Cellular functions of Ran GTPase

Ran GTPase 的细胞功能

• 批准号: 8157621
• 负责人: Petr Kalab
• 金额: $ 29.07万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/8157621
• 关键词: Cell physiology; Guanosine Triphosphate Phosphohydrolases; Running

Ran GTPase is a key regulator of macromolecular transport between nucleus and cytoplasm and has important role in several steps of cell division, including mitotic spindle assembly and nuclear envelope reformation at the exit from mitosis. Because RCC1, the guanine nucleotide exchange factor for Ran, binds to chromatin while RanGAP is cytoplasmic, the position of chromosomes is marked by the highest cellular concentration of RanGTP, the RanGTP gradient. Most, but not all, functions of Ran are mediated by its interactions with importin beta-related nuclear transport receptors (NTRs). Ran and NTRs functionally interact with nucleoporins (Nups) the components of NPCs. In interphase, step-wise RanGTP gradient across nuclear envelope provides direction and is also a source of energy for Ran-regulated transport of cargos carried by NTRs through the channels of nuclear pore complexes. In mitosis, diffusion limited RanGTP gradient induces localized release of spindle assembly factors (SAFs) from their inhibitory complexes with nuclear import receptors, importins. As a result, SAFs are preferably activated in mitotic cytoplasm surrounding chromosomes, providing essential spatial bias to mitotic spindle assembly. However, some SAFs are regulated by RanGTP in mitosis with no requirement for the existence of spatially resolved RanGTP gradient. Remarkably, most of the SAFs involved in Ran-regulated mitotic network are well known as cancer-related factors: TPX2, Aurora A, hTOG, HURP, BRCA1, RHAMM, NPM1, RASSF1a, TACC3/maskin, survivin, APC (adenoma polyposis coli) and others. In addition, more recently it was shown that upregulation of RanGTP gradient leads to transformation of NIH3T3 cells apparently through causing amplification of RanGTP-gradient dependent cytoplasmic decapping of mRNAs, which and thus inducing deregulated synthesis of growth promoting functions. In summary, multiple pieces of evidence suggest that potentially several different RanGTP gradient-regulated processes have an important role in cancer etiology. At present, we are focusing on the role of Ran in mitotic spindle assembly and our goal is to elucidate differences, if any, in the contribution of Ran to mitosis in cancer cells vs. normal cells. Many of the Ran-regulated mitotic mechanisms of spindle assembly are highly conserved between different organisms. Thus, Ran-regulated SAFs) carry similar functions in Xenopus laevis meiotic/embryonic egg extracts, in meiotic mouse oocytes and in human tissue culture cells, suggesting their evolutionary conservation. For example, TPX2 activates Aurora A in HeLa cells and in X. laevis egg extracts. However, the relative contribution to spindle assembly and cell division is dramatically between different types of cells, such as in comparison of meiotic vs. somatic cells. We use two approaches in addressing these important questions: 1) Quantitative analysis of RanGTP gradient in mitotic normal and cancer cells 2) Proteomic and functional reconstitution analysis of Ran-regulated mitotic spindle assembly. During 2009/10, in both areas we succeeded in developing reagents and techniques which are needed to carry the key experiments. In the first approach, we made a significant progress in developing a much improved FRET sensor for quantitative fluorescence lifetime imaging microscopy (FLIM) measurements of RanGTP gradient in live cells. This sensor, called Rango-3, displays about 2.5-fold increased sensitivity in live cell measurements of RanGTP gradient compared with our previously published Rango sensors. For the first time, with Rango-3 we are now able to quantitatively monitor the changes of RanGTP-regulated importin beta cargo gradient during the course of live cell division. Unexpectedly, these experiments revealed significant attenuation of RanGTP-regulated release of importin beta cargos during the reformation of nuclear envelope, and RanGTP gradient-independent first rounds of nuclear import in nascent nuclei in HeLa cells. Using Rango-3 and live FLIM/FRET measurements, we are now setting up experiments to quantitatively compare RanGTP regulated importin beta cargo gradients in normal and transformed tissue culture cells. In the second approach, we significantly improved our method to isolate highly purified RanGTP-induced microtubules from X. laevis egg extracts for proteomic analysis. The major reason for using this system in our project is the conservation of Ran-regulated mitotic pathways. In addition, the completed sequencing of highly similar X. tropicalis genome enables efficient application of high throughput proteomic approaches. Until recently, the major limitation in using X. laevis egg extract system for proteomic analysis were the variability between batches of eggs and the need to initiate the purification of spindles from fresh extracts. We solved this problem by developing a protocol yielding extracts which survive storage at -80C without loss of activity, and by optimizing our method of capturing spindle structures on magnetic beads coated with microtubule-binding proteins. Finally, in collaboration with the laboratory of Dr. J. Yates at Scripps institute we are now ready to carry the proteomic analysis of Ran-induced spindle assembly. In addition to the research performed at NCI, we continued in collaborative projects with several extramural laboratories. In collaboration with Professor Alexey Khodjakov (Wadsworth Center, Albany, NY), we showed that RanGTP gradient functions in parallel with kinetochore-dependent mechanisms to support bipolar spindle assembly in HeLa (J. Cell Biol., 2009, 187:43). In collaboration with Professor Iris Meier (The Ohio State University, Columbus, OH), we introduced Rango FRET sensor into transgenic A. thaliana plants, which allowed us to obtain first insights into the regulation of Ran in plant cells in vivo. Finally, in collaboration with Dr. Inke Nathke (University of Dundee, UK), we described and analyzed the RanGTP- and importin beta-regulated function of APC (Adenomatous polyposis coli) in mitotic microtubule assembly (J. Cell Sci, 2010, 123:736).

Ran GTPase是细胞核和细胞质之间大分子运输的关键调节因子，在细胞分裂的几个步骤中起重要作用，包括有丝分裂纺锤体组装和有丝分裂出口的核膜重组。由于Ran的鸟嘌呤核苷酸交换因子RCC1与染色质结合，而RanGAP在细胞质中，因此染色体的位置由RanGTP的最高细胞浓度（即RanGTP梯度）来标记。Ran的大部分（但不是全部）功能是通过其与输入蛋白β相关核转运受体（NTRs）的相互作用介导的。Ran和ntr在功能上与核孔蛋白（NPCs的组成部分）相互作用。在间期，跨核包膜的RanGTP梯度提供了方向，也是ntr通过核孔复合物通道携带的ran调控的货物运输的能量来源。在有丝分裂中，扩散受限的RanGTP梯度诱导纺锤体组装因子（SAFs）从其与核输入受体（进口蛋白）的抑制复合物中局部释放。因此，saf在染色体周围的有丝分裂细胞质中被激活，为有丝分裂纺锤体组装提供必要的空间偏倚。然而，在有丝分裂过程中，一些saf是由RanGTP调控的，而不需要存在空间分辨的RanGTP梯度。值得注意的是，大多数参与ran调控的有丝分裂网络的SAFs都是众所周知的癌症相关因子：TPX2、Aurora A、hTOG、HURP、BRCA1、RHAMM、NPM1、RASSF1a、TACC3/maskin、survivin、APC（腺瘤息肉病大肠杆菌）等。此外，最近的研究表明，RanGTP梯度的上调明显导致NIH3T3细胞的转化，其途径是引起依赖于RanGTP梯度的细胞质mrna脱帽扩增，从而诱导生长促进功能合成的解除调控。综上所述，多项证据表明，几种不同的RanGTP梯度调节过程在癌症病因学中可能发挥重要作用。目前，我们专注于Ran在有丝分裂纺锤体组装中的作用，我们的目标是阐明Ran在癌细胞和正常细胞有丝分裂中的贡献的差异，如果有的话。纺锤体组装的许多rna调控的有丝分裂机制在不同的生物体之间是高度保守的。因此，ran调控的SAFs在非洲爪蟾减数分裂/胚胎卵提取物、小鼠卵母细胞和人类组织培养细胞中具有相似的功能，表明它们具有进化保守性。例如，TPX2激活HeLa细胞和X. laevis蛋提取物中的Aurora A。然而，纺锤体组装和细胞分裂的相对贡献在不同类型的细胞之间是显著的，例如减数分裂细胞和体细胞的比较。我们使用了两种方法来解决这些重要的问题：1)定量分析有丝分裂正常细胞和癌细胞中的RanGTP梯度；2)蛋白质组学和功能重构分析ranp调节的有丝分裂纺锤体组装。在2009/10年度，我们在这两个领域成功开发了进行关键实验所需的试剂和技术。在第一种方法中，我们在开发一种改进的FRET传感器方面取得了重大进展，该传感器用于定量荧光寿命成像显微镜（FLIM）测量活细胞中的RanGTP梯度。这种传感器被称为Rango-3，在RanGTP梯度的活细胞测量中，与我们之前发表的Rango传感器相比，灵敏度提高了约2.5倍。利用Rango-3，我们首次能够定量监测活细胞分裂过程中由rangtp调控的输入β货物梯度的变化。出乎意料的是，这些实验显示，在HeLa细胞的新生细胞核中，在核膜重组过程中，RanGTP调控的β输入货物释放显著衰减，并且RanGTP与梯度无关的第一轮核输入。利用ranggo -3和活体FLIM/FRET测量，我们现在正在建立实验，定量比较RanGTP调节的正常和转化组织培养细胞的进口β货物梯度。在第二种方法中，我们显著改进了从野田鸡蛋提取物中分离高纯度rangtp诱导微管的方法，用于蛋白质组学分析。在我们的项目中使用该系统的主要原因是保存了ran调控的有丝分裂途径。此外，高度相似的热带蓟基因组的完成测序使高通量蛋白质组学方法的有效应用成为可能。直到最近，使用紫斑天竺葵蛋提取物系统进行蛋白质组学分析的主要限制是鸡蛋批次之间的差异以及需要从新鲜提取物中开始纯化纺锤体。我们通过开发一种方案来解决这个问题，该方案产生的提取物可以在-80℃下保存而不会失去活性，并优化了我们在涂有微管结合蛋白的磁珠上捕获纺锤体结构的方法。最后，在与Scripps研究所J. Yates博士实验室的合作下，我们现在准备对ran诱导的纺锤体组装进行蛋白质组学分析。除了在NCI进行的研究外，我们还继续与几个校外实验室进行合作项目。在与Alexey Khodjakov教授（Wadsworth中心，Albany， NY）的合作中，我们证明了RanGTP梯度与着丝点依赖机制并行运行，以支持HeLa的双极主轴组装（J. Cell Biol）。中文信息学报，2009,187:43)。我们与Iris Meier教授（The Ohio State University, Columbus， OH）合作，将Rango FRET传感器引入转基因拟南植物中，这使我们首次了解了Ran在植物细胞体内的调节。最后，与英国邓迪大学的Inke Nathke博士合作，我们描述并分析了APC（腺瘤性息肉病大肠杆菌）在有丝分裂微管组装中的RanGTP-和进口蛋白β调节功能（J.细胞科学，2010,123:736）。