TIME-RESOLVED STUDY OF PROTEIN:PROTEIN INTERACTIONS IN SINGLE CELLS

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们打算使用更有效的时间分辨技术来研究蛋白质：蛋白质相互作用的动力学。 此外，我们希望使用先进的成像技术来详细了解 FRET 中细胞中发生变化的位置。 我们的研究将继续主要集中于 IFN-γ 受体复合物，并扩展到 IL-10 和干扰素-γ (IFN-g) 受体复合物。 IFN-g受体复合物尤其重要，因为已提出其与IFN-g受体复合物的串扰和生物协同作用。 IFN-g 是一组称为 I 型 IFN 的蛋白质的代表，这些蛋白质在受到病毒感染时由细胞释放，并起到保护非造血细胞免受病毒感染的作用。 具体目标是： 1. 研究活细胞中受体链相互作用的动态。 2. 研究活细胞中Jak激酶与受体链相互作用的动态。 3. 研究受体运动、Jak激酶运动和Stat运动的动力学。 4. 分析受体复合物与配体结合时的相互作用。 此前，我们打算使用荧光共振能量转移（FRET）来确定活细胞天然环境中干扰素-γ（IFN-g）受体复合物的总体结构。 我们采用这种直接方法是因为从自然环境之外的受体复合物研究中获得了相互矛盾的结论。 区域激光生物技术实验室的系统设置非常适合 FRET 显微镜：共焦显微镜与单色仪耦合，无需任何额外的操作即可对生物样品进行频率分辨荧光测量。 频率分辨测量对于明确建立荧光发射中的 FRET 至关重要。 阐明真实细胞中存在的 IFN-g 信号转导级联的动力学对于表征 IFN-g 信号传导抑制剂或激活剂非常重要，这些抑制剂或激活剂可用于调节癌症、自身免疫性疾病、病毒性疾病、组织移植排斥和其他病例中的免疫系统。 我们跟踪了 IFN-g 受体复合物激活 Stat1 的动力学，发现该动力学非常不寻常：在配体结合几乎瞬时发生的条件下，除非 IFN-gR1 链或 IFN-gR2 链过量，否则 Stat1 激活存在潜伏期。 先前发表的观察结果支持了对该潜伏期的一种解释，即两个配体结合的 IFN-g 受体复合物正在相互作用。 实时荧光技术的使用对于建立受体激活和运动的动力学过程将很有价值。

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. We intend to use techniques that are more efficiently time-resolved to study the dynamics of protein:protein interactions. Additionally, we want to use advanced imaging techniques to detail where in the cell changes in FRET are occurring. Our studies will continue to focus primarily on the IFN-gamma receptor complex, with extensions into the IL-10 and interferon-gamma (IFN-g) receptor complexes. The IFN-g receptor complex is especially important as its crosstalk and biological synergy with the IFN-g receptor complex has been suggested. IFN-g is representative of a group of proteins called Type I IFNs that are released by cells when infected by virus, and function to protect nonhematopoietic cells from virus infection. The specific aims are: 1. To investigate the dynamics of receptor chain interactions in live cells. 2. To investigate the dynamics of interactions of Jak kinases with receptor chains in live cells. 3. To investigate the kinetics of receptor movements, Jak kinase movements and Stat movements. 4. To analyze the interactions among receptor complexes when bound by ligand. Previously we intended to use Fluorescence Resonance Energy Transfer (FRET) to ascertain the gross structure of the interferon-gamma (IFN-g) receptor complex in its native environment in live cells. We employed this direct approach because conflicting conclusions were obtained from studies of the receptor complex outside its natural environment. The system setup at the Regional Laser Biotechnology Laboratory is ideally suited for FRET microscopy: a confocal microscope is coupled to a monochromator, allowing frequency-resolved fluorescence measurements to be done on biological samples without any additional manipulations. Frequency-resolved measurements are essential in the unequivocal establishment of FRET in fluorescence emission. The elucidation of the kinetics of the IFN-g signal transduction cascade as it exists in real cells will be of great importance in the characterization of inhibitors or activators of IFN-g signaling that could have therapeutic use in modulating the immune system in cancer, autoimmune disease, viral disease, tissue transplant rejection, and other cases. We followed the kinetics of Stat1 activation by the IFN-g receptor complex and found that the kinetics were very unusual: under conditions where ligand-binding occurs almost instantaneously, a latency period in the activation of Stat1 exists unless either the IFN-gR1 chain or the IFN-gR2 chain is in excess. One explanation of this latency period that is supported by previously published observations is that two ligand-bound IFN-g receptor complexes are interacting. The use of real-time fluorescent techniques will be valuable in establishing the kinetic process of receptor activation and movement.