Cellular functions of Ran GTPase

Ran GTPase 的细胞功能

• 批准号: 8763339
• 负责人: Petr Kalab
• 金额: $ 64.83万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763339
• 关键词: Antimitotic Agents; Automobile Driving; Binding; Biosensor; Cell Cycle; Cell Nucleus; Cell division; Cell physiology; Cells; Chromatin; Chromosomal Gain; Chromosome Positioning; Chromosomes; Collaborations; Complex; Computer Simulation; Crowding; Cytoplasm; Data; Dependence; Diffusion; Energy-Generating Resources; Fluorescence; Fluorescence Resonance Energy Transfer; GTPase-Activating Proteins; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Human; Image; Importins; In Vitro; Interphase; Knowledge; Laboratories; Life; Maintenance; Malignant Neoplasms; Measurement; Measures; Mediating; Methods; Microscopy; Minnesota; Mitosis; Mitotic; Mitotic Cell Cycle; Mitotic spindle; Molecular; Mutate; Nuclear Envelope; Nuclear Import; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Production; Proliferating; Proteins; Publishing; Research; Role; Running; Somatic Cell; Somatic Mutation; TACC3 gene; Testing; Tumor-Derived; Universities; alpha Karyopherins; beta Karyopherins; cancer cell; cancer stem cell; chemotherapy; exportin 1 protein; follow-up; immortalized cell; nucleocytoplasmic transport; overexpression; pluripotency; receptor; sensor; survivin; tumor

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.

Ran GTPase 是细胞核和细胞质之间大分子运输的关键调节因子，在细胞分裂的多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处的核膜重组。由于 Ran 的鸟嘌呤核苷酸交换因子 RCC1 与染色质结合，而 RanGAP 位于细胞质中，因此染色体的位置由 RanGTP 的最高细胞浓度（RanGTP 梯度）来标记。 Ran 的大部分功能是通过其与输入 β 相关核转运受体 (NTR) 的相互作用介导的。 Ran 和 NTR 与 NPC 的成分核孔蛋白 (Nups) 发生功能性相互作用。在间期，跨核膜的逐步 RanGTP 梯度提供了方向，也是 NTR 携带的货物通过核孔复合体通道进行 Ran 调节运输的能量来源。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（输入蛋白）的抑制复合物中局部释放。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，至少一些 SAF 在有丝分裂中受到 RanGTP 的调节，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。专注于分析 Ran 调节的有丝分裂机制的癌细胞特异性改变，我们开发了使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) 来分析 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 诱导的自由导入 β 货物的梯度，对应于激活的 SAF 的梯度。使用这些传感器，我们测量了各种人类体细胞的两个梯度，包括正常原代细胞、永生化细胞、肿瘤衍生和肿瘤诱导的癌细胞。我们发现，虽然在快速增殖的永生化和/或癌症衍生的人类体细胞中表达陡峭的有丝分裂 RanGTP 梯度，但在生长缓慢的人类原代细胞中，该梯度强烈降低或检测不到。我们发现 RCC1 表达增加和染色体增益增加是有丝分裂 RanGTP 梯度陡峭的关键驱动因素 (Hasekawa et al., 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. Odde 博士（明尼苏达大学）的实验室建立了合作。在这项研究中，我们使用来自活细胞测量和计算模型的数据来检验以下假设：由于染色体拥挤而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，我们发现体外诱导的正常体细胞向癌症干细胞 (CSC) 前体的转化伴随着有丝分裂 RanGTP 梯度的急剧增加和 RCC1 表达的增加。因为我们在山中因子诱导多能性过程中观察到 RanGTP 产生的这种激活，所以我们假设增加的 RCC1 表达和陡峭的 RanGTP 梯度是维持 CSC 去分化状态所必需的。