Regulation of Mitotic Kinetochores by the Ran GTPase

Ran GTPase 对有丝分裂着丝粒的调节

• 批准号: 8149305
• 负责人: MARY C. DASSO
• 金额: $ 55.62万
• 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/8149305
• 关键词: Guanosine Triphosphate Phosphohydrolases; Kinetochores; Mitotic; Regulation; Running

The Ran GTPase is required for many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. The sole nucleotide exchange factor for Ran, RCC1, binds chromatin throughout the cell cycle. The GTPase activating protein for Ran, RanGAP1, localizes to the cytosolic face of the nuclear pore complex (NPC) during interphase through association with RanBP2, a large nucleoporin. The interphase distribution of Ran regulators leads to a high concentration of Ran-GTP in nuclei, and low Ran-GTP in cytosol. The major effectors for Ran are a family of Ran-GTP binding proteins that were discovered as nuclear transport receptors. These receptors are collectively called Karyopherins; those that mediate import are called Importins, and those that mediate export are called Exportins. Karyopherins transit the NPC in a Ran- and cargo-independent fashion. Their cargo loading is governed by Ran-GTP levels: Importins bind to their cargo in the cytoplasm. Import complexes traverse the NPC and dissociate upon Ran-GTP-Importin binding. Exportins bind their cargo inside nuclei in complexes that contain Ran-GTP. After passage through the NPC, export complexes dissociate upon Ran-GTP hydrolysis. To date, two nuclear transport receptors have been shown to act as Ran effectors during mitosis, Importin-beta and the exportin Crm1. Importin-beta binds and imports cargo with classical nuclear localization sequences (cNLSs) through an adaptor subunit, Importin-alpha. In mitotic metazoan cells, Importin-alpha/beta bind and inhibit spindle assembly factors (SAFs). Elevation of diffusible Ran-GTP concentrations near mitotic chromatin releases inhibition by Importin-alpha/beta, allowing localized activation of such factors. SCCR studies have been particularly concerned with Ran functions at kinetochores. Kinetochores are proteinaceous structures that assemble at the centromere of each sister chromatid during mitosis, and that serve as sites of spindle microtubule (MT) attachment. The kinetochore fibers (k-fibers) that link mammalian kinetochores to spindle poles contain both MTs that are directly attached to the kinetochores at their plus ends (kMTs) and MTs that are not. Kinetochore attachment is monitored through the spindle assembly checkpoint (SAC), which prevents mitotic exit by blocking anaphase promoting complex/cyclosome (APC/C) activation until all chromosomes are attached and aligned onto the metaphase plate. The APC/C is a ubiquitin ligase that regulates the destruction of key mitotic regulatory proteins. Components of the SAC include: Mad1, Mad2, Mps1, Bub1, Bub3, BubR1, and CENP-E. Elevated levels of Ran-GTP abrogate SAC-mediated mitotic arrest in Xenopus egg extracts (XEEs) and disrupt the kinetochore localization of SAC components, suggesting that the SAC is directly responsive to the overall concentration of Ran-GTP in that system. The effector for Ran in the SAC remains an unresolved issue, and this problem is a major focus of our current interests. (Our findings indicate that the effector is neither Importin-beta nor Crm1.) We have found that Crm1 localizes to kintochores, and that inhibition of Crm1 ternary complex formation blocks kinetochore recruitment of RanGAP1/RanBP2. Crm1 itself requires neither ternary complex assembly nor MTs for kinetochore binding. Under these circumstances, kinetochores dramatically failed to maintain discrete end-on attachments to single k-fibers and showed a resultant elevation in chromosome mis-segregation. The component(s) at kinetochores that is directly involved in Crm1 recruitment is a major focus of our ongoing studies. In addition, we hypothesize that Crm1 acts in mitosis through sequestration of its cargoes, as Importin-alpha/beta does. We have identified to cargos of Crm1 within lysates of HeLa cells arrested at different cell cycle stages. Western blot analysis revealed that the cargos could be categorized into two groups with their respect to Crm1 binding: low-affinity and high-affinity cargoes (LACs and HACs, respectively). LACs were defined as those that could bind to tagged Crm1 protein in the presence of Ran-GTP, but whose depletion from the lysate was limited to 1-2% of the total amount in HeLa extracts. All tested LACs showed accumulation in the nucleus upon Crm1 inhibition. HACs were defined by their depletion from lysates through affinity chromatography after incubation with tagged Crm1 and Ran-GTP. Notably, Crm1 should quantitatively sequester HACs during mitosis when it shares the same compartment with both Ran-GTP and HACs. These complexes would be dissociated after the nuclear envelope is re-established in telophase, allowing Crm1 to return to its role as an exportin. We are currently investigating the functions of HACs, the biochemistry of their high affinity binding and their mode of regulation through Crm1.

RAN GTPase是许多细胞功能所必需的，包括核质运输，主轴组件，核装配和细胞周期控制。 RAN RCC1的唯一核苷酸交换因子在整个细胞周期中结合染色质。 RANGAP1的GTPase激活蛋白质通过与大核孔RANBP2缔合，将其定位于核孔复合物（NPC）的胞质面（NPC）。 RAN调节剂的相间分布导致核中高浓度的RAN GTP，而细胞质中的RAN GTP低。 RAN的主要效应子是被发现为核转运受体的RAN GTP结合蛋白家族。这些受体被统称为核蛋白。介导的导入的人称为导入蛋白，介导导出的人称为导出。核蛋白以跑步和货物独立的方式过渡NPC。他们的货物负荷受RAN GTP水平的控制：进口素与其在细胞质中的货物结合。导入复合物穿越NPC并在RAN-GTP-rimettring结合上解离。导出蛋白将其货物在核中结合，其中包含RAN-GTP的复合物。通过NPC后，导出复合物将在RAN GTP水解后解离。迄今为止，已经显示两个核转运受体在有丝分裂，Importin-Beta和Exportin CRM1期间起效应子。 Importin-Beta通过适配器亚基Imptimin-Alpha结合和进口货物与经典的核定位序列（CNLS）。在有丝分裂的后生细胞中，进口蛋白 - α/β结合并抑制纺锤体组装因子（SAFS）。在有丝分裂染色质附近的可扩散RAN GTP浓度升高通过进口蛋白-Alpha/beta抑制，从而允许对此类因素进行局部激活。 SCCR研究特别关注动力学的RAN功能。动力学是蛋白质结构，在有丝分裂过程中在每个姐妹染色质剂的中心粒中组装，并且用作纺锤微管（MT）附着的位点。将哺乳动物动力学连接到主轴杆的动力学纤维（k纤维）都包含直接连接到其正末端（KMTS）和没有的MT的MTS。通过纺锤体组件检查点（SAC）监测动力学附件，该检查点（SAC）通过阻止后期促进复合物/循环体（APC/C）激活来防止有丝分裂出口，直到将所有染色体连接到中期板上。 APC/C是一种泛素连接酶，可调节关键有丝分裂调节蛋白的破坏。 SAC的组件包括：MAD1，MAD2，MP​​S1，BUB1，BUB3，BUBR1和CENP-E。 RAN-GTP水平升高消除了SAC介导的Xenopus卵提取物（XEES）中的有丝分裂停滞，并破坏了SAC成分的动力学定位，这表明该SAC直接响应该系统中RAN-GTP的整体浓度。 SAC中RAN的效应子仍然是一个尚未解决的问题，这个问题是我们当前利益的主要重点。 （我们的发现表明效应子既不是importin-beta也不是CRM1。） 我们发现CRM1定位于Kintochores，并且抑制CRM1三元复合物的形成阻碍了Rangap1/ranbp2的Kinetochore募集。 CRM1本身不需要三元复合物组件，也不需要MTS才能进行动力学结合。在这种情况下，动力学巨大未能保持对单个K纤维的离散末端附件，并显示出染色体错误分离的升高。直接参与CRM1募集的动力学的成分是我们正在进行的研究的主要重点。此外，我们假设CRM1通过隔离其货物的序列作用，就像Importin-Alpha/beta一样。我们已经确定了在不同细胞周期阶段捕获的HeLa细胞裂解物中CRM1的Cargos。 Western印迹分析表明，可以将Cargos归类为两组CRM1结合：低亲和力和高亲和力货物（分别是LAC和HACS）。 LAC被定义为在RAN GTP存在下可以与标记的CRM1蛋白结合的lAC，但其裂解物的耗竭限制为HELA提取物中总量的1-2％。所有经过测试的紫胶在CRM1抑制后均显示在核中积累。 HAC与标记的CRM1和RAN-GTP孵育后，通过裂解物从裂解物中的耗竭来定义。值得注意的是，当CRM1与RAN-GTP和HACS共享相同的隔室时，应在有丝分裂过程中定量隔离HAC。这些复合物将在核包膜重新建立后将其分离，从而使CRM1恢复其作为导出蛋白的作用。我们目前正在研究HAC的功能，其高亲和力结合的生物化学及其通过CRM1的调节方式。