REGULATION OF MICROTUBULE DYNAMIC INSTABILITY

我们的长期目标是了解细胞内的运输过程 如：有丝分裂过程中染色体的运动和染色体的连接 受精时的雄性和雌性原核。这些过程是 以微管为基础，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 流程运行不正常，导致严重的健康问题 包括癌症和非整倍体。 这项研究的重点是了解微管是如何组装在 细胞受微管相关蛋白(MAPs)的调节。该阵列 在细胞中发现的微管由两个亚群组成：一个 子群体是动态的，并与亚单位快速交换 而其他亚群的稳定性要高得多。装配的 动态细胞微管和纯化微管蛋白的特征 通过一种称为动态不稳定性的独特行为，微管 在两个阶段中的一个阶段之间过渡：伸长和快速缩短。这个 这些阶段之间的转变是突然的和随机的。与.相比 纯微管蛋白，细胞中必须存在的因子：增加延伸率 速度，增加过渡频率，块减去端部组件和 防止无核组装。此外，该组织的活动 转换频率调节器可能在单元期间被调节 周而复始。虽然这些类型的地图必须存在，但很少有地图 在所需单个微管的水平上进行识别和分析 测量动态不稳定性的参数(速率和转变 频率)。此外，负责的职能和机制 产生稳定的微管尚不清楚。我们的目标是利用 能够显示单个微管的功能分析 对：(1)描述先前确定的地图对 微管动态不稳定性；(2)MAP的分离和表征 调节海胆卵提取液中的动态不稳定性 (这个无细胞系统中的微管动态不稳定性类似于 在细胞中)和在人类单核细胞中；(3)发展无细胞系统 研究稳定的微管的形成。 对于目标(1)，我们将使用视频增强差分干扰 显微镜(DIC)实时记录动态不稳定性 在存在和不存在以下MAP的情况下纯化的微管蛋白：xMAP 来自非洲爪哇，核苷酸二磷酸激酶，和哺乳动物的大脑 地图、地图2和tau。对于目标(2)，我们将使用视频和 分级海胆卵的免疫荧光显微镜检测 提取并分离目前存在的调节动态的因素 不稳定。这些研究将集中在生化分级上。 包括微管亲和层析和底物分离 我们将分析活动丧失情况的实验。我们还将使用 本实验旨在从功能上筛选人单核细胞基因文库。为 目标(3)，我们将再次使用显微功能分析来检查 无细胞系统中稳定的微管。我们将首先开发一种细胞 在体外形成稳定微管的自由系统。接下来，我们将使用 这个系统来表征这些稳定的组装和拆卸 微管和分离负责生成的因素 稳定性。

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.