Assembly and Maintenance of the Axonal Cytoskeleton in Cultured Neurons

NB2a/dl神经母细胞瘤细胞为轴突细胞骨架动力学研究提供了一个模型系统。在dbcAMP分化后，NB2a/dl细胞表达和磷酸化所有三种神经丝蛋白亚基（NFPs），并将它们组装成神经丝，其事件序列与在体内观察到的非常相似。这些细胞的轴突微管含有乙酰化的α。微管蛋白，一个帖子。赋予微管稳定性的微管蛋白的翻译修饰。NB2a/dl细胞也表达钙蛋白酶。优先降解细胞骨架蛋白及其调控分子钙pastatin的活化蛋白酶。在这个项目中，NFP和alpha的事件序列。微管蛋白的合成、修饰、组装和降解，以及钙蛋白酶和钙pastatin的合成和活性将在分化过程中定义：1。免疫印迹分析。使用未修饰亚基特异性抗血清的细胞骨架和可溶性组分中存在的状态水平：放射标记免疫沉淀。J H.subunits;和3。内源性钙蛋白酶对分离亚基的蛋白水解。一个或多个事件的改变导致轴突萎缩的假设将通过撤销dbcAMP（诱导轴突吸收），然后通过上述方法分析培养物来验证。将钙蛋白酶抑制分子直接导入细胞将被用于进一步研究蛋白水解在轴突萎缩中的作用。

NB2a/dl neuroblastoma cells provide a model system for the study of axonal cytoskeletal dynamics. Following differentiation with dbcAMP, NB2a/dl cells express and phosphorylate all three neurofilament protein subunit (NFPs) and assemble them into neurofilaments in a sequence of events closely paralleling that observed in vivo. Axonal microtubules of these cells contain acetylated alpha.tubulin, a post.translational modification of tubulin that confers stability to microtubules. NB2a/dl cells also express calpain, the calcium.activated protease that preferentially degrades cytoskeletal proteins and its regulatory molecule, calpastatin. In this project, the sequence of events of NFP and alpha.tubulin synthesis, modification, assembly and degradation, and the synthesis and activity of calpain and calpastatin will be defined during differentiation by: 1. immunoblot analysis of steady.state levels present in cytoskeletal and soluble fractions utilizing antisera specific for unmodified subunits: 2. immunoprecipitation of radiolabeled . J H.subunits; and 3. proteolysis of isolated subunits by endogenous calpain. The hypothesis that alterations in one or more of the events is responsible for axonal atrophy will be tested by withdrawal of dbcAMP (which induces resorption of axons) followed by analysis of cultures by the above methods. Direct introduction into cells of calpain inhibitory molecules will be utilized to further examine the role of proteolysis in axonal atrophy.