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TRANSCRIPTION FACTOR INTERACTIONS REVEALED BY FRET

FRET 揭示的转录因子相互作用

基本信息

批准号：
11358013
负责人：
KAWATA Mitsuhiro
金额：
$ 24.7万
依托单位：
KYOTO PREFECTURAL UNIVERSITY OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

GFP-GR and GFP-MR plasmid was transfected into COS cells and brain cells from the hippocampus and time-lapse images were aquired by a cooled CCD camera. Colchicine treatment caused the disruption of microtubule but this did not affect the subsequent nuclear accumulation of GFP-GR. After blocking by geldanamycin you can see the inhibition of translocation of GR as well as MR from the cytoplasm to the nucleus, indicating that HSP 90 is necessary for the transport of adrenal corticosteroid receptors to the nucleus. CFP-GR was translocated to the nucleus and in accordance with this, YFP-importin a also changed from the cytoplasm to the nucleus, suggesting that importin a/b system is involved in GR nuclear transport. In order to examine whether GR and MR are colocalized in the nucleus, we analyzed the subnuclear localization of these two receptors by confocal microscope. Two receptors showed a partial colocalization at 30 min and nearly complete colocalization at 60 min. after addition of c … More articosterone. The occurrence of FRET between GR and MR was observed. FRAP analysis showed that cytoplasmic GR was very dynamic and nuclear GR was also mobile, but its mobility is different. Before estradiol treatment, both YFP-ER alfa and CFP-ER beta showed a diffuse distribution throughout the nucleus but was excluded from nucleolus. Upon the ligand treatment YFP-ER alfa and CFP-ER bata in the same cell was relocalized to show discrete pattern. These discrete cluster formation was appeared within 10 min. and reached in maximum within 30 min. and they were localized at the same discrete cluster, suggesing that both subtypes of ERs were bound to the same nucleuar sites. In the absence of the ligand, any significant relationship between the diffuse distribution of GFP-ERs and the discrete distribution of BRg-1. In the presence of the estradiol, however, the discrete staining pattern of GFP-ER alfa nad bata were mostly overlapped with BRG-1, indicating that most of the ERs clusters are involved in the chromatin remodeling machinery. Treatment by MAP kinase inhibitor, PD98059 did not block AR transport from the cytoplasm to the nucleus, suggesting that MAPkinase is not directly involved in the AR translocation. Nuclear receptors and their ligands are interplaying each other at the stage of the cells and visualization of nuclear receptors and the trandcriptional regulators in libing cells by using GFPs provided a number of findings that can not be detected by biochemial methods. Less

将GFP-GR和GFP-MR质粒转染到COS细胞和海马脑细胞中，通过冷却CCD相机获取延时图像。秋水仙碱处理引起微管破坏，但不影响GFP-GR随后的核积累。格尔达霉素阻断后，可以看到GR和MR从细胞质向细胞核的易位受到抑制，这表明HSP 90对于肾上腺皮质类固醇受体向细胞核的运输是必需的。CFP-GR向细胞核转运，与此相对应，yfp -输入蛋白a也从细胞质向细胞核转运，提示输入蛋白a/b系统参与了GR核转运。为了检验GR和MR是否在细胞核内共定位，我们用共聚焦显微镜分析了这两种受体的亚核定位。两个受体在加入c…More articosterone后30 min出现部分共定位，60 min几乎完全共定位。观察GR和MR之间FRET的发生情况。FRAP分析表明，胞质GR是动态的，细胞核GR也是可移动的，但其可移动性不同。在雌二醇治疗前，YFP-ER α和CFP-ER β均在细胞核内弥漫性分布，但核仁内不存在。在配体处理后，同一细胞中的YFP-ER α和CFP-ER β重新定位，呈现离散模式。这些离散簇的形成在10分钟内出现，在30分钟内达到最大值，并且它们定位在同一个离散簇上，这表明两种亚型的er结合在相同的核位点上。在没有配体的情况下，GFP-ERs的弥散分布与BRg-1的离散分布之间没有显著的关系。然而，在雌二醇存在的情况下，GFP-ER α和β的离散染色模式大多与BRG-1重叠，表明大多数er簇参与染色质重塑机制。经MAP激酶抑制剂处理后，PD98059没有阻断AR从细胞质到细胞核的转运，这表明map激酶不直接参与AR易位。核受体及其配体在细胞阶段相互作用，利用gfp可视化libing细胞中的核受体和转录调节因子提供了许多生物化学方法无法检测到的发现。少