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、结构要求和调控 组装：体外组装的特定结构要求， 交换将通过蛋白质生化和 分子生物学方法。 具体而言，分离的结构域的 核心蛋白、选定残基的化学修饰及其影响 对应于亚结构域的合成肽将在 组装和交换的动力学。 的构建和表达 具有亚结构域缺失的遗传改变的NF-L核心蛋白，位点- 特定的突变，或神经丝之间构建的嵌合体， 将测试神经胶质丝在体外的组装和交换， 鉴定负责神经丝装配那些结构域。 磷酸化在神经丝组装和相互作用中的作用将 通过荧光光漂白恢复和深度冷冻蚀刻进行检查 电镜 进一步详细研究了 神经丝特异性蛋白激酶的表征将是 继续，激酶抗体将用于免疫细胞化学 在正常和阿尔茨海默氏症的大脑中， 已经观察到神经丝。 此外，自杀抑制肽 基于磷酸化序列将被用来评估的作用， 磷酸化对轴突生长和稳定性的影响。 二. 神经丝的细胞动力学：荧光标记 将神经丝蛋白和亚片段显微注射到培养的 神经元，以确定体内是否存在NF-L的动态平衡 以及NF-L的哪些部分 负责体内组装和交换。 此外， 轴浆基质的轴突运输，新合成的 轴突生长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 将通过荧光直接在单细胞水平上进行检测 光漂白恢复和低光视频显微术。

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.