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Real-time imaging of steroid hormone receptor dynamics in living cells with GFP

使用 GFP 对活细胞中类固醇激素受体动态进行实时成像

基本信息

批准号：
10680748
负责人：
NISHI Mayumi
金额：
$ 2.18万
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 (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680748/
关键词：
steroid hormone receptor transcription factor primary neural culture GFP fusion protein real time imaging CCD camera nuclear translocation

项目摘要

Corticosteroids exert markedly diverse effects on development, differentiation, aging and regeneration in the nervous system. These effects are mediated via two receptor system in the brain, glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). The expression of GR occurs in most neurons and glial cells throughout the brain, while MRs are localized mainly in the hippocampus. Although it is mostly accepted that GR is translocated into the nucleus from the cytoplasm only after ligand binding, the subcellular localization of MR is still quite controversial. To elucidate the intracellular trafficking of GR and MR in response to various conditioned in living cells, a chimera construct of a green fluorescent protein (GFP) and rat GR (GFP-GR) and MR (GFP-MR) were prepared. We transiently transfected the chimera construct into primary cultured rat hippocampal neurons, cortical glial cells, and non-neural cells such as COS-1 cells, and examined dynamic changes in subcellular localiz … More ation of GFP-GR and GFP-MR in these cells. GFP-GR was predominantly localized in the cytoplasm in the absence of ligand, while GRP-MR was distributed in both cytoplasm and nucleus in the majority of the transfected cells without ligand. Corticosterone (CORT), the principle corticosteroid of the rat, induced a rapid nuclear accumulation of GFP-GR and GFP-MR in all cell types used. Furthermore, we investigated the differential effects of CORT on the nuclear translocation pattern of corticosteroid receptors in single living cells co-transfected with cyan fluorescent protein (CFP)-GR and yellow fluorescent protein (YFP)-MR.Our findings clearly showed that the subcellular distributions of GFP-GR and GFP-MR are dynamically changed in response to extracellular and intracellular conditions, and that there are no conspicuous variations in the manner of trafficking of GFP-GR and GFP-MR between neural cells and non-neural cells. We also demonstrated that CORT-induced nuclear accumulation of CFP-GR and YFP-MR exhibited differential time-course under different ligand concentrations and cell types. Less

皮质类固醇在神经系统的发育、分化、衰老和再生中发挥着显著的不同作用。这些作用是通过大脑中的糖皮质激素受体（GR）和矿皮质激素受体（MR）两种受体系统介导的。GR在全脑大多数神经元和神经胶质细胞中表达，而MRs主要局限于海马。虽然人们普遍认为GR只有在配体结合后才会从细胞质转运到细胞核中，但MR的亚细胞定位仍然存在很大争议。为了阐明GR和MR在活细胞中对各种条件的响应，我们构建了绿色荧光蛋白（GFP）与大鼠GR （GFP-GR）和MR （GFP-MR）的嵌合体。我们将嵌合体构建体瞬时转染到原代培养的大鼠海马神经元、皮质胶质细胞和非神经细胞（如COS-1细胞）中，观察其亚细胞定位的动态变化，以及GFP-GR和GFP-MR在这些细胞中的表达。在没有配体的转染细胞中，GFP-GR主要分布在细胞质中，而在没有配体的转染细胞中，大部分GRP-MR分布在细胞质和细胞核中。皮质酮（CORT）是大鼠的主要皮质类固醇，在所有使用的细胞类型中诱导GFP-GR和GFP-MR的快速核积累。此外，我们研究了CORT对共转染青色荧光蛋白(CFP)-GR和黄色荧光蛋白(YFP)-MR的单个活细胞中皮质类固醇受体核易位模式的差异影响。我们的研究结果清楚地表明，GFP-GR和GFP-MR的亚细胞分布随细胞外和细胞内条件的变化而动态变化，并且GFP-GR和GFP-MR在神经细胞和非神经细胞之间的运输方式没有明显变化。我们还证明，在不同的配体浓度和细胞类型下，cort诱导的CFP-GR和YFP-MR的核积累表现出不同的时间过程。少