NUCLEAR ORGANIZATION & STEROID RECEPTORS FUNCTION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。核受体是一类大型的超家族配体依赖性转录因子，其功能机制已经在分子和生化水平上被研究了几十年。我们的方法，聚焦于核驻留或易位受体（雌激素受体α， ER和雄激素受体，AR分别），集中在细胞内运输使用高分辨率光学显微镜。对于内质网和一个关键的辅激活因子SRC-1，最近发现了配体加入后的核内快速运动，这表明受体的大范围移动和与不溶性核结构的关联取决于受体的激动剂或拮抗剂结合、ATP水平，以及令人惊讶的蛋白酶功能。我们寻求利用NCMIR来帮助我们在两个关键层面上推进我们的研究：1)利用多光子显微镜提高核内运动的时间分辨率，2)利用ReAsH表位标记系统鉴定活细胞中大量受体/共调节因子，获得相关的高分辨率/优秀超微结构电镜，3)利用ReAsH系统标记DNA阵列靶向受体和共激活因子，并通过TEM观察配体依赖性染色质原纤维的缩合/脱脱。Mancini博士的研究工作正在继续进行，以光转化带有各种四胱氨酸基元标记的核因子，并标记有ReAsH。到目前为止，还没有整理出手稿。我们正试图将大部分工作转移到曼奇尼的实验室。他将准备转染的细胞，用ReAsH标记它们，然后将它们送过去进行光转化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nuclear receptors are a large super family ligand-dependent transcription factors, whose mechanism(s) of function have been studied at the molecular and biochemical level for decades. Our approach, focusing upon nuclear resident or translocating receptors (estrogen receptor alpha, ER, and androgen receptor, AR, respectively), has centered upon the intracellular trafficking using high-resolution light microscopy. For ER and a key coactivator, SRC-1, have recently identified rapid intranuclear movements directly following addition of ligand that indicate a wide range of receptor mobilities and association with insoluble nuclear structure dependent upon the agonist or antagonist binding to the receptor, ATP levels, and, suprisingly, proteaomse function. We seek to utilize the NCMIR to aid us in furthing our studies at two key levels: 1) improved time resolution of intranculear movements using the videorate multiphoton microscope, 2)use the ReAsH epitope-labeling system to identify bulk receptor/coregulators in live cells for correlative high res/excellent ultrastructure electron microscopy, and 3) use the ReAsH system to label DNA array targeted receptors and coactivators and observe ligand dependent condensation/decondesation of chromatin fibrils by TEM. The research work is continuing with Dr. Mancini to photoconvert nuclear factors tagged with various tetracysteine motifs and labeled with ReAsH. So far, no manuscript has been put together. We are trying to transfer most of the work to Mancini's laboratory. He will prepare transfected cells, labeled them with ReAsH and send them over for photoconversion.