Cellular functions of Ran GTPase

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，至少有一些saf在有丝分裂中是由RanGTP调控的，而不需要存在空间分辨的RanGTP梯度。ran调控的SAFs是众所周知的癌症相关因子：TPX2、HURP、TACC3、survivin、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对癌细胞和正常细胞有丝分裂的贡献的差异。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。我们采用了两种方法：1)分析Ran调控的有丝分裂机制的癌细胞特异性改变2)分析Ran调控的有丝分裂途径的生化和功能。在第一种方法中，我们利用活细胞中表达的FRET生物传感器的荧光寿命成像显微镜（FLIM）开发了高灵敏度定量测量有丝分裂细胞中Ran功能的方法。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，对应于活化的SAFs梯度。在2010- 2012年，我们使用这些传感器测量了14种不同人类体细胞的两种梯度，包括正常原代细胞、永生化细胞、肿瘤来源和肿瘤诱导的癌细胞。我们发现，尽管在快速增殖的人体细胞（包括正常乳腺上皮细胞MCF10a和癌源性HeLa细胞）中，有丝分裂的RanGTP梯度表达陡峭，但在缓慢生长的人原代细胞（如HFF-1成纤维细胞）中，梯度强烈降低或无法检测到。与RanGTP在加速纺锤体组装中的作用一致，RanGTP梯度的破坏导致了前期中期进展的延迟。体外细胞融合诱导HFF-1细胞的有丝分裂RanGTP梯度增大，MCF10a和HeLa细胞的RanGTP梯度增大，表明非整倍体癌细胞的染色体增益可以通过Ran促进其纺锤体组装。《细胞生物学》杂志回顾了一篇描述我们发现的手稿。目前，我们正在对大多数正面评价做出回应。在第二种方法中，我们关注的是有丝分裂纺锤体组装机制，该机制由输入蛋白家族的复合物或输入蛋白β及其衔接蛋白调节。虽然在人类基因组中表达了7种进口蛋白α，但其中只有一种，即进口蛋白α 1 （KPNA2）被鉴定为SAF调节因子。有趣的是，在几种形式的癌症中，高水平的KPNA2表达与不良预后密切相关。我们感兴趣的是确定输入蛋白α 1是否确实是专门用于有丝分裂功能的输入蛋白α异构体，并全面确定其有丝分裂靶点的完整列表。为此，我们对非洲爪蟾卵提取物进行了检测，以比较不同的人类输入蛋白α在有丝分裂纺锤体组装中的作用。这些分析是相关的，因为先前的研究确定了非洲爪蟾和人类输入蛋白以及不同的saf在有丝分裂纺锤体组装中的作用具有高度的功能同源性。令人惊讶的是，到目前为止，我们的研究结果表明，输入蛋白α 3的显性负突变体（KPNA4）比输入蛋白α 1的类似突变形式更有效地抑制有丝分裂纺锤体组装，这表明输入蛋白α 3存在靶向SAF。目前，研究人员正在利用质谱法鉴定α - 3结合蛋白，并利用细胞生物学方法分析它们在有丝分裂纺锤体组装中的功能。

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.