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的大多数功能是通过其与importin β相关的核转运受体（NTR）的相互作用来介导的。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。专注于分析癌细胞特异性的RAN调节的有丝分裂机制的改变，我们开发的方法，RAN功能使用荧光寿命成像显微镜（FLIM）的FRET生物传感器在活的有丝分裂细胞中表达。我们的FRET传感器称为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，我们测量了各种人体细胞，包括正常原代细胞，永生化细胞，肿瘤衍生和肿瘤诱导的癌细胞的两个梯度。我们发现，虽然陡峭的有丝分裂RanGTP梯度在快速增殖的永生化和/或癌症衍生的人体细胞中表达，但该梯度在缓慢生长的人原代细胞中强烈降低或不可检测。我们发现，RCC 1表达增加和大的染色体获得是陡峭的有丝分裂RanGTP梯度的关键驱动因素（Hasegawa等人，细胞生物学杂志，200（2）151-6，2013）。为了分析染色体增益驱动的有丝分裂RanGTP梯度上升的机制，我们与D。Odde（明尼苏达大学）。在这项研究中，我们使用来自活细胞测量和计算建模的数据来检验这一假设，即由于染色体拥挤而减少的扩散足以驱动染色体数目增加的细胞中陡峭的有丝分裂RanGTP梯度。在体细胞中筛选有丝分裂RanGTP梯度的另一个后续研究中，我们发现正常体细胞体外诱导转化为癌症干细胞（CSC）的前体细胞伴随有丝分裂RanGTP梯度的急剧增加和RCC 1表达的增加。因为我们在Yamanaka因子诱导多能性期间观察到RanGTP产生的这种激活，所以我们假设增加的RCC 1表达和陡峭的RanGTP梯度是维持CSC的去分化状态所必需的。

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 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. Focusing on the analysis of cancer cell-specific alterations of Ran-regulated mitotic mechanisms, we developed methods to of Ran function using fluorescence lifetime imaging microscopy (FLIM) of FRET biosensors expressed in live mitotic cells. 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 we measured the two gradients in variety of 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 immortalized and/or cancer-derived human somatic cells, the gradient was strongly reduced or not detectable in slow growing human primary cells. We found that increased expression of RCC1 and large chromosomal gain are the key drivers of steep mitotic RanGTP gradients (Hasegawa et al., J. Cell Biol., 200(2)151-6, 2013). To analyze the mechanism responsible for the chromosome gain-driven rise of mitotic RanGTP gradients, we set up collaboration with the laboratory of Dr. D. Odde (University of Minnesota). In this study we use data derived from live cell measurements and computational modeling to test the hypothesis that reduced diffusion owing to chromosomal crowding is sufficient to drive steep mitotic RanGTP gradient in cells with increased chromosome number. In another follow-up to the screen for mitotic RanGTP gradients in somatic cells, we found that the in vitro-induced transformation of normal somatic cells into precursors of cancer stem cells (CSCs) is accompanied by a dramatic increase of mitotic RanGTP gradient and increased RCC1 expression. Because we observed such activation of RanGTP production during the induction of pluripotency by the Yamanaka factors, we hypothesize that increased RCC1 expression and steep RanGTP gradients are required for the maintenance of de-differentiated state of CSCs.