NUCLEAR ORGANIZATION & STEROID RECEPTORS FUNCTION

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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.