GROWTH CONE SURFACE PROTEINS THAT MEDIATE TARGET CONTACT

介导目标接触的生长锥表面蛋白质

• 批准号: 3412581
• 负责人: RICHARD T AMBRON
• 金额: $ 16.12万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 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 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3412581
• 关键词: Gastropoda; cell membrane; chemical binding; chromatography; electron microscopy; electrophoresis; glycoproteins; growth cones; histochemistry /cytochemistry; immunologic techniques; ligands; membrane proteins; molecular weight; monoclonal antibody; motor neurons; muscle cells; neurochemistry; oligosaccharides; proteins; radioassay; receptor; saltwater environment; synaptogenesis; tissue /cell culture; western blottings

During embryonic development, neurons extend growth cones that seek out and synapse on the appropriate target. The culmination of such contacts is the neuronal circuitry that underlies behaviors. The goal of our research is to determine how neurons recognize, and are recognized, by their targets. Any attempt to define recognition at the molecular level requires the ability to isolate and characterize molecules on the growth cone surface and the means to assess the function of putative recognition ligands isolated from the growth cones. These prerequisites are difficult to attain in the complex and heterogenous vertebrate system. They can be attained, however, by using the nervous system of Aplysia california where many neurons can be recognized as individuals and are large enough to study using biochemical, immunological and electrophysio- logical approaches. Growth cones, for example, can be isolated in pure form RUQ neurons growing in vitro and their polypeptide composition can be examined. Also, neuron L7 synapses on auricle and gill vein muscles to elicit initiation of the heartbeat and gill withdrawl, respectively. This simple circuit has been assembled in vitro and synaptogenesis between L7 and its target muscles has been quantitated, thereby providing a statistically meaningful way to assay components of the growth cone that block synapse formation. One of the constituents enriched at RUQ growth cones is a 75kd membrane glycoprotein that is exposed on the surface. This glycoprotein is one member of a class of similar glycoproteins (GP-75) that are rapidly transported to the terminals of many Aplysia neurons, including L7. GP-75 is heterogenous since it contains a glycopeptide composed of several oligosaccharides that bind to wheat germ agglutinin. It is significant, therefore, that one or more of these oligosacch-arides bind specifically to the muscle cells that are targets of L7. WE will test two hypotheses: first, that the GP-75 found ont eh growth cones of motor neurons has oligosaccharides that participate in recognition of muscle cells during synaptogenesis. Second, that muscle cells have receptors on their surface that recognize GP-75 oligosaccharide. These ideas will be investigated by: 1) isolating and characterizing the GP-75 oligosaccharides and identifying the constituents that block the binding of GP-75 to isolated muscle membranes; 2) generating monoclonal antibodies against the various species of GP-75 to determine their distribution among identified neurons; 3) identifying the antibodies and GP-75 oligosaccharides that prevent synaptogenesis between L7 and its targets; and 4), using the oligosaccharides as affinity ligands to isolate the receptors from each muscle type. In this way we will define the ligands and receptors involved in the formation of simple circuit.

在胚胎发育期间，神经元延伸生长锥体，寻找和 在适当的目标上进行突触。这种接触的高潮是 构成行为基础的神经回路。我们的研究目标是 以确定神经元如何识别以及如何被它们的目标识别。 任何在分子水平上定义识别的尝试都需要 在生长锥体表面分离和表征分子的能力 以及评估假定识别配体功能的方法 从生长锥体中分离出来。这些前提条件很难 在复杂而异质的脊椎动物系统中达到。他们可以是 然而，通过使用加利福尼亚州海兔的神经系统来实现 许多神经元可以被识别为个体，并且足够大，可以研究 使用生化、免疫学和电生理方法。 例如，生长锥体可以在生长的RUQ神经元中分离出来 在体外，可以检测它们的多肽组成。还有，神经元 耳廓和鳃静脉肌肉上L7突触的启动 心跳和吉尔分别退出。这条简单的电路已经被 L7与靶肌肉之间的体外组装和突触发生 被量化，从而提供了一种统计上有意义的方法来分析 阻止突触形成的生长锥体的成分。 在RUQ生长锥富集的一种成分是75kd的膜。 暴露在表面的糖蛋白。这种糖蛋白是一种 一类类似的糖蛋白(GP-75)的成员，它们迅速地 运输到许多海兔神经元的终末，包括L7。GP-75 是异质性的，因为它含有一种由几个 与小麦胚芽凝集素结合的低聚糖。这很重要， 因此，这些寡糖中的一个或多个与 作为L7靶点的肌肉细胞。我们将检验两个假设： 首先，GP-75在运动神经元生长锥上发现的 参与肌细胞识别的低聚糖 突触发生。第二，肌肉细胞表面有受体 可以识别GP-75低聚糖。这些想法将通过以下方式进行调查： 1)GP-75寡糖的分离、鉴定及鉴定 阻断GP-75与离体肌结合的成分 膜；2)产生针对不同物种的单抗 确定其在已鉴定神经元中的分布；3) 鉴定抗体和GP-75寡糖可预防 L7与其靶点之间的突触发生；以及4)使用 低聚糖作为亲和配体从每个受体中分离出来 肌肉类型。通过这种方式，我们将定义所涉及的配体和受体 在形成简单的电路。