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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，并通过冷却 CCD 相机获取延时图像。秋水仙碱处理导致微管破坏，但这并不影响随后 GFP-GR 的核积累。用格尔德霉素封闭后可以看到GR和MR从细胞质到细胞核的易位受到抑制，表明HSP 90对于肾上腺皮质类固醇受体转运到细胞核是必需的。 CFP-GR 易位至细胞核，相应地，YFP-importin a 也从细胞质转移至细胞核，表明 importin a/b 系统参与了 GR 的核转运。为了检查GR和MR是否在细胞核中共定位，我们通过共聚焦显微镜分析了这两种受体的亚核定位。两个受体在 30 分钟时表现出部分共定位，在 60 分钟时表现出几乎完全共定位。添加c … More 洋蓟酮后。观察GR和MR之间FRET的发生。 FRAP分析表明，细胞质GR非常动态，核GR也具有移动性，但其移动性不同。在雌二醇治疗之前，YFP-ER alfa 和 CFP-ER beta 均显示出整个细胞核的弥漫分布，但被排除在核仁之外。配体处理后，同一细胞中的 YFP-ER alfa 和 CFP-ER bata 重新定位以显示离散模式。这些离散的簇形成在 10 分钟内出现。并在30分钟内达到最大值。并且它们位于相同的离散簇，表明两种 ER 亚型都与相同的核位点结合。在没有配体的情况下，GFP-ER 的弥散分布和 BRg-1 的离散分布之间存在任何显着关系。然而，在雌二醇存在的情况下，GFP-ER alfa nad bata 的离散染色模式大部分与 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 PD98059 的治疗并没有阻断 AR 从细胞质到细胞核的转运，表明 MAP 激酶并不直接参与 AR 易位。核受体及其配体在细胞阶段相互作用，通过使用 GFP 对解放细胞中的核受体和转录调节因子进行可视化，提供了许多生化方法无法检测到的发现。较少的