REGULATION OF MICROTUBULE DYNAMIC INSTABILITY

微管动态不稳定性的调节

• 批准号: 2187298
• 负责人: LYNNE CASSIMERIS
• 金额: $ 10.51万
• 依托单位: LEHIGH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187298
• 关键词: Xenopus; acidity /alkalinity; alternatives to animals in research; cell free system; cellular polarity; colchicine; egg /ovum; enzyme mechanism; genetic library; guanosine triphosphate; human genetic material tag; interference microscopy; intermolecular interaction; intracellular transport; microtubules; monocyte; nucleoside diphosphate kinase; paclitaxel; phosphorylation; protein biosynthesis; sea urchins; tau proteins; tissue /cell culture; tubulin

Our long term goal is to understand intracellular transport processes such as: the movement of chromosomes during mitosis and the joining of male and female pronuclei at fertilization. These processes are microtubule-based and require proper assembly and reorganization of the microtubule cytoskeleton. When these basic intracellular transport processes do not function properly, serious health problems result including cancer and aneuploidy. The focus of this study is to understand how microtubule assembly in cells is regulated by microtubule associated proteins (MAPs). The array of microtubules found in cells consists of two subpopulations: one subpopulation is dynamic and exchanges subunits rapidly with the subunit pool, while the other subpopulation is much more stable. Assembly of the dynamic cellular microtubules and of purified tubulin is characterized by a unique behavior termed dynamic instability where microtubules transit between one of two phases: elongation and rapid shortening. The transitions between these phases are abrupt and stochastic. Compared to pure tubulin, factors must exist in the cell that: increase elongation velocity, increase transition frequencies, block minus end assembly and prevent non-nucleated assembly. In addition, the activities of transition frequency regulators are likely regulated during the cell cycle. Although these types of MAPs must be present, few MAPs have been identified and analyzed at the level of individual microtubules required to measure the parameters of dynamic instability (rates and transition frequencies). In addition, the functions and mechanisms responsible for generating stable microtubules are not known. Our goals are to use functional assays with the ability to visualize individual microtubules to: (1) characterize the effects of previously identified MAPs on microtubule dynamic instability; (2) isolate and characterize MAPs regulating dynamic instability both in sea urchin egg extracts (microtubule dynamic instability in this cell free system is similar to that in the cell) and in human monocytes; (3) develop a cell free system to study the formation of stable microtubules. For goal (1) we will use video enhanced differential interference microscopy (DIC) to record, in real time, the dynamic instability of purified tubulin in the presence and absence of the following MAPs: XMAP from Xenopus, nucleotide diphosphate kinase, and the mammalian brain MAPs, MAP2 and tau. For goal (2), we will use video and immunofluorescent microscopic assays to fractionate sea urchin egg extracts and isolate the factors present that regulate dynamic instability. These studies will focus on biochemical fractionations including microtubule affinity chromatography and substraction experiments where we will assay for loss of activity. We will also use these assays to functionally screen a human monocyte cDNA library. For goal (3), we will again use microscopic functional assays to examine stable microtubules in a cell free system. We will first develop a cell free system where stable microtubules form in vitro. Next we will use this system to characterize the assembly and disassembly of these stable microtubules and to isolate the factors responsible for generating stability.

我们的长期目标是了解细胞内运输过程 例如：有丝分裂过程中染色体的运动和连接 施肥时男性和女性。 这些过程是 基于微管，需要适当的组装和重组 微管细胞骨架。 当这些基本的细胞内运输 过程无法正常运行，严重的健康问题结果 包括癌症和非整倍性。 这项研究的重点是了解微管如何在 细胞由微管相关蛋白（地图）调节。 数组 细胞中发现的微管由两个亚群组成：一个 亚群是动态的，并与亚基迅速交换亚基 池，而另一个亚群更加稳定。 组装 表征动态细胞微管和纯化的小管蛋白 通过微管的独特行为称为动态不稳定性 两个阶段之一之间的过境：伸长和快速缩短。 这 这些阶段之间的过渡是突然的和随机的。 相比 纯微管蛋白，细胞中必须存在因素：增加伸长率 速度，增加过渡频率，块减去末端组件和 防止非无核组件。 此外， 过渡频率调节器可能在细胞期间受到调节 循环。 尽管必须存在这些类型的地图，但几乎没有地图 在所需的单个微管的水平上识别和分析 测量动态不稳定性的参数（速率和过渡 频率）。 另外，功能和机制负责 生成稳定的微管尚不清楚。 我们的目标是使用 具有可视化单个微管的能力的功能测定 至：（1）表征先前识别的地图对 微管动态不稳定性； （2）孤立和表征地图 调节动态不稳定性在海胆卵提取物中 （此无单元系统中的微管动态不稳定性类似于 在细胞中）和人类单核细胞中； （3）开发一个无细胞系统 研究稳定微管的形成。 对于目标（1），我们将使用视频增强差异干扰 显微镜（DIC），以实时记录动态不稳定性 在存在和不存在以下地图的情况下纯化的小管蛋白：xmap 来自爪蟾，核苷酸二磷酸激酶和哺乳动物脑 地图，map2和tau。 对于目标（2），我们将使用视频和 免疫荧光显微镜测定分级海胆卵 提取物并隔离了调节动态的因素 不稳定。 这些研究将集中于生化分离 包括微管亲和色谱和缩写 我们将在其中测定损失活动的实验。 我们还将使用 这些测定在功能上筛选人类单核细胞cDNA库。 为了 目标（3），我们将再次使用显微镜功能测定 无细胞系统中的稳定微管。 我们将首先开发一个单元格 自由系统在体外形成稳定的微管。 接下来我们将使用 该系统来表征这些稳定的组装和拆卸 微管并隔离负责产生的因素 稳定。