Cellular functions of Ran GTPase

A. Ran的有丝分裂功能利用FRET传感器的定量荧光寿命成像显微镜（FLIM），我们研究了正常细胞和癌源细胞中有丝分裂RanGTP梯度的功能。我们成功地建立了一个功能时间相关单光子计数（TCSPC） FLIM系统（Becker-Hickl），该系统连接在蔡司710共聚焦显微镜上，具有2光子激发。为了方便在相对非常小的正常人成纤维细胞有丝分裂细胞中进行Ran梯度成像，我们利用非荧光猝灭受体sREACH开发了FLIM/FRET传感器。在我们的第一次测试中，这些传感器的性能明显优于我们之前最好的一代Ran传感器。使用新的Zeiss 710系统，我们成功地成像了活HeLa细胞末期核包膜形成过程中ran调控的进口β货物梯度的重组。这些结果证实了我们之前在野鸡蛋提取物中获得的数据，并提出了一个意想不到的RanGTP梯度在核膜形成中的作用的新模型。预计本研究将在3-4个月内完成并提交发表。我们研究了核转运系统的调节在复制诱导的细胞衰老和癌症的发生中所起的重要作用，两者都是本质上与年龄相关的现象。我们推测，虽然衰老细胞程序触发核转运系统的整体减少，但在永生癌细胞中可能发生相反的趋势。我们在年轻和年老的正常人成纤维细胞和Hela细胞系中获得的蛋白质水平和定位数据证实了我们的这部分预测。我们现在正在建立实验来检查我们的测试细胞系中的RanGTP梯度，并看看是否可以使用核运输系统的特定操作来加速或防止细胞衰老的发生。这是4-5个月前才开始的项目的早期阶段。

A. Mitotic functions of Ran Using quantitative fluorescence lifetime imaging microscopy (FLIM) of FRET sensors we investigate the functions of the mitotic RanGTP gradient in normal and cancer-derived cells. We succeeded in building a functional time correlated single photon counting (TCSPC) FLIM system (Becker-Hickl) which is attached to Zeiss 710 confocal microscope with 2 photon excitation. To facilitate the Ran gradient imaging in relatively very small normal human fibroblast mitotic cells, we developed FLIM/FRET sensor utilizing non-fluorescent quenching acceptor sREACH. In our first tests, these sensors significantly outperformed our previous best generation of Ran sensors. Using the new Zeiss 710 system we succeeded in imaging the reorganization of Ran-regulated importin beta cargo gradient during the formation of the nuclear envelope in telophase in live HeLa cells. These results confirmed our previous set of data obtained in X. laevis egg extracts and suggested an unexpected new model of RanGTP gradient role in nuclear envelope formation. We expect that this study will be completed and submitted for publication within 3-4 months. B. Ran in senescence and cancer We examine the the role of the regulation of nuclear transport system has important function in the replication-induced cell senescence vs. the onset of cancer, both being intrinsically age-related phenomena. We hypothesized that while senescent cell program triggers overall decrease of nuclear transport system, possibly an opposite trend takes place in immortalized cancer cells. Our data on protein levels and localization obtained with young and aged normal human fibroblasts and Hela cell lines confirm this part of our prediction. We are now setting up experiments to examine the RanGTP gradient in our test cell lines and to see whether specific manipulation of the nuclear transport system could be used to either accelerate or prevent the cell senescence onset. This is an early stage of the project that started only 4-5 months ago.