Studying 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。本提案中描述的主要实验方法利用了从非洲爪蟾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。这提供了一个很好的模型系统来表征有丝分裂产物的成分和功能，表征有丝分裂梯度，并开发新的重建分析。我们的目标是：(1)阐明存在于需要RNA才能发挥其活性的复合体中的ran调控货物Rae1的功能，并研究纺锤体相关RNA。(2)利用荧光传感器可视化和表征rcc1在提取物和各种细胞类型的整个细胞周期中诱导的梯度。(3)用rcc1涂覆的珠子重建RanGTP梯度和主轴组件。(4)利用新开发的Ran通路小分子抑制剂，进一步剖析RanGTP在细胞分裂过程中的功能。在所有真核生物中，纺锤体的组装和功能对于细胞分裂过程中遗传信息的准确分布至关重要，而这一过程中的错误与出生缺陷和癌症有关。有丝分裂Ran通路中因子的鉴定和表征不仅是我们理解有丝分裂和减数分裂纺锤体组装的基础，而且可能为我们理解人类疾病提供重要的新见解。

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 laevis that can be studied using biochemical and functional assays. This provides an excellent model system to characterize the components and function of mitotic cargoes, to characterize mitotic gradients, and to develop novel reconstitution assays. Our aims are: (1) To elucidate the function of a Ran-regulated cargo, Rae1 that exists in a complex requiring RNA for its activity, and investigate spindle-associated RNAs. (2) To use fluorescent sensors to visualize and characterize RCC1-induced gradients throughout the cell cycle in extracts and in a variety of cell types. (3) To reconstitute RanGTP gradients and spindle assembly using RCC1-coated beads. (4) To use a newly developed small molecule inhibitor of the Ran pathway to further dissect the functions of RanGTP during cell division. In all eukaryotes, assembly and function of the spindle apparatus is essential to accurately distribute the genetic information during cell division, and errors in this process are associated with birth defects and cancer. The identification and characterization of factors in the mitotic Ran pathway is not only fundamental to our understanding of mitotic and meiotic spindle assembly, but may also provide important new insight into our understanding of human diseases.