INTERMEDIATE FILAMENT-CELL SURFACE INTERACTIONS

本提案的总体目标是确定 中间丝（IF）细胞骨架网络的性质， 成纤维细胞和上皮细胞，以及确定的性质， IF与细胞表面的相互作用。 如果动力学将成为 在IF系统解聚的有丝分裂细胞中以及在 其中IF被重新聚合的子细胞。 具体而言，研究 III型IF网络广泛重塑的分子基础 将进行含有波形蛋白或波形蛋白/结蛋白的试验。 爆炸 解聚，其代表早中期前中期和快速解聚， 在后期-末期发生的IF重组可以是 在很大程度上，这是由IF的过度磷酸化引起的。 结构蛋白 在BHK细胞中发现了两种有丝分裂激酶 参与磷酸化反应。 我们干 对其中一种称为波形蛋白的激酶的广泛分析 激酶（VPK）。 VPK是三种蛋白质的复合物：65 kD组分， 110 kD组分和成熟的催化亚基p34 cdc 2 促进因子（MPF），并已知在触发 分裂。 我们计划进行旨在确定 被VPK磷酸化的特异性位点的功能意义， 使用多种细胞因子作用于III型IF蛋白的其它激酶 生理的（例如，显微注射），形态学（免疫荧光， 共聚焦显微镜和电子显微镜）、生物化学和分子生物学 (the使用细菌表达的蛋白质和瞬时转染 培养的哺乳动物细胞）技术。 我们还将尝试研究 IF的相间动力学性质。 在这些研究中，我们将利用 微注射衍生的IF蛋白，例如生物素化的 波形蛋白和角蛋白进入活细胞，并确定它们的命运在原位 使用共聚焦和电子显微镜。 类似的方法使用 X-罗丹明标记的IF蛋白将用于确定是否 在相间细胞中存在稳态或动态平衡， 使用光漂白后荧光恢复（FRAP）实验。 还描述了旨在确定生物化学的研究。 IF与细胞表面相关桥粒结合的基础 上皮细胞 这些研究涉及分离和生化 牛舌桥粒组分的表征和 确定角蛋白多肽如何与它们结合。 的信息 从我们的调查中得出的结果应该会对这些性质有新的认识。 和IF在哺乳动物细胞中的功能。 更好地理解IF 结构和功能是理解A 多种疾病中，IF发生变化，如阿尔茨海默氏症 疾病，帕金森病，各种癌症和酒精性肝硬化。

The overall goals of this proposal are to determine the dynamic properties of the intermediate filament (IF) cytoskeletal networks in fibroblasts and epithelial cells as well as to determine the nature of the interaction between IF and the cell surface. IF dynamics will be studied in mitotic cells in which the IF system is depolymerized and in daughter cells in which IF are repolymerized. Specifically, studies of the molecular basis of the extensive remodeling of Type III IF networks containing vimentin or vimentin/desmin will be undertaken. The explosive depolymerization which typifies early-mid prometaphase and the rapid reassembly of IF which takes place during anaphase-telophase can be explained, for the most part, by the hyperphosphorylation of IF structural proteins. Two mitotic kinases have been found in BHK cells which are involved in the phosphorylation reaction. We are undertaking an extensive analysis of one of these kinases termed vimentin protein kinase (VPK). VPK is a complex of three proteins: a 65kD component, a 110kD component, and p34cdc2 which is the catalytic subunit of maturation promoting factor (MPF) and is known to play a major role in triggering mitosis. We plan to carry out experiments aimed at determining the functional significance of the specific site(s) phosphorylated by VPK and other kinases which act on Type HI IF proteins using a variety of cell physiological (e.g., microinjection), morphological (immunofluorescence, confocal, and electron microscopy), biochemical and molecular biological (the use of bacterially expressed proteins and transient transfection of cultured mammalian cells) techniques. We will also attempt to study the interphase dynamic properties of IF. For these studies we will utilize the microinjection of derivatized IF proteins such as biotinylated vimentin and keratin into live cells and to determine their fate in situ using confocal and electron microscopy. Similar approaches using x-rhodamine labelled IF proteins will be used to determine whether or not a steady state or dynamic equilibrium exists in interphase cells through the use of fluorescence recovery after photobleaching (FRAP) experiments. Studies are also described which are aimed at determining the biochemical basis of the binding of IF to cell surface associated desmosomes in epithelial cells. These studies involve the isolation and biochemical characterization of bovine tongue desmosome components and the determination of how keratin polypeptides bind to them. The information derived from our investigations should shed new light on the properties and functions of IF in mammalian cells. A better understanding of IF structure and function is basic to understanding the pathogenesis of a variety of diseases in which changes in IF occur, such as Alzheimer's disease, Parkinson's disease, various cancers, and alcoholic cirrhosis.