Core C: Cell Phenotyping and Molecular Imaging Core

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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.