERACTIONS AND SURFACE MOLECULES

我们已经确定并开始从分子和功能上 两种新的细胞表面分子的特性 神经发生 Lazarillo和Conulin是通过制备单克隆抗体发现的 专门针对发育中的神经系统， mAb作为生化试剂，用于纯化和表征 抗原的 我们克隆了拉扎里洛的基因 在本申请中提出克隆编码锥花蛋白的基因。 Lazarillo代表了一类新的分子，脂质运载蛋白， 在神经元通路的发现，并提出了一种新的分子信号 通路 我们提出实验来描述 拉扎里洛调节生长锥和丝状伪足的行为， 沿着一条特定的通路。 这将需要制定一个 “分子敲除”反义技术特异性抑制 Lazarillo在已识别神经元中的表达。 我们将测试 Lazarillo介导生长锥调节假说 行为通过信号转导途径，包括一个小的 亲脂性配体和蛋白质配体。 结构和功能 提出实验来鉴定小的亲脂性配体 结合在脂质运载蛋白疏水口袋和蛋白质配体中 与蛋白质相互作用域结合 这是我们的模型研究所提出的。 我们预测， 拉扎里洛存在于其他动物，包括脊椎动物， 在神经系统的发育过程中也是如此。 我们提出了一种简单的基于PCR的方法， 鉴定这些同源物并将使用序列信息， 原位杂交以验证同源性。 芋螺蛋白独特的空间结构 和暂时定位到神经生长锥的子集 使其成为新分子功能的良好候选者。 鉴定Conulin的实验包括免疫亲和性和 生化纯化和表达克隆。 我们假设 锥状蛋白在神经元路径发现中起着生长锥的作用， 在中央神经节中从一个轴突束转换到另一个 神经系统疾病 我们将通过观察 确定了已经从其生长锥中去除了锥蛋白的生长锥， 表面。 很明显，许多基本的分子机制 在发育过程中是高度保守的 神经系统的评估，如线虫，苍蝇和 老鼠. 我们建议拉扎里洛和科努林将带领我们找到新的 所有发育中的神经系统所使用的分子机制。

We have identified and begun the molecular and functional characterization of two new cell surface molecules involved in neurogenesis. Lazarillo and Conulin were found by making mAbs specific to the developing nervous system and then using those mAbs as biochemical reagents to purify and characterize each antigen. We have cloned the gene encoding Lazarillo and we are proposing to clone the gene encoding Conulin in this application. Lazarillo represents a new class of molecule, a lipocalin, involved in neuronal path findings and suggests a new molecular signaling pathway. We are proposing experiments to characterize how Lazarillo regulates growth cone and filopodial behavior to guide neurons along a specific pathway. This will entail developing a "molecular knockout" anti-sense technology to specifically inhibit the expression of Lazarillo in identified neurons. We will test the hypothesis that Lazarillo mediates its regulation of growth cone behavior via a signal transduction pathway that includes a small lipophilic ligand and a protein ligand. Structural and functional experiments are proposed to identify both the small lipophilic ligand that binds in the lipocalin hydrophobic pocket and the protein ligand that binds to the putative protein protein interaction domain suggested by our modeling studies. We predict that homologues of Lazarillo exist in other animals, including vertebrates, and function in a similar way during the development of their nervous systems. We have proposed a straightforward PCR based approach to identify these homologues and will use sequence information and in situ hybridization to verify the homology. Conulin's unique spacial and temporal localization to a subset of neuronal growth cones makes it a good candidate for a novel molecular function. Experiments to identify Conulin include immunoaffinity and biochemical purification, and expression cloning. We hypothesize that Conulin functions in neuronal path finding as growth cones switch from one axonal fascicle to another in the central ganglionic neuropil. We will test this hypothesis by observing the behavior of identified growth cones that have had conulin removed from their surfaces. It is clear that many of the basic molecular mechanisms in developmental processes have been highly conserved during the evaluation of nervous systems as diverse as nematode, fly and mouse. We propose that Lazarillo and Conulin will lead us to new molecular mechanisms used by all developing nervous systems.