NUCLEAR ORGANIZATION & STEROID RECEPTORS FUNCTION

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。核受体是一个配体依赖性转录因子超家族，其作用机制已在分子和生物化学水平上研究了几十年。我们的方法，专注于核居民或易位受体（雌激素受体α，ER和雄激素受体，AR，分别），集中在细胞内的交通使用高分辨率光学显微镜。对于ER和关键的辅激活因子SRC-1，最近已经鉴定出在添加配体后直接的快速核内运动，其表明广泛的受体迁移性和与不溶性核结构的缔合，这取决于与受体结合的激动剂或拮抗剂、ATP水平以及令人惊讶的蛋白酶功能。 我们寻求利用NCMR来帮助我们在两个关键层面上推进我们的研究：1）使用视频多光子显微镜提高了细胞内运动的时间分辨率，2）使用ReAsH表位标记系统鉴定活细胞中的大量受体/辅调节剂，用于相关的高分辨率/优异的超微结构电子显微镜，3）用ReAsH系统标记DNA阵列靶向的受体和辅激活因子，并通过TEM观察染色质原纤维的配体依赖性凝聚/去凝聚。Mancini博士的研究工作正在继续进行，以光转化标记有各种四半胱氨酸基序并标记有ReAsH的核因子。到目前为止，还没有手稿被整理出来。我们正试图把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，并将它们送去进行光转换。

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.