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: 2266014
• 负责人: RICHARD T AMBRON
• 金额: $ 16.66万
• 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 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2266014
• 关键词: Aplysia; alternatives to animals in research; 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; 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， 在合适的目标上建立突触 这种接触的高潮是 行为背后的神经回路。 我们的研究目标是 以确定神经元如何识别和被其目标识别。 任何在分子水平上定义识别的尝试都需要 分离和表征生长锥表面上的分子的能力 以及评估推定的识别配体的功能的方法 从生长锥中分离出来。 这些先决条件很难 在复杂和异质的脊椎动物系统中实现。 它们可以 然而，通过使用加州州的神经系统， 许多神经元可以被识别为个体，并且足够大以进行研究。 使用生物化学、免疫学和电生理学方法。 生长锥，例如，可以分离纯形式的RUQ神经元生长 并可检测其多肽组成。 另外，neuron 耳廓和鳃静脉肌肉上的L7突触，以引起 心跳和鳃回缩。 这个简单的电路 在体外组装，L7和它的靶肌肉之间的突触发生， 定量，从而提供了一种有统计学意义的方法来测定 生长锥中阻止突触形成的成分。 在RUQ生长锥中富集的成分之一是75 kd膜 暴露在表面的糖蛋白。 这种糖蛋白是一种 一类类似糖蛋白（GP-75）的成员， 运输到许多失神经元的末梢，包括L7。 GP-75 是异质的，因为它含有由几个糖肽组成的糖肽， 与麦胚凝集素结合的低聚糖。 它是重要的， 因此，这些寡糖中的一种或多种特异性结合至 肌肉细胞是L7的目标。 我们将检验两个假设： 首先，GP-75在运动神经元的生长锥中发现， 低聚糖参与识别肌肉细胞， 突触发生 第二，肌肉细胞表面有受体 能够识别GP-75寡糖 这些想法将由以下人员进行研究： 1)分离和表征GP-75寡糖并鉴定 阻断GP-75与离体肌肉结合的成分 膜; 2）产生针对各种物种的单克隆抗体 GP-75，以确定它们在识别的神经元中的分布; 3） 鉴定抗体和GP-75寡糖， 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，以从每一个 肌肉型 通过这种方式，我们将定义所涉及的配体和受体 形成简单的电路。