Cellular functions of Ran GTPase

Ran GT3是细胞核与细胞质之间大分子运输的关键调节剂，在细胞分裂的几个步骤中具有重要作用，包括有丝分裂纺锤体组装和有丝分裂出口处的核膜重组。由于RCC 1（Ran的鸟嘌呤核苷酸交换因子）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（RanGTP梯度）标记。Ran的大多数但不是全部功能是通过其与importin β相关核转运受体（NTR）的相互作用介导的。Ran和NTR在功能上与核孔蛋白（Nups）（NPC的组分）相互作用。在间期，跨核膜的逐步RanGTP梯度提供方向，也是通过核孔复合物的通道的Ran-regulated运输NTR携带的货物的能量来源。在有丝分裂中，扩散限制的RanGTP梯度诱导纺锤体组装因子（SAF）从其与核输入受体（importins）的抑制性复合物中局部释放。因此，SAF优选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差。然而，一些SAF在有丝分裂中受RanGTP调节，不需要空间分辨RanGTP梯度的存在。值得注意的是，参与Ran调控的有丝分裂网络的大多数SAF都是众所周知的癌症相关因子：TPX 2，Aurora A，hTOG，HURP，BRCA 1，RHAMM，NPM 1，RASSF 1a，TACC 3/maskin，生存素，APC（腺瘤息肉病结肠）等。此外，最近显示RanGTP梯度的上调明显地通过引起mRNA的RanGTP梯度依赖性细胞质去帽的扩增而导致NIH 3 T3细胞的转化，这因此诱导生长促进功能的失调合成。总之，多项证据表明，潜在的几种不同的RanGTP梯度调节过程在癌症病因学中具有重要作用。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对癌细胞与正常细胞有丝分裂的贡献的差异（如果有的话）。Ran调控的纺锤体组装的有丝分裂机制在不同生物体之间是高度保守的。因此，Ran调节的SAF在非洲爪蟾减数分裂/胚胎卵提取物、减数分裂小鼠卵母细胞和人组织培养细胞中具有相似的功能，表明它们在进化上是保守的。例如，TPX 2在HeLa细胞和X.鸡蛋提取物。然而，对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，例如在减数分裂与体细胞的比较中。我们使用两种方法来解决这些重要的问题：1）定量分析RanGTP梯度在有丝分裂的正常和癌细胞2）蛋白质组学和功能重建分析RAN调节有丝分裂纺锤体组装。在第一种方法中，在2009/10年，我们开发了改进的FRET传感器，用于活细胞中RanGTP梯度的定量荧光寿命成像显微镜（FLIM）测量。使用这些传感器，在2010年秋天，我们发现，在显着的对比，HeLa，有丝分裂RanGTP梯度几乎是不存在的正常原代人成纤维细胞。通过两种不同的FRET成像方法证实了这一令人惊讶的发现，每种方法具有两种不同的FRET传感器：RanGTP结合传感器和RanGTP调节的输入β货物传感器。此外，我们很快还确定，转移性乳腺癌MCF 10 CA 1a细胞显示出比同基因正常或非恶性永生化乳腺癌细胞系更强的RCC 1与染色质结合和更陡的有丝分裂RanGTP梯度。由于这一发现的潜在重要性，2011年我们将实验室的大部分研究集中在研究正常细胞与癌细胞中RanGTP梯度差异的分子机制上。我们发现，除了Ran浓度增加外，导致RanGTP梯度变陡的关键因素是RCC 1与染色质结合增加。使用RCC 1-mCherry进行的光漂白后活细胞荧光恢复（FRAP）测量显示，在整个细胞周期中，RCC 1与染色质的结合在HeLa中比在成纤维细胞中更强，需要NRMT的N-末端甲基化，并得到RCC 1丝氨酸10磷酸化的支持。而NRMT耗竭导致减少RCC 1结合染色质和纺锤体缺陷的HeLa细胞，同样的治疗没有显着影响纺锤体组装在正常成纤维细胞。与有丝分裂纺锤体组装中提出的RAN功能一致，原代细胞中没有陡峭的有丝分裂RanGTP梯度与延长的前中期相关。这些发现表明，RanGTP梯度和它的作用在有丝分裂纺锤体组装衰减在正常体组织的机制，包括减少RCC 1甲基化。另一方面，陡峭的有丝分裂RanGTP梯度是快速分裂的正常细胞和癌细胞的常见标志。我们写了一份手稿描述这些发现，并计划在未来几周内提交出版（目标是2011年10月1日）。

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. 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. In the first approach, in 2009/10 we developed improved FRET sensors for quantitative fluorescence lifetime imaging microscopy (FLIM) measurements of RanGTP gradient in live cells. Using these sensors, in the fall of 2010 we discovered that in striking contrast to HeLa, mitotic RanGTP gradient is virtually absent in normal primary human fibroblasts. This surprising finding was confirmed by two different FRET imaging methods, each with two different FRET sensors: RanGTP- binding sensor and sensor for RanGTP-regulated importin beta cargos. Moreover, we soon also determined that metastatic breast cancer MCF10CA1a cells displayed stronger RCC1 binding to chromatin and steeper mitotic RanGTP gradient than isogenic normal or non-malignant immortalized breast cancer cell lines. Because of the potentially high significance of this finding, in 2011 we focused most of the research in the lab on investigating the molecular mechanism underlying the differences in RanGTP gradient in normal vs. cancer cells. We found addition to increased concentration of Ran, the key factor responsible for steeper RanGTP gradients was increased RCC1 binding to chromatin. Live-cells fluorescence recovery after photobleaching (FRAP) measurements with RCC1-mCherry showed that throughout the cell cycle, the binding of RCC1 to chromatin was stronger in HeLa than in fibroblasts, required N-terminal methylation by NRMT and was supported by RCC1 phosphorylation on Serine 10. While NRMT depletion caused decreased RCC1 binding to chromatin and spindle defects in HeLa, the same treatment had no significant effect on spindle assembly in normal fibroblasts. Consistent with proposed Ran functions in mitotic spindle assembly, the absence of a steep mitotic RanGTP gradient in primary cells correlated with extended prometaphase. These findings suggest that RanGTP gradient and its role in mitotic spindle assembly are attenuated in normal somatic tissues by mechanisms including decreased RCC1 methylation. On the other hand, steep mitotic RanGTP gradient is a commonly expressed hallmark of rapidly dividing normal and cancer cells. We wrote a manuscript describing these findings and plan submitting it for publication within the next few weeks (aiming for October 1, 2011).