Studing the Role of Ran in Mitosis

描述（由申请人提供）：对细胞分裂过程至关重要的是将一套完整的染色体准确地传递给每个子细胞，这在真核生物中是通过有丝分裂纺锤体的功能实现的。本研究计划的目的是阐明高度保守的GTPase Ran在细胞分裂中的作用。目前的模型表明，RanGTP在真核细胞中起着空间标记的作用，表明了基因组的位置。在有丝分裂过程中，RanGTP被认为通过刺激染色体附近的微管聚合和组织来促进纺锤体组装。这种“局部效应”源于RCC1的染色体定位，RCC1是产生RanGTP的鸟嘌呤核苷酸交换因子，它与运输因子结合，导致它们释放纺锤体组装所需的货物。然而，这些转运蛋白的性质、它们的有丝分裂功能以及rcc1 - ran -微管信号级联的分子机制仍然知之甚少。本提案中描述的一种主要实验方法利用了从非洲爪蟾（Xenopus/aevis）卵中制备的复杂细胞提取物，可以使用生化和功能分析进行研究。这为分馏和重构实验识别和表征下游因素提供了一个很好的模型系统。此外，我们建议使用体细胞来检查有丝分裂Ran途径在体内的保护。我们的目标是：(1)鉴定和表征rangtp -微管级联中的中间体。(2)利用Ran核苷酸状态的荧光传感器可视化和表征萃取物和分子定义反应中的RanGTP梯度。(3)利用培养的哺乳动物细胞系，结合显微注射和RNA干扰技术，确定RanGTP/cargo梯度在体内的存在及其作用。正常的纺锤体组装和功能是维持基因组稳定性所必需的，而这一过程中的缺陷与出生缺陷和癌症有关。有丝分裂Ran途径中因子的鉴定和表征是我们理解真核细胞中有丝分裂和减数分裂纺锤体组装的基础，并支持我们使用纯化组分重建细胞分裂的复杂形态发生事件的长期目标。

DESCRIPTION (provided by applicant): Of central importance to the process of cell division is the accurate transmission of a complete set of chromosomes to each daughter cell, which in eukaryotes is achieved through the function of the mitotic spindle. The goal of this research proposal is to elucidate the role of the highly conserved GTPase Ran in cell division. The current model suggests that RanGTP functions as a spatial marker that signals the position of the genome in eukaryotic cells. During mitosis, RanGTP is thought to promote spindle assembly by stimulating microtubule polymerization and organization in the vicinity of chromosomes. This "local effect" results from the chromosomal localization of RCC1, the guanine nucleotide exchange factor that generates RanGTP, which binds to transport factors causing them to release cargoes required for spindle assembly. However, the nature of the cargoes, their mitotic function and the molecular mechanisms underlying the RCC1-Ran-microtubule signaling cascade are still poorly understood. A major experimental approach described in this proposal takes advantage of complex cellular extracts prepared from eggs of the African frog Xenopus/aevis that can be studied using biochemical and functional assays. This provides an excellent model system for fractionation and reconstitution experiments to identify and characterize downstream factors. In addition, we propose to examine conservation of the mitotic Ran pathway in vivo using somatic cells. Our aims are: (1) To identify and characterize intermediates in the RanGTP-microtubule cascade. (2) To use fluorescent sensors of the Ran nucleotide state to visualize and characterize the RanGTP gradient in extracts and in molecularly-defined reactions. (3) To determine the existence and role of RanGTP/cargo gradients in vivo using cultured mammalian cell lines combined with microinjection and RNA interference techniques. Proper spindle assembly and function is required to maintain genome stability, and defects in this process are associated with birth defects and cancer. The identification and characterization of factors in the mitotic Ran pathway is fundamental to our understanding of mitotic and meiotic spindle assembly in eukaryotic cells, and supports our long term goal of reconstituting the complex morphogenetic events of cell division using purified components.