1 Z01 HD008740-05 LGRD

1 Z01 HD008740-05 LGRD

• 批准号: 7334118
• 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/7334118
• 关键词: Z01; HD008740; 05; LGRD

The Ran GTPase is required for many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. Ran?s nucleotide exchange factor is called RCC1, and it remains chromatin associated throughout the cell cycle. Ran?s GTPase activating protein is called RanGAP1. Ran-GTP nucleotide hydrolysis also requires a family of Ran-GTP binding proteins, which act as RanGAP1 accessory factors. This family includes the RanBP1 protein and the nucleoporin RanBP2. SUMO-1 conjugation of RanGAP1 promotes its association with the interphase nuclear pore complex (NPC) through binding to RanBP2. RanBP2 is found on kinetochores in mitosis, and RanGAP1 also concentrates at kinetochores in a microtubule and SUMO-1-dependent fashion. Our work suggests that Ran has two important roles at mitotic kinetochores. Namely, it is essential for regulation of the spindle assembly checkpoint and for assembly of microtubule fibers that attach kinetochores to spindle poles. We have shown that RanBP2 and RanGAP1 are targeted during mitosis as a single complex that is both regulated by and important for stable kinetochore-MT association in mitotic spindles. We have found that Crm1, a Ran-GTP-binding nuclear export receptor, localizes to kinetochores in mammalian cells. Inhibition of Crm1 using the drug Leptomycin B (LMB) causes release of RanGAP1/RanBP2 from kinetochores and the formation of spindles in which continuous MT bundles span the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibers. These findings demonstrate that proper localization of RanGAP1/RanBP2 is critical for definition of kinetochore fibers and for chromosome segregation at anaphase. Additional data supporting a role of the RanGAP1/RanBP2 complex at kinetochores have also been obtained by our collaborator (Roberge). In particular, they have identified a small molecular inhibitor, ent-15-oxokaurenoic acid (EKA), which binds to RanBP2. EKA blocks association of the mitotic motor protein CENP-E with kinetochores and inhibits chromosome movement, consistent with these a role of RanBP2 in these processes. Notably, the targeting of RanGAP1 to spindles is conserved between animals and plants, although the molecular mechanims that are utilized are completely distinct (Meier). We are currently attempting to identify kinetochore components required for the Crm1-mediated recruitment of the RanGAP1/RanBP2 complex, and to understand this mechanism at the molecular level. The spindle assembly checkpoint monitors spindle formation and prevents the onset of the metaphase-anaphase transition until chromosomes are correctly attached and aligned on the metaphase plate. In previous experiments, we documented that the spindle assembly checkpoint can be regulated through Ran-GTP in Xenopus egg extracts. In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the NPCs during interphase, and we have examined the relationship of these proteins to the Ran pathway in budding yeast. We found that deletion of yeast MAD1 or MAD2 did not grossly affect steady-state nucleocytoplasmic trafficking or Ran localization. However, yeast with conditional mutations in the yeast Ran GTPase pathway that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. These observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC. Both the targeting of the RanGAP1/RanBP2 complex to mitotic kinetochores and the association of Mad1 and Mad2 to the interphase NPC suggests an intimate relationship between kinetochores and nuclear pores. Work of our collaborators (D. Forbes, B. Fontoura) has shown that the Nup107-160 complex, which includes nine nucleoporins, localizes at mitotic spindles and kinetochores in mammalian cells. In collaborative studies, they have further shown that the Nup107-160 complex is required for spindle assembly in Xenopus egg extracts. On the other hand, the capacity of egg extracts to polymerize microtubules in response to elevated Ran-GTP levels appears to be intact in the absence of the Nup107-160 complex. These findings do not exclude the possibility that the Nup107-160 complex may be an upstream regulator of Ran-GTP, but argue against the notion that it works as a Ran effector in spindle formation. Notably, the Nup107-160 complex is not required for activation of the spindle checkpoint in egg extracts, nor for checkpoint silencing by increased levels of Ran-GTP. We are extending these studies through the examination of the mitotic roles of other nucleoporin complexes, and investigation of their possible function as mitotic Ran effectors.

Ran GTPase 是许多细胞功能所必需的，包括核细胞质运输、纺锤体组装、核组装和细胞周期控制。 Ran 的核苷酸交换因子称为 RCC1，它在整个细胞周期中始终与染色质相关。 Ran 的 GTP 酶激活蛋白称为 RanGAP1。 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 蛋白和核孔蛋白 RanBP2。 RanGAP1 的 SUMO-1 缀合通过与 RanBP2 结合促进其与间期核孔复合物 (NPC) 的结合。 RanBP2 存在于有丝分裂的着丝粒上，RanGAP1 也以微管和 SUMO-1 依赖性方式集中在着丝粒上。我们的工作表明 Ran 在有丝分裂着丝粒中发挥两个重要作用。也就是说，它对于纺锤体组装检查点的调节以及将动粒附着到纺锤体极的微管纤维的组装至关重要。 我们已经证明，RanBP2 和 RanGAP1 在有丝分裂过程中作为单一复合物被靶向，该复合物既受有丝分裂纺锤体中稳定的着丝粒-MT 关联的调节，又对其至关重要。我们发现 Crm1 是一种 Ran-GTP 结合核输出受体，定位于哺乳动物细胞的动粒。使用药物 Leptomycin B (LMB) 抑制 Crm1 会导致 RanGAP1/RanBP2 从着丝粒释放，并形成纺锤体，其中连续的 MT 束跨越着丝粒，表明它们的着丝粒不维持与单个着丝粒纤维的离散端接附着。这些发现表明 RanGAP1/RanBP2 的正确定位对于着丝粒纤维的定义和后期染色体分离至关重要。我们的合作者 (Roberge) 还获得了支持 RanGAP1/RanBP2 复合物在着丝粒中的作用的其他数据。特别是，他们已经鉴定出一种小分子抑制剂，ent-15-oxokaurenoic Acid (EKA)，它与 ​​RanBP2 结合。 EKA 阻断有丝分裂运动蛋白 CENP-E 与动粒的关联并抑制染色体运动，这与 RanBP2 在这些过程中的作用一致。值得注意的是，RanGAP1 对纺锤体的靶向在动物和植物之间是保守的，尽管所利用的分子机制是完全不同的 (Meier)。我们目前正在尝试鉴定 Crm1 介导的 RanGAP1/RanBP2 复合物募集所需的动粒成分，并在分子水平上了解这一机制。 纺锤体组装检查点监测纺锤体形成并防止中期-后期转变的开始，直到染色体正确附着并在中期板上对齐。在之前的实验中，我们记录了爪蟾卵提取物中的纺锤体组装检查点可以通过 Ran-GTP 进行调节。在酵母和哺乳动物细胞中，纺锤体组装检查点蛋白 Mad1p 和 Mad2p 在分裂间期定位于 NPC，我们研究了这些蛋白与芽殖酵母中 Ran 途径的关系。我们发现酵母 MAD1 或 MAD2 的缺失并不会严重影响稳态核质运输或 Ran 定位。然而，在酵母 Ran GTPase 途径中发生条件突变的酵母会破坏细胞核中 Ran 的浓度，从而取代了 NPC 中的 Mad2p，但没有取代 Mad1p。 M 期细胞中 Mad2p 的位移与纺锤体检查点的激活相关。这些观察结果表明，Mad2p 在 NPC 中的定位对 Ran 的核水平敏感，并表明 Mad2p 从 NPC 中的释放与酵母中纺锤体装配检查点的激活密切相关。这是第一个证据表明 Ran 影响 Mad2p 向 NPC 的定位。 RanGAP1/RanBP2 复合物靶向有丝分裂着丝粒以及 Mad1 和 Mad2 与间期 NPC 的关联都表明着丝粒与核孔之间存在密切关系。我们的合作者（D. Forbes、B. Fontoura）的工作表明，包含九个核孔蛋白的 Nup107-160 复合物定位于哺乳动物细胞的有丝分裂纺锤体和着丝粒。在合作研究中，他们进一步表明，爪蟾卵提取物中的纺锤体组装需要 Nup107-160 复合物。另一方面，在缺乏 Nup107-160 复合物的情况下，鸡蛋提取物响应 Ran-GTP 水平升高而聚合微管的能力似乎是完整的。这些发现并不排除 Nup107-160 复合物可能是 Ran-GTP 上游调节因子的可能性，但反驳了它在纺锤体形成中作为 Ran 效应子的观点。值得注意的是，Nup107-160 复合物不是激活鸡蛋提取物中纺锤体检查点所必需的，也不是通过增加 Ran-GTP 水平来沉默检查点所必需的。我们正在通过检查其他核孔蛋白复合物的有丝分裂作用并研究它们作为有丝分裂 Ran 效应器的可能功能来扩展这些研究。