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.

项目摘要 该计划赠款提案的目标是对核心细胞周期蛋白D-CDK4/6-RB-的更好理解 E2F途径调节G1/S转变控制细胞分裂。该计划的总体假设 是，对G1/S控制的机械理解越来越多将提供基本进步 了解人类细胞分裂的控制，以促进新型癌症治疗剂的发展。我们的 项目旨在使用生物化学，遗传学和质谱法来生成分子的假设 控制细胞周期蛋白D-CDK4/6-RB-E2F途径中相互作用的机制。为了在体内检验这些假设， 所有项目都将依靠细胞表型和分子成像核心。成像是测试的关键技术 活细胞中的分子机制。特别是，时间流失单细胞分析是评估的黄金标准 突变对G1/s转变下细胞周期进程的影响。除了时间衰变成像外，核心C 将提供访问荧光跨相关光谱法（FCS和FCC）的访问权限 测量摩尔单元中蛋白质浓度的测量，并测量的分离常数 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的明确测试 我们的体外和遗传研究产生的沿细胞周期蛋白D-CDK4/6-RB-E2F轴调节的机制 在每个项目中。为了服务项目，该核心的目的是1）使用CRIPSR/CAS9基因组编辑来产生突变体 细胞系； 2）单细胞分辨率下的图像基因表达，细胞生长和细胞周期过渡动力学；和 3）构建和维护软件，以分析时间衰变成像数据的trabytes。这些细胞表型 分析将反馈和完善我们的机械假设，我们期望这将导致进一步的基因组 编辑和测试。