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的细胞功能由RanGTP与输入素β相关核转运受体（NTR）的相互作用介导。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梯度提供了方向性的Ran调节的NTR通过核孔复合物携带的货物运输。在有丝分裂期间，染色体被RanGTP的陡峭扩散梯度包围，RanGTP继续调节有丝分裂细胞质中NTR上的货物的装载和卸载。因为一些有丝分裂调节剂同时是NTR货物，它们的活性或与有丝分裂结构的结合由RanGTP控制，有助于有丝分裂纺锤体组装和染色体分离到子细胞。以前，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。为此，我们开发了定量测量Ran功能在有丝分裂细胞中使用荧光寿命成像显微镜（FLIM）的FRET生物传感器在活细胞中表达的方法。使用这种方法，我们发现有丝分裂RanGTP梯度在快速增殖的正常细胞和癌细胞中更稳健。此外，我们发现，增加RCC 1的表达和非整倍体细胞中有丝分裂染色体的数量增加是决定有丝分裂RanGTP梯度的陡度的两个最重要的因素。作为2013年发表的这项研究的后续行动，我们采用了两种方法。首先，我们着手确定有丝分裂RanGTP梯度的染色体增益驱动激活的机制。我们假设，强调机制取决于有丝分裂细胞中染色体-细胞质界面的生物物理特性，并涉及RCC 1的染色体结合位点数量增加和有丝分裂细胞中心的扩散减少，染色体数量增加。为了验证这一假设，我们计划与明尼苏达大学大卫奥德博士的实验室合作，将活细胞测量与有丝分裂RanGTP梯度的计算建模相结合。虽然我们很快就获得了最初的测量结果并开发了有丝分裂模型，但由于长谷川博士于2013年底离开NIH，这一进展被中断。在第二种方法中，我们研究了Ran在细胞周期调控中的作用。在本项目的第一阶段，我们分析了Ran在衰老细胞退出细胞周期中的作用。我们发现，由于染色质的不可逆胞质加工，衰老细胞中的永久性G1/S停滞与RCC 1蛋白水平的强烈下降有关。这一结果与我们最初的假设一致，即RanGTP水平的耗尽通过减少细胞周期因子的Ran调节的核质转运来强制稳定退出细胞周期。不幸的是，由Ryu博士在实验室进行的这个项目的进展被严重的技术挫折所打断，使他无法在2013年9月底离开实验室前往新的工作之前获得明确的结果。因此，实验室的两个主要项目都受到严重干扰，并在2013年底未发表，大约比2014年6月实验室实地考察提前了半年。Cekan博士，最近聘请的博士后研究员，和我专注于细胞周期项目。我们获得的证据表明，RCC 1驱动的Ran激活加速了DNA损伤后重新进入细胞周期，导致正常细胞和癌细胞逃避细胞衰老。强调的机制涉及DNA损伤修复的激活，需要输出蛋白1功能，并涉及RCC 1表达诱导的主要致癌转录调节因子的激活。描述这些结果的手稿将在几天内提交出版。然而，由于2014年现场访问审查的不利结果，该实验室将在大约一年后关闭。

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. The cellular functions of Ran are mediated by RanGTP 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 directionality to the Ran-regulated transport of cargos carried by NTRs through nuclear pore complexes. During mitosis, chromosomes are surrounded by a steep diffusional gradient of RanGTP which continues to regulate the loading and unloading of cargos on NTRs in the mitotic cytoplasm. Because some mitotic regulators are at the same time NTR cargos, their activities or binding to mitotic structures are controlled by RanGTP, contributing to mitotic spindle assembly and chromosome segregation to daughter cells. Previously, we focused on the role of Ran in mitotic spindle assembly and our goal was to elucidate differences in the contribution of Ran to mitosis in cancer cells vs. normal cells. To that end, we developed methods for quantitative measurements of Ran function in mitotic cells using fluorescence lifetime imaging microscopy (FLIM) of FRET biosensors expressed in live cells. Using this approach, we found that the mitotic RanGTP gradients were more robust in rapidly proliferating normal and cancer cells. Furthermore, we showed that increased RCC1 expression and increased number of mitotic chromosomes in aneuploid cells were the two most important factors determining the steepness of mitotic RanGTP gradients. As a follow up to this study, which was published in 2013, we pursued two approaches. First, we set out to determine the mechanisms of chromosome gain-driven activation of mitotic RanGTP gradients. We hypothesized that the underlining mechanism depends on the biophysical properties of the chromosome-cytoplasm interface in mitotic cells and involves the increased number of chromosome binding sites for RCC1 and reduced diffusion in the center of mitotic cells with increased chromosome number. To test this hypothesis, we planned combining live cell measurements with computational modeling of the mitotic RanGTP gradient, in collaboration with the laboratory of Dr. David Odde at the University of Minnesota. Although we soon obtained the initial set of measurements and developed the mitotic models, the progress was interrupted by the departure of Dr. Hasegawa from NIH at the end of 2013. In the second approach, we studied the role of Ran in the regulation of cell cycle. In the first phase of this project we analyzed role of Ran in the exit from cell cycle in senescent cells. We found that, as a result of irreversible cytoplasmic processing of chromatin, the permanent G1/S arrest in senescent cells was associated with a strong decline of RCC1 protein levels. That result was consistent with our initial hypothesis that the depletion of RanGTP levels enforces the stable exit from cell cycle via reducing the Ran-regulated nuclear-cytoplasmic transport of cell cycle factors. Unfortunately, the progress of this project, which was carried by Dr. Ryu in the lab, was disrupted by serious technical setback that prevented him to obtain definitive results before leaving the lab for a new job at the end of September 2013. As a result, both main projects of the lab were severely disrupted and unpublished at the end of 2013, about half a year before the Site Visit of the lab in June 2014. Dr. Cekan, the remaining recently hired postdoctoral researcher, and I have focused on the cell cycle project. We obtained evidence that RCC1-driven activation of Ran accelerates the reentry to cell cycle following DNA damage, leading to the evasion of cell senescence in normal and cancer cells. The underlining mechanism involved the activation of DNA damage repair, required exportin 1 function and involved the RCC1 expression-induced activation of prominent oncogenic transcriptional regulators. A manuscript describing these results will be submitted for publication in a few days. However, as a result of the unfavorable outcome of the Site Visit 2014 review, the lab will be closing in about a year from now.