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。因为RCC 1（Ran的鸟嘌呤核苷酸交换因子）结合于染色质，而RanGAP是细胞质，所以染色体的位置由RanGTP的最高细胞浓度（RanGTP梯度）标记。Ran的大多数（但不是全部）功能是通过其与输入蛋白β相关的核转运受体（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，至少有一些SAF在有丝分裂中受RanGTP调节，不需要空间分辨RanGTP梯度的存在。 RAN调节的SAF是众所周知的癌症相关因子：TPX 2、HURP、TACC 3、生存素、APC等。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。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：1）Ran调节的有丝分裂机制的癌细胞特异性改变的分析2）Ran调节的有丝分裂途径的生物化学和功能分析在第一种方法中，我们开发了使用活细胞中表达的FRET生物传感器的荧光寿命成像显微镜（FLIM）对有丝分裂细胞中的Ran功能进行高灵敏度定量测量的方法。 我们的FRET传感器之一，称为RBP-4，直接测量RanGTP梯度，另一个，称为Rango-4，测量RanGTP诱导的自由输入β货物的梯度，对应于激活的SAF的梯度。使用这些传感器，在2010-12年，我们测量了14种不同人体细胞的两个梯度，包括正常原代细胞，永生化细胞，肿瘤衍生和肿瘤诱导癌细胞。我们发现，虽然陡峭的有丝分裂RanGTP梯度在快速增殖的人体细胞中表达，包括正常乳腺上皮MCF 10a细胞和癌症来源的HeLa细胞，但在缓慢生长的人原代细胞中，如HFF-1成纤维细胞，梯度强烈降低或检测不到。与RanGTP在加速纺锤体组装中的作用一致，RanGTP梯度的中断导致前中期进展延迟。体外细胞融合诱导HFF-1细胞中陡峭的有丝分裂RanGTP梯度，并增加MCF 10a和HeLa细胞中梯度的陡度，表明非整倍体癌细胞中的染色体获得可以通过Ran促进其纺锤体组装。描述我们发现的手稿由J. Cell Biol.审查， 我们目前正在努力对大多数积极评价作出回应。 在第二种方法中，我们专注于有丝分裂纺锤体组装机制的复杂或importin β和它的适配器蛋白的importin α家族的调节。虽然在人类基因组中有7种importins α表达，但其中只有一种importin α 1（KPNA 2）被鉴定为SAF调节因子。有趣的是，高水平的KPNA 2表达与几种癌症的预后不良密切相关。我们有兴趣确定输入素α 1是否确实是专门用于有丝分裂功能的输入素α亚型，并全面确定其有丝分裂靶点的完整列表。为此，我们在非洲爪蟾卵提取物中开发了测定方法，以比较不同的人类importins α在有丝分裂纺锤体组装中的作用。这些测定是相关的，因为先前的研究确定了非洲爪蟾和人输入蛋白的作用与有丝分裂纺锤体组装中不同SAF之间的高水平功能同源性。令人惊讶的是，迄今为止我们的结果表明，输入蛋白α 3（KPNA 4）的显性负突变体比输入蛋白α 1的类似突变形式作为有丝分裂纺锤体组装的甚至更有效的抑制剂起作用，这表明存在输入蛋白α 3靶向SAF。通过质谱鉴定importin α 3结合的SAFs，然后通过细胞生物学方法分析它们在有丝分裂纺锤体组装中的有丝分裂功能，工作正在进行中。

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, at least some SAFs are regulated by RanGTP in mitosis with no requirement for the existence of spatially resolved RanGTP gradient. Ran-regulated SAFs are well known as cancer-related factors: TPX2, HURP, TACC3, survivin, APC and others. We are focusing on the role of Ran in mitotic spindle assembly and our goal is to elucidate differences 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: 1) The analysis of cancer cell-specific alterations of Ran-regulated mitotic mechanisms2) Biochemical and functional analysis of Ran-regulated mitotic pathways In the first approach, we developed methods for highly sensitive quantitative measurements of Ran function in mitotic cells using fluorescence lifetime imaging microscopy (FLIM) of FRET biosensors expressed in live cells. One of our FRET sensors, called RBP-4, measures directly the RanGTP gradient and another, called Rango-4, measures the RanGTP-induced gradient of free importin beta cargos, corresponding to the gradient of activated SAFs. Using these sensors, in 2010-12 we measured the two gradients in a panel of 14 different human somatic cells, including normal primary cells, immortalized cells, tumor-derived and tumor-inducing cancer cells. We found that while a steep mitotic RanGTP gradient was expressed in rapidly proliferating human somatic cells, including normal breast epithelial MCF10a cells and cancer-derived HeLa cells, the gradient was strongly reduced or not detectable in slow growing human primary cells, such as HFF-1 fibroblasts. Consistent with the role of RanGTP in accelerating the spindle assembly, disruptions of RanGTP gradient caused delays in prometaphase progression. In vitro cell fusion induced steep mitotic RanGTP gradient in HFF-1 cells and increased the steepness of the gradient in MCF10a and HeLa cells, indicating that chromosomal gain in aneuploid cancer cells can promote their spindle assembly via Ran. A manuscript describing our findings was reviewed by J. Cell Biol., and we are currently working on the response on mostly positive reviews. In the second approach, we focused on the mitotic spindle assembly mechanisms regulated by the complex or importin beta and its adaptor proteins of the importin alpha family. Although there are 7 importins alpha expressed in human genome, only one of them, importin alpha 1 (KPNA2) was identified as SAF regulator. Interestingly, high levels of KPNA2 expression strongly correlate with poor prognosis in several forms of cancer. We are interested to determine whether importin alpha 1 is indeed the importin alpha isoform specialized for mitotic functions and to comprehensively determine the complete list of its mitotic targets. To that end, we developed assays in Xenopus laevis egg extracts to compare the roles of different human importins alpha in mitotic spindle assembly. These assays are relevant because previous studies determined high level of functional homology between the role of Xenopus and human importins and different SAFs in mitotic spindle assembly. Surprisingly, our results so far indicate a dominant negative mutant of importin alpha 3 (KPNA4) functions as even more potent inhibitor of mitotic spindle assembly than a similar mutated form of importin alpha 1, indicating the existence of importin alpha 3 targeted SAF(s). Work is in progress to identify the importin alpha 3-binding SAFs by mass spectrometry and then to analyze their mitotic function in mitotic spindle assembly by cell biological approaches.