Cellular functions of Ran GTPase

RAN GTP酶是核质间大分子运输的关键调节因子，在细胞分裂的几个步骤中发挥重要作用，包括有丝分裂纺锤体组装和有丝分裂出口核膜重塑。由于RCC1是RAN的鸟嘌呤核苷酸交换因子，与染色质结合，而RanGAP是细胞质的，染色体的位置由RanGTP的最高细胞浓度，RanGTP梯度来标记。RAN的大部分功能是通过与Importinβ相关的核运输受体(NTRs)的相互作用来实现的。RAN和NTRs在功能上与核孔蛋白(NUP)相互作用，这些核孔蛋白是NPC的组成部分。在间期，跨核膜的逐步RanGTP梯度提供了方向，也是RAN调节的NTRs通过核孔复合体通道运输货物的能量来源。在有丝分裂中，扩散受限的RanGTP梯度诱导纺锤体组装因子(SAF)从其与核输入受体Importins的抑制复合体中局部释放。因此，SAF最好在染色体周围的有丝分裂细胞质中被激活，为有丝分裂纺锤体的组装提供必要的空间偏向。然而，至少有一部分SAF在有丝分裂过程中受到RanGTP的调控，而不需要存在空间分辨的RanGTP梯度。RAN调节的SAF是众所周知的癌症相关因子：TPX2、HURP、TACC3、Survivin、APC等。着眼于分析RAN调控有丝分裂机制的癌细胞特异性改变，我们开发了利用活有丝分裂细胞中表达的FRET生物传感器的荧光寿命成像显微镜(FLIM)来研究RAN功能的方法。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诱导的自由进口贝塔货物的梯度，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体细胞的两个梯度，包括正常原代细胞、永生化细胞、肿瘤来源的癌细胞和诱导肿瘤的癌细胞。我们发现，尽管在快速增殖的永生化和/或癌症来源的人体细胞中表达了陡峭的有丝分裂RanGTP梯度，但在生长缓慢的人的原代细胞中，这种梯度显著降低或检测不到。我们发现，RCC1的表达增加和大的染色体增益是陡峭的有丝分裂RanGTP梯度的关键驱动因素(Hasegawa等人，J.Cell Biol.，200(2)151-6,2013)。为了分析染色体增益驱动有丝分裂RanGTP梯度上升的机制，我们与D·奥德博士(明尼苏达大学)的实验室建立了合作关系。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样的假设，即染色体拥挤导致的扩散减少足以在染色体数量增加的细胞中驱动陡峭的有丝分裂RanGTP梯度。在体细胞有丝分裂RanGTP梯度筛选的另一项后续研究中，我们发现，在体外诱导正常体细胞向肿瘤干细胞(CSCs)前体转化的过程中，伴随着有丝分裂RanGTP梯度的急剧增加和RCC1表达的增加。由于我们在山中因子诱导多能性的过程中观察到了RanGTP产物的激活，我们假设RCC1的表达增加和RanGTP梯度陡峭是维持CSCs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.