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 相机拍摄延时图像。秋水仙碱处理导致微管破裂，但这并不影响随后 GFP 的核积累。 -GR。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对于转运是必需的。肾上腺皮质类固醇受体 CFP-GR 转移到细胞核，相应地，YFP-importin a 也从细胞质转移到细胞核，这表明 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，两种受体在 30 分钟时显示出部分共定位，并且几乎完全共定位。添加c… More articosterone后，观察到GR和MR之间发生FRET，结果显示细胞质GR非常动态，细胞核GR也具有移动性，但其移动性与雌二醇治疗前不同。 YFP-ER alfa 和 CFP-ER beta 显示在整个细胞核中弥漫分布，但在配体处理后，同一细胞中的 YFP-ER alfa 和 CFP-ER bata 被排除在外。重新定位以显示离散模式。这些离散簇形成在 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上，这表明 ER 的两种亚型都结合在相同的核位点上。没有配体时，GFP-ER 的弥散分布与 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 的离散染色模式没有显着关系。 GFP-ER alfa nad bata 大部分与 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 PD98059 处理不会阻断 AR 从细胞质到细胞核的转运。 MAPkinase 不直接参与 AR 易位。核受体及其配体在细胞阶段以及核受体和转录的可视化过程中相互作用。通过使用 GFP 在 libing 细胞中调节调节剂提供了许多生化方法无法检测到的发现。