Assembly and Maintenance of the Axonal Cytoskeleton in Cultured Neurons

NB2a/dl神经母细胞瘤细胞为研究提供了模型系统 轴突细胞骨架动力学。 分化后， dbcAMP，NB2a/dl细胞表达并磷酸化所有三种 神经丝蛋白亚基（NFP），并将它们组装成 一系列事件中的神经丝， 在体内观察。 这些细胞的轴突微管含有 乙酰化α微管蛋白，一种翻译后修饰， 赋予微管稳定性的微管蛋白。 NB2a/dl细胞 也表达钙蛋白酶，钙激活蛋白酶， 优先降解细胞骨架蛋白及其调节 分子，钙蛋白酶抑制素。 在这个项目中， NFP和α微管蛋白的合成、修饰、组装和 钙蛋白酶的合成和活性， 钙蛋白酶抑制蛋白在分化过程中定义为：1. 免疫印迹分析中存在稳态水平 利用特异性抗血清的细胞骨架和可溶性部分 对于未修饰的亚基：2.放射性标记的免疫沉淀 . J H.亚基;和3.分离的亚基的内源性蛋白水解 钙蛋白酶 假设一个或多个基因的改变 事件是轴突萎缩的原因，将通过 dbcAMP的撤回（其诱导轴突的再吸收） 然后通过上述方法分析培养物。 直接 将钙蛋白酶抑制分子引入细胞中， 用于进一步检查轴突中蛋白水解的作用， 萎缩

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.