Cellular functions of Ran GTPase

Ran GTPase 的细胞功能

• 批准号: 7966041
• 负责人: Petr Kalab
• 金额: $ 62.52万
• 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/7966041
• 关键词: Area; BRCA1 gene; Binding; Biosensor; CENP-E protein; Cell Aging; Cell Cycle; Cell Line; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromosomes; Cytoplasm; Data; Data Set; Development; Drug Delivery Systems; Embryo; Fibroblasts; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Generations; Genome; Guanosine Triphosphate Phosphohydrolases; HMMR gene; Hela Cells; Human; Image; Interphase; Life; Malignant Neoplasms; Mediating; Meiosis; Microscope; Microscopy; Mitosis; Mitotic; Mitotic spindle; Modeling; NPM1 gene; Nuclear; Nuclear Envelope; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Organism; Photons; Plants; Positioning Attribute; Proteins; Publications; Regulation; Research; Role; Running; Staging; System; Testing; Time; Undifferentiated; Work; age related; cancer cell; cancer cell differentiation; design; egg; human STK6 protein; macromolecule; normal aging; nucleocytoplasmic transport; prevent; programs; receptor; research study; senescence; sensor; survivin; telophase; trend

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.

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肿瘤来源细胞中有丝分裂的RanGTP梯度的功能。我们成功地建立了功能时间相关单光子计数(TCSPC)薄膜系统(Becker-Hickl)，该系统安装在蔡司710共焦显微镜上，采用双光子激发。为了便于在相对较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，这些传感器的性能远远超过了我们上一代最好的RAN传感器。使用新的Zeiss 710系统，我们成功地对活的HeLa细胞在末期核膜形成过程中RAN调控的导入蛋白β货物梯度的重组进行了成像。这些结果证实了我们之前在莱维氏X.laevis卵提取液中获得的一组数据，并提出了一种意想不到的RanGTP梯度在核膜形成中的作用模型。我们预计这项研究将在3-4个月内完成并提交出版。B.RAN在衰老和癌症中的作用我们研究了核运输系统的调节在复制诱导的细胞衰老和癌症的发生中具有重要作用，两者都是本质上与年龄相关的现象。我们推测，虽然衰老的细胞程序触发了核运输系统的全面下降，但永生化的癌细胞可能出现了相反的趋势。我们在年轻和老年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一部分预测。我们现在正在建立实验，以检查我们的测试细胞系中的RanGTP梯度，并看看是否可以使用对核运输系统的特定操作来加速或防止细胞衰老的开始。这是该项目的早期阶段，仅在4-5个月前开始。