Studing the Role of Ran in Mitosis

研究 Ran 在有丝分裂中的作用

• 批准号: 6769416
• 负责人: Rebecca W Heald
• 金额: $ 29.96万
• 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/6769416
• 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(申请人提供)：在细胞分裂过程中最重要的是将一套完整的染色体准确地传递到每个子细胞，这在真核生物中是通过有丝分裂纺锤体的功能实现的。本研究的目的是阐明高度保守的GTP酶RAN在细胞分裂中的作用。目前的模型表明，RanGTP在真核细胞中起到了空间标记的作用，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的组装。这种“局部效应”源于RCC1的染色体定位，RCC1是一种产生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，货物的性质、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。这项建议中描述的一种主要实验方法利用了从非洲爪蛙/埃及斑蛙卵中提取的复杂细胞提取物，可以用生化和功能分析进行研究。这为识别和表征下游因素的分馏和重组实验提供了一个很好的模型系统。此外，我们建议使用体细胞在体内检查有丝分裂RAN途径的保守性。我们的目标是：(1)识别和表征RanGTP-微管级联中的中间体。(2)使用RAN核苷酸状态的荧光传感器来可视化和表征提取物和分子定义的反应中的RanGTP梯度。(3)利用体外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梯度的存在及其在体内的作用。正确的纺锤体组装和功能是维持基因组稳定所必需的，而这一过程中的缺陷与出生缺陷和癌症有关。鉴定和鉴定有丝分裂RAN途径中的因子是我们理解真核细胞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的成分重建细胞分裂的复杂形态发生事件的长期目标。