Core C: Cell Phenotyping and Molecular Imaging Core

核心 C：细胞表型和分子成像核心

• 批准号: 10332385
• 负责人: Jan M Skotheim
• 金额: $ 27.65万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-25 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10332385
• 关键词: Applications Grants; Biochemical; Biochemistry; CRISPR/Cas technology; Cell Cycle; Cell Cycle Progression; Cell Line; Cell division; Cells; Cellular Assay; Communities; Computer software; Cyclin D1; Data; Development; Dissociation; Dyes; Feedback; Fluorescence; G1/S Transition; Gene Expression; Generations; Genes; Genetic; Genetic study; Goals; Gold; Human; Image; Image Analysis; In Vitro; Laboratories; Ligation; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methods; Microscope; Microscopy; Mitotic Cell Cycle; Molecular; Mutate; Mutation; Pathway interactions; Phenotype; Phosphorylation Site; Process; Protein Overexpression; Proteins; Regulation; Research Assistant; Resolution; Rest; Science; Site; Spectrum Analysis; Structure of thyroid parafollicular cell; System; Technology; Testing; Therapeutic; Time; cancer cell; digital repositories; experience; experimental study; genome editing; imaging facilities; imaging software; improved; in vivo; interest; live cell imaging; molecular imaging; mutant; novel; programs; protein protein interaction; single cell analysis; terabyte

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.

项目概要 该计划拨款提案的目标是更好地理解核心 Cyclin D-Cdk4/6-Rb- E2F 通路调节 G1/S 转变控制细胞分裂。该计划的总体假设 增加对 G1/S 控制的机械理解将带来根本性的进步 了解人类细胞分裂的控制，以促进新型癌症疗法的开发。我们的 项目目标全部使用生物化学、遗传学和质谱来生成分子假设 控制 Cyclin D-Cdk4/6-Rb-E2F 通路中相互作用的机制。为了在体内测试这些假设， 所有项目都将依赖于细胞表型分析和分子成像核心。成像是检测的关键技术 活细胞中的分子机制。特别是，延时单细胞分析是评估的黄金标准 突变对 G1/S 期细胞周期进展的影响。除了延时成像之外，Core C 将提供荧光互相关光谱方法（FCS 和 FCCS），允许 以摩尔单位测量蛋白质浓度，以及测量解离常数 活细胞中的特定蛋白质（例如，Rb 和 E2F）。这些功能将允许对生化进行明确的测试 我们的体外和遗传学研究产生的 Cyclin D-Cdk4/6-Rb-E2F 轴调节机制 在每个项目中。为了服务于该项目，该核心目标是 1) 使用 CRIPSR/Cas9 基因组编辑来生成突变体 细胞系； 2) 以单细胞分辨率对基因表达、细胞生长和细胞周期转变动态进行成像；和 3) 构建和维护用于分析 TB 级延时成像数据的软件。这些细胞表型 分析将反馈并完善我们的机制假设，我们预计这将导致进一步的基因组研究 编辑和测试。