Studing the Role of Ran in Mitosis

研究 Ran 在有丝分裂中的作用

• 批准号: 6614139
• 负责人: Rebecca W Heald
• 金额: $ 24.75万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6614139
• 关键词: SDS polyacrylamide gel electrophoresis; Xenopus oocyte; affinity chromatography; biological signal transduction; cell cycle; fluorescence microscopy; fluorescence resonance energy transfer; guanine nucleotide binding protein; guanine nucleotide exchange factors; guanosinetriphosphatases; matrix assisted laser desorption ionization; microinjections; microtubule associated protein; microtubules; mitotic spindle apparatus; nucleotides; protein structure function; recombinant proteins; tissue /cell culture

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.

描述（由申请人提供）：对细胞分裂过程至关重要的是将一组完整的染色体的准确传输到每个子细胞中，在真核生物中，通过有丝分裂纺锤体的功能实现了这一目标。该研究建议的目的是阐明高度保守的GTPase在细胞分裂中的作用。当前的模型表明，RangTP充当一个空间标记，它标志着基因组在真核细胞中的位置。在有丝分裂期间，人们认为RANGTP通过刺激染色体附近的微管聚合和组织来促进纺锤体组件。这种“局部效应”是由RCC1的染色体定位引起的，RCC1是产生RANGTP的鸟嘌呤核苷酸交换因子，该核苷酸交换因子与运输因子结合，导致它们释放纺锤体组装所需的货物。然而，货物的性质，它们的有丝分裂功能和RCC1-RAN微键传导级联的基础的分子机制仍然很少了解。该提案中描述的一种主要的实验方法利用了从非洲青蛙武士/AEVI的卵中制备的复杂细胞提取物，可以使用生化和功能分析研究。这为分馏和重构实验提供了出色的模型系统，以识别和表征下游因素。此外，我们建议使用体细胞在体内检查有丝分裂RAN途径的保护。我们的目标是：（1）识别和表征Rangtp-Microutule级联的中间体。 （2）使用RAN核苷酸态的荧光传感器可视化和表征提取物和分子定义的反应中的RANGTP梯度。 （3）使用培养的哺乳动物细胞系与微注射和RNA干扰技术相结合，确定RangTP/货物梯度在体内的存在和作用。需要适当的主轴组件和功能来维持基因组稳定性，并且此过程中的缺陷与先天缺陷和癌症有关。有丝分裂RAN途径中因子的鉴定和表征是我们对真核细胞中有丝分裂和减数分裂纺锤体组装的理解至关重要的，并支持了我们使用纯化的成分重新建立细胞分裂的复杂形态发生事件的长期目标。