MOLECULAR ANALYSIS OF A NOVEL MAP/MTOC COMPONENT

这项提案的目的是研究一种新型蛋白质的功能 与微管和微管组织中心相互作用 （MTOC）。 微管参与重要的细胞过程，包括 细胞分裂和形态发生，并阐明其机制 微管的功能及其调节对于理解 生物的生长、分化和病理学。 关键 微管功能调节元件是MAP （微管相关蛋白）和MTOC，它们调节 微管和直接组织的更高层次的结构， 对微管功能的发挥至关重要。 这项建议 将重点关注一种最近被一个 单克隆抗磷蛋白抗体AX3，针对来自 细胞黏鼹鼠，盘基网骨脂鲤。 AX3抗原是一种 在真菌等多种生物体中与MTOC相关的新型MAP 变成人类 不同生物学中编码蛋白质的基因 系统（Dictyosteoblasts和HeLa细胞）将被克隆， 将比较核苷酸序列。 磷酸化性质 在细胞周期和细胞增殖过程中检测抗原， 分化，以及磷酸化对其关联的影响 将在蛋白质相互作用的能力中进行分析 将通过构建亲和力来检查与其他MTOC组件的关系 用重组DNA技术生产的融合蛋白。 在网骨藻中对应于AX3抗原的基因将是 使用基因靶向方法破坏，发现在这方面是成功的， 转化细胞的表型和细胞内的变化， 将分析微管组织和有丝分裂纺锤体的形成 通过显微镜。 该蛋白质在HeLa细胞中的作用将通过以下方法进行评估： 采用体外抑制微管组装到分离的 中心体，并通过显微注射抗体到活细胞中， 不同的细胞周期阶段。 蛋白质的细胞内浓度 将通过引入反义RNA来调节，或者 显微注射融合蛋白，或用DNA转染细胞 表达AX3抗原。 关于地图及其在 MTOC指导的微管组织调节使我们能够 分析微管依赖性细胞的分子基础 过程，这将是重要的了解机制， 不受控制的细胞分裂和分化。

The purpose of this proposal is to study the function of a novel protein interacting with microtubules and microtubule-organizing centers (MTOCs). Microtubules are involved in vital cell processes including cell division and morphogenesis, and elucidation of the mechanism of microtubule functions and its regulation is crucial to an understanding of biological growth, differentiation and its pathologies. The key elements for regulation of microtubule functions are MAPs (Microtubule-associated proteins) and MTOCs which modulate properties of microtubules and direct organization of higher ordered structures which are essential for performance of microtubule functions. This proposal will focus on a molecule that has recently been identified by a monoclonal anti-phosphoprotein antibody, AX3, raised against MTOCs from the cellular slime mole, Dictyostelium discoideum. The AX3 antigen is a novel MAP associated with MTOCs in a wide range of organisms from fungi to human. The genes coding for the protein in different biological systems (Dictyostelium and HeLa cells) will be cloned and their nucleotide sequences will be compared. Phosphorylation properties of the antigen will be examined during the process of cell cycle and cell differentiation, and the effect of phosphorylation on its association with microtubules will be analyzed in The proteins' capacity to interact with other MTOC components will be examined by constructing affinity columns with fusion proteins produced using recombinant DNA technology. The gene corresponding to the AX3 antigen in Dictyostelium will be disrupted using the gene targeting method found to be successful in this organism, and the phenotype of transformed cells and changes in the microtubule organization and mitotic spindle formation will be analyzed by microscopy. The protein's role in HeLa cells will be evaluated by employing in vitro inhibition of microtubule assembly onto isolated centrosomes, and by microinjection of antibodies into living cells at different cell cycle stages. Intracellular concentration of the protein will be modulated either by introduction of antisense RNA, or microinjection of fusion proteins, or transfection of cells with DNAs expressing the AX3 antigen. Such studies on MAPs and their role in MTOC-directed regulation of microtubule organization enable us to analyze the molecular basis of the microtubule-dependent cellular processes, which will be important for understanding the mechanism of uncontrolled cell division and differentiation.