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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质粒分别转染COS细胞和海马脑细胞，用冷CCD摄像机采集延时图像。秋水仙素处理引起微管断裂，但不影响随后GFP-GR在细胞核内的积聚，格尔德霉素阻断后，GR和MR从胞质向细胞核的转位受到抑制，表明HSP 90是肾上腺皮质激素受体向细胞核转运所必需的。CFP-GR转运至细胞核，与此相应，YFP-importin a也从细胞质转运至细胞核，提示importin a/B系统参与GR的核转运。为了研究GR和MR是否共定位于细胞核内，我们用共聚焦显微镜分析了这两种受体的亚核定位。两种受体在30 min时出现部分共定位，60 min时几乎完全共定位 ...更多信息洋蓟甾酮观察到GR和MR之间发生FRET。FRAP分析表明，胞浆GR是动态的，核GR也是移动的，但其移动性不同。在雌二醇处理之前，YFP-ER α和CFP-ER β均显示弥漫性分布于整个细胞核，但被排除在核仁之外。在配体处理后，同一细胞中的YFP-ER α和CFP-ER β重新定位以显示离散模式。这些离散簇的形成在10分钟内出现，并在30分钟内达到最大值，它们位于同一个离散簇上，表明两种亚型的ER结合在同一个核位点上。在不存在配体的情况下，GFP-ER的扩散分布和BRg-1的离散分布之间存在任何显著关系。然而，在雌二醇的存在下，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。MAP激酶抑制剂PD 98059不能阻断AR从胞浆向胞核的转运，提示MAP激酶不直接参与AR的转运。核受体及其配体在细胞发育阶段就相互作用，利用GFP对细胞核受体及其转录调节因子的可视化，提供了许多生物化学方法无法检测到的结果。少