Core C: Cell Phenotyping and Molecular Imaging Core

项目摘要 该计划拨款提案的目标是更好地了解核心细胞周期蛋白D-Cdk 4/6-Rb的机制。 E2 F途径调节G1/S转换控制细胞分裂。该计划的首要假设是 对G1/S控制机制的进一步理解将为以下方面提供根本性的进展： 了解人类细胞分裂的控制，以促进新的癌症治疗方法的发展。我们 该项目旨在利用生物化学、遗传学和质谱分析法来产生分子生物学的假设。 在细胞周期蛋白D-Cdk 4/6-Rb-E2 F途径中控制相互作用的机制。为了在体内验证这些假设， 所有项目都将依靠细胞表型分析和分子成像核心。成像是测试的关键技术 活细胞中的分子机制。特别是，时间推移单细胞分析是评估 突变对G1/S期细胞周期进程的影响。除了延时成像，核心C 将提供荧光互相关光谱方法（FCS和FCCS）， 测量蛋白质浓度（摩尔单位），以及测量 活细胞中的特定蛋白质（例如，Rb和E2 F）。这些能力将允许对生物化学物质进行明确的测试， 通过我们的体外和遗传研究产生的沿着细胞周期蛋白D-Cdk 4/6-Rb-E2 F轴的调节机制 在每个项目中。为了服务于项目，该核心旨在1）使用CRIPSR/Cas9基因组编辑来产生突变体， 细胞系; 2）以单细胞分辨率成像基因表达、细胞生长和细胞周期转换动力学;以及 3)建立和维护软件，分析TB级的时间推移成像数据。这些细胞表型 分析将反馈和完善我们的机制假设，我们预计这将导致进一步的基因组 编辑和测试。

PROJECT SUMMARY The goal of this program grant proposal is a better mechanistic understanding of the core Cyclin D-Cdk4/6-Rb- E2F pathway regulating the G1/S transition controlling cell division. The overarching hypothesis of the program is that an increased mechanistic understanding of G1/S control will provide fundamental advances in understanding the control of human cell division to facilitate development of novel cancer therapeutics. Our project aims all use biochemistry, genetics, and mass spectrometry to generate hypotheses for molecular mechanisms controlling interactions in the Cyclin D-Cdk4/6-Rb-E2F pathway. To test these hypotheses in vivo, all projects will rely on the cell phenotyping and molecular imaging core. Imaging is a key technology for testing molecular mechanisms in live cells. In particular, time lapse single cell analysis is the gold standard for assessing effects of mutations on cell cycle progression at the G1/S transition. In addition to time lapse imaging, Core C will provide access to fluorescence cross correlation spectroscopy methods (FCS and FCCS) that allow the measurement of protein concentrations in molar units, and the measurement of the dissociation constants of specific proteins in live cells (e.g., Rb and E2F). These capabilities will allow definitive testing of biochemical mechanisms for regulation along the Cyclin D-Cdk4/6-Rb-E2F axis generated by our in vitro and genetic studies in each Project. To serve the Projects, this core aims to 1) use CRIPSR/Cas9 genome editing to generate mutant cell lines; 2) image gene expression, cell growth, and cell cycle transition dynamics at single cell resolution; and 3) build and maintain software that analyzes the terabytes of time lapse imaging data. These cell phenotyping analyses will feedback and refine our mechanistic hypotheses, which we expect will lead to further genome editing and testing.