Core C: Cell Phenotyping and Molecular Imaging Core

项目总结 该计划拨款提案的目标是更好地从机制上理解核心Cyclin D-CDK4/6-Rb- E2F途径调控G1/S过渡，控制细胞分裂。该计划的首要假设是 增加对G1/S控制机制的理解将在 了解人类细胞分裂的控制，以促进新的癌症疗法的开发。我们的 该项目的目标都是使用生物化学、遗传学和质谱学来生成分子假说 细胞周期蛋白D-CDK4/6-Rb-E2F途径相互作用的调控机制为了在体内检验这些假说， 所有项目都将依赖于细胞表型和分子成像核心。成像是测试的一项关键技术 活细胞的分子机制。特别是，时间推移单细胞分析是评估的黄金标准 突变对G1/S期细胞周期进程的影响除了时间推移成像，核心C 将提供对荧光互相关光谱方法(FCS和FCC)的访问，这些方法允许 用摩尔单位测量蛋白质浓度，并测量蛋白质的离解常数 活细胞中的特定蛋白质(例如，Rb和E2F)。这些能力将允许对生物化学进行最终测试 我们体外和遗传学研究产生的沿Cyclin D-CDK4/6-Rb-E2F轴的调节机制 在每个项目中。为了服务于这些项目，这个核心的目标是1)使用CRIPSR/Cas9基因组编辑来产生突变 单细胞分辨率下的图像基因表达、细胞生长和细胞周期转变动力学；以及 3)构建和维护分析TB级延时成像数据的软件。这些细胞表型 分析将反馈和完善我们的机械假说，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.