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的细胞功能是通过RanGTP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梯度为ntr通过核孔复合物携带的ran调控的货物运输提供了方向性。在有丝分裂过程中，染色体被RanGTP的陡峭扩散梯度所包围，RanGTP继续调节有丝分裂细胞质中ntr上货物的装载和卸载。由于一些有丝分裂调节因子同时是NTR的货物，它们的活性或与有丝分裂结构的结合受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。之前，我们关注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。为此，我们开发了使用活细胞中表达的FRET生物传感器的荧光寿命成像显微镜（FLIM）定量测量有丝分裂细胞中Ran功能的方法。使用这种方法，我们发现有丝分裂的RanGTP梯度在快速增殖的正常细胞和癌细胞中更加强大。此外，我们发现非整倍体细胞中RCC1表达的增加和有丝分裂染色体数量的增加是决定有丝分裂RanGTP梯度陡峭度的两个最重要因素。作为2013年发表的这项研究的后续研究，我们采用了两种方法。首先，我们着手确定染色体增益驱动有丝分裂RanGTP梯度激活的机制。我们假设潜在的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，并涉及随着染色体数量的增加，RCC1的染色体结合位点数量增加，有丝分裂细胞中心的扩散减少。为了验证这一假设，我们计划与明尼苏达大学David Odde博士的实验室合作，将活细胞测量与有丝分裂RanGTP梯度的计算模型结合起来。我们执行了最初的测量集，并根据我们的目的修改了现有的计算模型。然而，在2013年期间，Hasegawa博士决定离开NIH，并将他未来的职业生涯重点放在大学水平的物理和生物物理学教学上。在成功找到工作后，长谷川博士接受了阿默斯特大学（Amherst University）的一个教学职位，并在2013年底离开了实验室，同时也离开了他未完成的项目。在第二种方法中，我们研究了Ran在细胞周期调节中的作用。在该项目的第一阶段，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，由于染色质不可逆的细胞质加工，衰老细胞中永久G1/S停滞与RCC1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水平的降低通过减少ran调控的细胞周期因子的核质转运来强制稳定退出细胞周期。这项研究主要由Ryu博士完成，实验室其他成员也提供了帮助。不幸的是，Ryu博士的进展受到严重技术问题的影响，使他无法在2013年9月底离职前完成这项工作。因此，在2014年6月实验室Site Visit之前的半年左右，实验室的两个主要项目都没有发表，也没有人员。不出所料，不利的Site Visit审查导致科学顾问委员会在2016年2月建议永久关闭该实验室。在剩下的时间里，我和最近聘用的博士后Cekan博士一起专注于细胞周期项目。我们获得的证据表明，rcc1驱动的Ran激活加速了DNA损伤后细胞周期的重新进入，导致正常细胞和癌细胞逃避细胞衰老。2014年，我们提交了一份描述这些结果的手稿（Nature Communications），但收到了负面的编辑反馈，表明需要对Ran在衰老中的功能进行机械洞察。2015年，由于医学原因，Cekan博士离开实验室三个月，我们在这方面的进展被放慢了。尽管如此，我们目前正在完成一份更新的手稿，以提交新的出版物。

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. We performed initial sets of measurements and modified the existing computational models for our purposes. However, during 2013 Dr. Hasegawa decided to leave NIH and focus his future career on teaching physics and biophysics at college level. After a successful job search, Dr. Hasegawa accepted a teaching position at Amherst University and left the lab, as well as his unfinished project 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. This research was performed primarily by Dr. Ryu, with the help of other members of the lab. Unfortunately, Dr. Ryu's progress was compromised by a serious technical problem that prevented him to complete this work before leaving for a new job at the end of September 2013. As a result, both mainprojects in the lab were left unpublished and unstaffed about half a year before the Site Visit of the lab in June 2014. Not surprisingly, the unfavorable Site Visit review led to the recommendation of the Board of Scientific Advisors to permanently close the lab in February 2016. In the remaining time, 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. In 2014, we submitted a manuscript describing those result for publication (Nature Communications) but received a negative editorial feedback that indicated a need for mechanistic insight into the Ran's function in senescence. In 2015, our progress to that end was slowed by three months absence of Dr. Cekan from the lab, for medical reasons. Nevertheless, we are currently finalizing an updated manuscript for a new submission for publication.