MOLECULAR CYTOLOGY OF INTERMEDIATE FILAMENTS IN NERVE

这项研究计划的总体目标是阐明 神经丝组装的分子和细胞机制。 I.神经丝的机制、结构要求和调节 组装：体外组装的特殊结构要求 交换将通过结合蛋白质生化和 分子生物学方法。具体地说， 核心蛋白、选定残基的化学修饰及其影响 与亚区相对应的合成肽的数量将在 组装和交换的动力学。的构建和表达 亚域缺失的转基因核因子-L核心蛋白，位点- 特定的突变，或构建在神经丝和 胶质细丝将进行体外组装和交换测试，以 确定那些负责神经丝组装的结构域。 磷酸化在神经丝组装和相互作用中的作用将 用荧光光漂剂回收和深度冷冻蚀刻进行检测 电子显微镜。关于分子的进一步详细研究 神经细丝特异性蛋白激酶的特征将是 继续，该激酶的抗体将用于免疫细胞化学 在正常和阿尔茨海默病的大脑中， 已观察到神经丝。此外，自杀抑制肽 根据磷酸化序列将被用来评估其作用 轴突生长和稳定性中的磷酸化。 II.神经细丝的细胞动力学：荧光标记 神经丝蛋白和亚片段将被显微注射到培养的 确定神经细胞中是否存在体内动态平衡的核因子-L 在组装和未组装状态之间以及核因子-L的哪些部分 负责体内组装和交换。此外，模型还包括 新合成的轴浆基质的轴突运输 神经突起生长、亚单位周转过程中的神经丝蛋白 成熟轴突和再生轴突中的重组神经丝 将直接在单细胞水平上进行荧光检测 光漂白回收和微光视频显微镜。

The overall objective of this research program are to elucidate the molecular and cellular mechanisms of neurofilament assembly. I. Mechanism, Structural Requirements, and Regulation of Neurofilament Assembly: The specific structural requirement for in vitro assembly and exchange will be examined by a combination of protein biochemical and molecular biological approaches. Specifically, isolated domains of the core protein, chemical modification of selected residues, and the effects of synthetic peptides corresponding to subdomains will be examined on the kinetics of assembly and exchange. The construction and expression of genetically altered NF-L core protein with subdomain deletions, site- specific mutations, or chimeras constructed between neurofilaments and glial filaments will be tested for assembly and exchange in vitro to identify those structural domains responsible for neurofilament assembly. The role of phosphorylation in neurofilament assembly and interactions will be examined by fluorescence photobleach recovery and deep freeze etch electron microscopy. Further detailed studies on the molecular characterization of the neurofilament specific protein kinase will be continued and antibodies to the kinase will be used for immunocytochemistry in both normal and Alzheimer's brain where abnormal phosphorylation of neurofilaments has been observed. In addition, suicide inhibitory peptides based on the phosphorylation sequence will be used to assess the role of phosphorylation in axonal growth and stability. II. Cellular Dynamics of Neurofilaments: Fluorescently labeled neurofilament proteins and subfragments will be microinjected into cultured neurons to determine whether in vivo there is a dynamic equilibrium of NF-L between assembled and unassembled states and what portions of NF-L are responsible for assembly and exchange in vivo. In addition, models of axonal transport of the axoplasmic matix, desposition of newly synthesized neurofilament proteins during neurite outgrowth, turnover of subunits in mature axons, and the reorganization neurofilaments in regenerating axons will be directly tested at the single cell level by fluorescence photobleach recovery and low light video microscopy.