Mitotic Roles Of Ran GTPase

Ran GTPase 的有丝分裂作用

• 批准号: 6993681
• 负责人: MARY C. DASSO
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6993681
• 关键词: Xenopus oocyte; cell cycle; cell growth regulation; centromere; cyclins; cytogenetics; guanine nucleotide exchange factors; guanosinetriphosphatase activating protein; guanosinetriphosphatases; mitotic spindle apparatus; protein localization; protein structure function

Ran is a small GTPase required for nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. The nucleotide exchange factor for Ran, RCC1, is a chromatin-associated protein. The GTPase activating protein for Ran, RanGAP1, is cytoplasmic during interphase. During mitosis, the bulk of RanGAP1 is broadly distributed, although a significant fraction of RanGAP1 becomes associated with kinetochores in a SUMO-1 dependent fashion (see Z01 HD001902-10). Ran-GTP nucleotide hydrolysis also requires a family of Ran-GTP binding proteins, which act as RanGAP1 accessory factors. This family includes RanBP1 and RanBP2. The distribution of Ran?s regulators has been widely hypothesized to modulate local concentrations of Ran-GTP within cells, spatially directing processes in which Ran has been implicated. We have been examining the mechanisms through which key Ran regulators are localized within mitotic metazoan cells and the functional consequences to cells when such distribution patterns are disrupted. To look at the mitotic fate of RCC1, we examined its chromosomal association in cycling Xenopus egg extracts. Remarkably, the amount of chromatin-associated RCC1 increased drastically at anaphase onset. In order to determine the significance of this finding, we assayed whether the Ran pathway has a role in mitotic progression or checkpoint control in Xenopus egg extracts. Prior to each anaphase, chromosomes are aligned onto the metaphase through attachment between spindle microtubules and kinetochores, proteinaceous structures that assemble over the centromere of each chromosome. The spindle assembly checkpoint is a cell cycle regulatory pathway that monitors spindle assembly in all eukaryotic cells and prevents the onset of anaphase and the dissolution of sister chromatin cohesion in the presence of unattached on inappropriately attached kinetochores. Remarkably, a five- to seven-fold elevation of RCC1 concentration was sufficient to abrogate spindle checkpoint arrest in extracts containing nuclei plus the microtubule depolymerizing agent nocodazole. While assembly of centromeric structures occurred normally under these circumstances, we found that many checkpoint components were mis-localized away from kinetochores after RCC1 addition, indicating that increased RCC1 levels abolish checkpoint arrest by altering interactions between kinetochores and checkpoint regulators. Together with additional data, these observations suggest that the spindle checkpoint is directly responsive to Ran-GTP levels. Notably, the capacity of RCC1 to reverse spindle checkpoint arrest is specific, since increased RCC1 does not compromise other modes of M phase arrest (e.g. CSF arrest). Our results suggest a model wherein complete chromosome alignment on the metaphase plate triggers the increased binding of RCC1 to chromosomes, resulting in the local elevation of Ran-GTP levels and the ejection of the final population of kinetochore-associated checkpoint components. In parallel to our studies on RCC1, we have investigated the mitotic behavior and function of RanGAP1. In metazoans, RanGAP1 is conjugated with SUMO-1. Studies by this group and others have shown that SUMO-1 modification causes RanGAP1 to associate during interphase with Ubc9 and RanBP2, a large nuclear pore protein with multiple Ran-GTP binding domains and a SUMO E3 ligase domain. Through further investigation of the mitotic behavior and interactions of RanGAP1, we have found that RanGAP1 associates with kinetochores in a SUMO-1 dependent manner. Notably, RanBP2 co-localized with RanGAP1 on spindles and kinetochores. Recently, we have examined the structural requirements for targeting RanGAP1 and RanBP2, as well as their function in mitosis. We found that elimination of RanBP2 expression through RNA interference (RNAi) displaced RanGAP1 from kinetochores, supporting the notion that these proteins target to kinetochores as part of a single complex. Both proteins were displaced after RNAi of integral kinetochore components, suggesting that they require intact kinetochore structures to localize appropriately. By contrast, peripheral kinetochore proteins were not essential for correct targeting of either protein. Cells depleted of RanBP2 show abnormalities in both spindle formation and mitotic progression, substantiating the importance of correct targeting of the RanGAP1/RanBP2 complex during mitosis.

Ran 是核细胞质运输、纺锤体组装、核组装和细胞周期控制所需的小型 GTP 酶。 Ran 的核苷酸交换因子 RCC1 是一种染色质相关蛋白。 Ran 的 GTP 酶激活蛋白 RanGAP1 在分裂间期存在于细胞质中。在有丝分裂期间，尽管 RanGAP1 的很大一部分以 SUMO-1 依赖性方式与动粒相关，但大部分 RanGAP1 分布广泛（参见 Z01 HD001902-10）。 Ran-GTP 核苷酸水解还需要 Ran-GTP 结合蛋白家族，它们充当 RanGAP1 辅助因子。该家族包括 RanBP1 和 RanBP2。 Ran 调节因子的分布已被广泛假设为调节细胞内 Ran-GTP 的局部浓度，从而在空间上引导 Ran 所涉及的过程。我们一直在研究关键 Ran 调节因子在有丝分裂后生动物细胞内定位的机制，以及当这种分布模式被破坏时对细胞的功能影响。 为了研究 RCC1 的有丝分裂命运，我们检查了其在循环爪蟾卵提取物中的染色体关联。值得注意的是，染色质相关 RCC1 的数量在后期开始时急剧增加。为了确定这一发现的意义，我们检测了 Ran 途径是否在爪蟾卵提取物的有丝分裂进展或检查点控制中发挥作用。在每个后期之前，染色体通过纺锤体微管和动粒之间的附着排列到中期，动粒是在每条染色体的着丝粒上组装的蛋白质结构。纺锤体组装检查点是一种细胞周期调节途径，可监测所有真核细胞中的纺锤体组装，并防止后期的发生以及在未附着在不适当附着的着丝粒上的情况下姐妹染色质内聚力的溶解。值得注意的是，在含有细胞核和微管解聚剂诺考达唑的提取物中，RCC1 浓度升高 5 至 7 倍足以消除纺锤体检查点停滞。虽然在这些情况下着丝粒结构的组装正常发生，但我们发现在添加 RCC1 后，许多检查点组件错误地定位在远离着丝粒的位置，这表明 RCC1 水平的增加通过改变着丝粒和检查点调节因子之间的相互作用来消除检查点停滞。结合其他数据，这些观察结果表明纺锤体检查点直接响应 Ran-GTP 水平。值得注意的是，RCC1 逆转纺锤体检查点停滞的能力是特定的，因为 RCC1 的增加不会损害其他 M 期停滞模式（例如 CSF 停滞）。我们的结果提出了一个模型，其中中期板上的完整染色体排列会触发 RCC1 与染色体的结合增加，导致 Ran-GTP 水平局部升高，并排出动粒相关检查点成分的最终群体。 在研究 RCC1 的同时，我们还研究了 RanGAP1 的有丝分裂行为和功能。在后生动物中，RanGAP1 与 SUMO-1 结合。该小组和其他人的研究表明，SUMO-1 修饰导致 RanGAP1 在间期与 Ubc9 和 RanBP2 结合，RanBP2 是一种大核孔蛋白，具有多个 Ran-GTP 结合域和 SUMO E3 连接酶域。通过进一步研究 RanGAP1 的有丝分裂行为和相互作用，我们发现 RanGAP1 以 SUMO-1 依赖性方式与动粒相关。值得注意的是，RanBP2 与 RanGAP1 在纺锤体和动粒上共定位。最近，我们研究了靶向 RanGAP1 和 RanBP2 的结构要求，以及它们在有丝分裂中的功能。我们发现，通过 RNA 干扰 (RNAi) 消除 RanBP2 的表达，可以将 RanGAP1 从着丝粒中取代，这支持了这些蛋白质作为单一复合物的一部分以着丝粒为目标的观点。两种蛋白质在对完整着丝粒成分进行 RNAi 后均被取代，表明它们需要完整的着丝粒结构才能适当定位。相比之下，外周动粒蛋白对于正确定位任一蛋白都不是必需的。 RanBP2耗尽的细胞显示纺锤体形成和有丝分裂进展异常，证实了有丝分裂期间正确靶向RanGAP1/RanBP2复合物的重要性。