Regulating the Coordination of Microtubule Organization and Cell Cycle State

项目摘要 微管组织中心（MTOC）产生特定的微管排列， 对许多细胞功能至关重要，包括细胞分裂、极化和迁移。不同亚细胞 为了适应这些不同的过程，研究中心可以发挥MTOC的作用，在这里，我们寻求 了解在开发过程中如何建立不同的MTOC位置。为了分裂，动物细胞 中心体作为MTOC，定位特定的蛋白质以生长和锚微管，以建立 有丝分裂纺锤体;微管然后促进两个子细胞的正确形成。在鉴别 细胞，像一些上皮细胞一样，MTOC的位置发生了变化：微管组织蛋白离开了细胞。 中心体并移动到膜表面，一个“MTOC开关”将膜建立为MTOC。 上皮细胞具有独特的形状，并紧密地粘在一起形成细胞片， 由膜MTOC产生的微管构型对于细胞的结构和功能是重要的。 这些细胞。然而，微管的这种排列不允许分裂，这就提出了一个重要的问题： 当一个上皮细胞分裂时，它是如何改变MTOC的位置来重组微管， 有丝分裂纺锤体微管重组对于调节正常细胞的分裂和形状都是至关重要的。 发展和癌症。中心体缺陷是许多癌症的典型标志[1]，最近的研究 已经表明，增加中心体的微管组织活性会导致上皮细胞 更容易分裂和显示入侵行为[11，12]。尽管它很重要，但人们对它是如何运作的知之甚少。 细胞控制它们的微管组织以及对细胞分裂和形状的影响。 本提案的目标是了解上皮细胞如何建立特定的MTOC位置， 不同的功能以及MTOC位置的变化如何与细胞分裂相协调。细胞周期是一个 严格控制的过程，使用一系列检查点来确保细胞为分裂做好准备。这里 我们提出了一系列的遗传和生物化学实验在肠道上皮细胞的模式生物 C. elegans来确定细胞周期和MTOC位置是如何协调的。我们的实验室最近优化了 一种靶向蛋白质降解的新策略。这种方法，加上最近的基因组编辑方法， 将使我们能够揭示细胞周期调节因子和微管组织之间的分子联系。 Aim 1将使用新的和经典的遗传学方法来确定哪些细胞周期基因调节细胞周期。 MTOC位置。目标2将使用互补的遗传学和生物化学方法来确定这些 蛋白质将微管束缚在特定的位置。在Aim 3中，基因筛选将发现新的调节因子， MTOC定位和细胞周期的协调。由于大多数癌症起源于上皮，我们预计， 了解上皮细胞如何协调其MTOC与细胞分裂将与癌症生物学相关 并可能导致发现有用的癌症生物标志物或治疗靶点。

Project Summary Microtubule organizing centers (MTOCs) generate specific arrangements of microtubules that are essential for many cellular functions, including cell division, polarization, and migration. Different subcellular sites can function as the MTOC in order to accommodate these different processes, and here we seek to understand how different MTOC locations are established during development. To divide, animal cells use centrosomes as MTOCs, localizing specific proteins to grow and anchor microtubules in order to build the mitotic spindle; the microtubules then promote the correct formation of two daughter cells. In differentiating cells, like some epithelial cells, the location of the MTOC changes: microtubule-organizing proteins leave the centrosome and move to a membrane surface, an “MTOC switch” establishing the membrane as the MTOC. Epithelial cells have distinctive shapes and stick together tightly to form cellular sheets, and the microtubule configuration generated by the membrane MTOC is important for the structure and function of these cells. However, this arrangement of microtubules does not permit division, raising an important question: when an epithelial cell divides, how does it change the location of its MTOC to reorganize its microtubules into the mitotic spindle? Microtubule reorganization is critical for regulating cell division and shape both in normal development and in cancer. Centrosomal defects are classic hallmarks of many cancers [1], and recent studies have shown that increasing the microtubule organizing activity of the centrosome causes epithelial cells to divide more readily and display invasive behavior [11,12]. Despite its importance, little is known about how cells control their microtubule organization and the consequent effect on cell division and shape. The goal of this proposal is to understand how epithelial cells establish specific MTOC locations for different functions and how the change in MTOC location is coordinated with cell division. The cell cycle is a tightly controlled process that uses a series of checkpoints to ensure that a cell is prepared for division. Here we propose a series of genetic and biochemical experiments in intestinal epithelial cells in the model organism C. elegans to determine how the cell cycle and MTOC location are coordinated. Our lab has recently optimized a new strategy for targeted protein degradation. This method, coupled with recent methods for genome editing, will allow us to uncover the molecular connection between cell cycle regulators and microtubule organization. Aim 1 will use both new and classic genetic approaches to determine which cell cycle genes regulate the MTOC location. Aim 2 will use complementary genetic and biochemical methods to identify which of these proteins tether microtubules to specific locations. In Aim 3, a genetic screen will uncover new regulators of the coordination of MTOC location and cell cycle. As the majority of cancers are epithelial in origin, we expect that understanding how epithelial cells coordinate their MTOC with cell division will be relevant to cancer biology and may lead to the discovery of useful cancer biomarkers or therapeutic targets.