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质粒转化为COS细胞和海马的脑细胞，而冷却的CCD摄像头需要延时图像。秋水仙碱治疗引起了微管的破坏，但这不会影响GFP-GR的随后核积累。在通过Geldanamycin封闭后，您可以看到抑制GR的翻译以及MR从细胞质到核的抑制作用，这表明HSP 90对于将肾上腺皮质类固醇受体转运到细胞核中是必不可少的。 CFP-gr被转化为核，据此，YFP-Irmentin A也从细胞质变为细胞核，这表明Importin A/B系统参与GR核转运。为了检查GR和MR是否在核中共定位，我们通过共聚焦显微镜分析了这两个受体的亚核定位。两个受体在30分钟时显示部分共定位，在60分钟时几乎完全共定位。添加了C…更多的关轴酮。观察到GR和MR之间的FRET发生。 FRAP分析表明，细胞质GR非常动态，核GR也是可移动的，但其迁移率不同。在雌二醇治疗之前，YFP-ER AlfA和CFP-ERβ均显示出整个核的弥漫性分布，但被排除在核olus中。在配体处理后，将同一细胞中的YFP-ER Alfa和CFP-ER BATA重新定位以显示离散的模式。这些离散的簇形成在10分钟内出现。并在30分钟内最多达到。它们位于同一离散集群中，这表明ER的两个亚型都与同一核位点结合。在没有配体的情况下，GFP-ers的扩散分布与BRG-1的离散分布之间的任何显着关系。然而，在雌二醇的存在下，GFP-ER Alfa NAD BATA的离散染色模式主要与BRG-1重叠，这表明大多数ERS簇都参与染色质重塑机械。通过MAP激酶抑制剂的处理，PD98059并未阻止AR从细胞质到细胞核的转运，这表明MAPKINASE与AR易位无直接参与。核接收器及其配体在细胞的阶段相互互动，核接收器的可视化和使用GFP的转录调节剂的可视化提供了许多无法通过生化方法检测到的发现。较少的