Time and spacial analyses for mechanisms of intracellular traffic and localization of nuclear receptors using imaging analyses including FRET

1 .两例雄激素不敏感综合征（AIS）患者突变雄激素受体（AR）细胞内动力学分析。使用gfp融合突变ARs分析了来自AIS患者的两种不同AR突变（AR- c579f, AR- f582y）的细胞内动力学。野生型AR依赖于配体转移到细胞核中，形成精细的亚核病灶。令人惊讶的是，在加入双氢睾酮（DHT）后，两个突变ar最初形成了大的细胞质点，其中许多在电镜下被发现靠近线粒体。随后，与野生型AR相比，一部分配体结合的突变型AR逐渐进入细胞核，形成数量更少的大点。光漂白后荧光恢复（FRAP）分析显示，突变型AR的核内迁移率与野生型AR相比降低。在这些受试者中，更多的突变ARs可能是AIS的原因。对新型共阻遏物TZF和TobWe转录抑制机制的分析表明，睾丸锌指蛋白TZF是AR的新型共阻遏物。TZF依赖于配体，与AR共定位，并与AR的n端相互作用。TZF的中心部分（512-663氨基酸）TZF（512-663）既与AR结合，又抑制AR的交互激活。TZF招募内源性组蛋白去乙酰化酶2 (HDAC2)，并与激动剂结合的AR形成复合物。影像学分析显示，TZF和TZF（512-663）被AR招募，同时损害不同AR灶的形成。利用三维成像方法量化病灶数，发现核内AR病灶的数量与其交互激活活性有关。此外，TZF水平的增加使辅激活因子TIF2从AR灶中分离出来，反之亦然。这些结果表明，AR的配体依赖的转激活功能在数量上与其核内病灶的形成有关，并表明TZF等共抑制因子与共激活因子竞争性地作用于这些核内事件。我们还发现Tob被认为是成骨细胞功能的负调节因子，对AR、ER和GR的交互激活功能的抑制作用较小

I. Analyses of intracellular dynamics of mutant androgen receptors (AR) derived from two androgen insensitivity syndrome (AIS) patients.Intracellular dynamics of two different AR mutants (AR-C579F, AR-F582Y) derived from AIS patients were analysed using GFP-fused mutant ARs. The wild-type AR was ligand-dependently translocated into the nucleus where it formed fine subnuclear foci. Surprisingly, after the addition of dihydrotestosterone (DHT), the two mutant ARs initially formed large cytoplasmic dots, many of which were found to be close to mitochondria by electron microscopy. Subsequently, a proportion of the ligand-bound mutant ARs gradually entered the nucleus to form a smaller number of larger dots compared with the wild-type AR. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the intranuclear mobilities of the mutant ARs were decreased compared with that of the wild-type AR. These results suggest that the abnormal translocation, localization and mobility o … More f the mutant ARs may be the cause of AIS in these subjects.II. Analyses of transcriptional repression mechanisms by novel corepressors, TZF and TobWe showed that testicular zinc finger protein, TZF, was a novel corepressor for AR. TZF was ligand-dependently colocalized with AR and interacted with the N-terminus of AR. A central portion (amino acids 512-663) of TZF, TZF(512-663), is responsible for both binding to AR and repressing the transactivation. TZF recruited endogenous histone deacetylase 2 (HDAC2) and formed a complex with agonist-bound AR. Imaging analyses showed that TZF and TZF(512-663) were recruited by AR and simultaneously impaired distinct AR foci formation. Quantification of the foci number using a three-dimensional imaging method revealed that the number of intranuclear AR foci was related to its transactivation activity. Moreover, increased levels of TZF dissociated a coactivator, TIF2, from the AR foci and vice versa. These results indicate that the ligand-dependent transactivation function of AR is quantitatively related to its intranuclear foci formation, and suggest that corepressors, such as TZF, act on these intranuclear events competitively with coactivators.We also found that Tob, which was regarded as a negative regulator of osteoblast function, repressed transactivation function of AR, ER and GR. Less