INTERMEDIATE FILAMENT-CELL SURFACE INTERACTIONS

中间丝状细胞表面相互作用

• 批准号: 2178531
• 负责人: ROBERT D GOLDMAN
• 金额: $ 21.39万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1995-08-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178531
• 关键词: active sites; affinity chromatography; animal tissue; cell cycle; cell membrane; confocal scanning microscopy; cow; cytoskeleton; electron microscopy; enzyme complex; enzyme structure; fibroblasts; fibrous protein; fluorescent dye /probe; immunofluorescence technique; intercellular connection; intermediate filaments; ion exchange chromatography; keratin; laboratory mouse; laboratory rabbit; microinjections; monoclonal antibody; phosphorylation; polymerization; protein biosynthesis; protein engineering; protein kinase; protein purification; protein structure function; site directed mutagenesis; tissue /cell culture; transfection; vimentin

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.

这项提案的总体目标是确定动态 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。如果动态将是 在有丝分裂细胞中进行的研究，在有丝分裂细胞中IF系统被解聚，并且在 其中IF被重新聚合的子细胞。具体地说，研究 III型IF网络广泛重构的分子基础 将进行含有Vimentin或Vimentin/desmin的实验。爆炸品 解聚，以早中期、早中期和快速的 在后期-末期发生的IF重组可以是 在很大程度上，可以通过IF的过度磷酸化来解释 结构蛋白。在BHK细胞中发现了两种有丝分裂酶 它们参与了磷酸化反应。我们正在进行 其中一种名为波形蛋白的蛋白的广泛分析 蛋白激酶(VPK)。VPK是由三种蛋白质组成的复合体：65kD组分， 110kD组分，成熟催化亚基p34cdc2 促进因子(MPF)，已知在触发 有丝分裂。我们计划进行实验，以确定 VPK和VPK磷酸化的特定位点(S)的功能意义 作用于使用多种细胞的HI型If蛋白的其他激酶 生理(例如，微量注射)、形态(免疫荧光、 共聚焦和电子显微镜)、生物化学和分子生物学 (利用细菌表达的蛋白和瞬时转基因技术 培养的哺乳动物细胞)技术。我们还将尝试研究 中频的相间动力学特性。对于这些研究，我们将利用 微量注射衍生的IF蛋白，如生物素 将波形蛋白和角蛋白导入活细胞，并在原位确定它们的命运 使用共聚焦显微镜和电子显微镜。类似的方法使用 X-罗丹明标记的蛋白质是否将被用于确定 间期细胞中存在稳态或动态平衡，通过 利用荧光漂白后恢复(FRAP)实验。 还描述了旨在确定生物化学的研究。 细胞表面相关桥粒与IF结合的基础 上皮细胞。这些研究涉及到分离和生化。 牛舌桥粒组分的特征及其与蛋白质的关系 角蛋白多肽如何与它们结合的测定。这些信息 从我们的调查中得出的结论应该会对这些财产有新的了解 以及IF在哺乳动物细胞中的功能。更好地理解IF 其结构和功能是了解其发病机制的基础。 发生IF变化的各种疾病，如阿尔茨海默氏症 疾病、帕金森氏症、各种癌症和酒精性肝硬变。