REGULATION OF MICROTUBULE DYNAMIC INSTABILITY

我们的长期目标是了解细胞内转运过程 例如：有丝分裂过程中染色体的运动和 受精时的雄性和雌性原核。 这些过程 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 流程不能正常运行，导致严重的健康问题 包括癌症和非整倍体。 本研究的重点是了解微管组装是如何在 微管相关蛋白（MAPs）是细胞的重要调节因子。 阵列 细胞中发现的微管由两个亚群组成： 亚群是动态的，与亚基迅速交换亚基 池，而另一个亚群则稳定得多。 组装 动态细胞微管和纯化的微管蛋白的特征在于， 一种叫做动态不稳定性的独特行为， 在两个阶段之一之间过渡：伸长和快速缩短。 的 这些相位之间的转换是突然的和随机的。 相比 纯微管蛋白，因子必须存在于细胞中：增加伸长 速度，增加过渡频率，阻止端部组装， 防止非成核组装。 此外， 过渡频率调节器可能在小区期间被调节 周期 虽然这些类型的地图必须存在，但很少有地图被发现。 在所需的单个微管水平上进行鉴定和分析 测量动态不稳定性参数（速率和转变 频率）。 此外，负责以下方面的职能和机制： 产生稳定的微管尚不清楚。 我们的目标是利用 具有可视化单个微管能力的功能测定 （1）描述先前确定的MAP对 微管动力学不稳定性;（2）分离和表征MAPs 调节海胆卵提取物中的动态不稳定性 （这种无细胞系统中的微管动态不稳定性类似于 （3）开发无细胞系统 来研究稳定微管的形成 对于目标（1），我们将使用视频增强差分干扰 用DIC来记录真实的时间， 在存在和不存在以下MAP的情况下纯化的微管蛋白：XMAP 从非洲爪蟾，核苷酸二磷酸激酶，和哺乳动物大脑 MAP、MAP 2和tau。 对于目标（2），我们将使用视频和 裂殖海胆卵免疫荧光显微分析 提取并分离出调节动态的因子， 不稳定 这些研究将集中在生化分馏 包括微管亲和层析和消减 实验中我们将检测活性的丧失。 我们还将使用 这些测定用于功能性筛选人单核细胞cDNA文库。 为 目标（3），我们将再次使用显微功能测定来检查 无细胞系统中稳定的微管。 我们首先要培育一个细胞 自由系统，其中稳定的微管在体外形成。 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 并分离出负责产生 稳定

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.