STUDIES OF CYTOPLASMIC MICROTUBULE HETEROGENEITY

细胞质微管异质性研究

• 批准号: 3283303
• 负责人: WILLIAM C THOMPSON
• 金额: $ 9.94万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1987-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283303
• 关键词: affinity chromatography; aminoacid analyzer; antiantibody; antibody formation; antibody specificity; biological polymorphism; cell growth regulation; cell migration; chemotaxis; drug adverse effect; fluorescence microscopy; gel electrophoresis; hybridomas; immunofluorescence technique; immunoprecipitation; messenger RNA; microinjections; neoplastic transformation; reagent /indicator; structural genes; tissue /cell culture

The overall goal of this research is to determine how cytoplasmic microtubules are regulated and how they in turn participate in regulation of cell proliferation and other cell functions. We have rencently shown that a unique monoclonal anti-tubulin antibody (Ab 1-6.1) binds to only a subset of microtubules within single fibroblastic cells. This discovery indicates that certain microtubule subsets are configured to differentially express certain antigenic sites or that isotubulin molecules are segregated into these microtubule subsets. The proposed studies will determine the physical and biochemical differences between antigenically dissimilar cytoplasmic microtubules. We will generate a "library" of monoclonal anti-tubulin antibodies which we will use to immunologically dissect the cytoplasmic microtubule complex into antigenic subsets and then determine the nature of the antigenic differences and any role of these subsets in controlling various physiological states of the cells. Specific antigenic determinants on tubulin molecules will be characterized by immunohistochemical modification of antigenic sites, Western blot antibody binding to electrophoresed isotubulins, immunoprecipitation or affinity chromatography of isotubulins and tubulin fragments and peptide mapping and amino acid analysis of unique peptides. To determine the role of microtubule subsets we will microinject monoclonal antibodies into individual living cells and observe the effects of these antibodies on physiological properties including mitogenesis, induced cell migration and maintenance of cell morphology. We will also microinject isolated tubulin mRNA into cells which do not express these subsets to examine the processing and effects of subset tubulin. The effects of micro-injected antibodies or the appearance of new microtubule subsets will be examined by immunofluorescent staining of fixed cells or by directly observing the fate of fluorescently labeled antibodies in living cells. To establish any correlations between altered microtubule subsets and various cell functions, the immunofluorescent patterns of various microtubule subsets will be examined in fixed cells which have been treated with a variety of agents to induce chemotaxis, mitogenesis or transformation. Because microtubules appear to be involved in initiation of normal cell proliferation and many transformed cells have been reported to have altered microtubules, the proposed investigation of microtubule subsets may provide a key to understanding the regulation of normal cell function and the molecular lesions which lead to neoplasia.

这项研究的总体目标是确定细胞质如何 微管是受调节的，以及它们如何反过来参与调节 细胞增殖和其他细胞功能。 我们最近发现 独特的单克隆抗微管蛋白抗体（Ab 1-6.1）仅结合 单个成纤维细胞内的微管亚群。 这一发现 表明某些微管亚群被配置为 表达某些抗原位点或异微管蛋白分子被分离 这些微管亚群。 拟议的研究将确定 不同抗原之间的物理和生化差异 细胞质微管 我们将产生一个单克隆抗体的“文库”， 抗微管蛋白抗体，我们将用它来免疫解剖 细胞质微管复合物转化为抗原亚群，然后确定 抗原差异的性质以及这些亚群在 控制细胞的各种生理状态。 特定抗原 微管蛋白分子上的决定簇的特征在于： 抗原位点免疫组织化学修饰，Western印迹抗体 结合到经标记的异微管蛋白，免疫沉淀或亲和 异微管蛋白和微管蛋白片段的层析和肽图谱， 独特肽的氨基酸分析。 确定的作用 我们将显微注射单克隆抗体到微管亚群中 单个活细胞，并观察这些抗体对 生理特性，包括有丝分裂、诱导细胞迁移和 维持细胞形态。 我们还将微量注射分离的微管蛋白 mRNA进入不表达这些亚群的细胞中，以检查细胞中的细胞分化。 亚类微管蛋白的加工和作用。 微量注射的效果 抗体或新的微管亚群的出现将通过 固定细胞的免疫荧光染色或通过直接观察命运 荧光标记的抗体在活细胞中。 建立任何 改变的微管亚群与各种细胞之间的相关性 功能，各种微管亚群的免疫荧光模式 将在固定的细胞中进行检查，这些细胞已经用各种 诱导趋化性、有丝分裂或转化的试剂。 因为 微管似乎参与了正常细胞的启动 增殖和许多转化细胞已被报道改变 微管，微管亚群的拟议调查可能提供 这是理解正常细胞功能调节的关键， 导致肿瘤形成的分子损伤。