PROJECT SUMMARY This proposal aims to determine how cell growth triggers cell division, which is a fundamental question in cell and developmental biology. Its understanding will also greatly impact our knowledge of cancer, where this process is misregulated. It has long been known that cell growth triggers human cell division at the G1/S transition before DNA is replicated. But, although many key regulatory proteins linking cell growth to cell division are known, the molecular mechanisms mammalian cells use to control their size have remained poorly understood and have been based solely on the study of cells growing in culture. My laboratory recently made a breakthrough advance in understanding how growth triggers division. Contrary to expectations that growth would increase Cyclin D-Cdk4,6 activity, we found instead that cell growth dilutes the cell cycle inhibitor Rb to trigger division in cultured cells. Our discovery of the Rb dilution mechanism in cell culture raises three key questions which are the focus of this grant: 1. What is the molecular mechanism regulating Rb’s concentration dynamics that control cell size? 2. What is the function of Rb-based cell size control? 3. Do Rb dilution or other cell size control mechanisms link cell growth to cell division in vivo. We have begun to address the first question and our preliminary data indicate that the mechanism regulating the size-dependence of Rb concentration is translational. To further determine how this molecular mechanism works we will take an approach using reporters to identify the DNA-sequence element responsible and then the corresponding proteins regulating its function. To address the second question, we will take a mass spectrometry-based approach to measure how protein concentrations change with cell size across the proteome. Preliminary data indicate that proteins associated with senescence phenotypes increase in concentration in large cells. This suggests that cell size may be causal for senescence and cell size control functions to avoid this deleterious outcome. To address the final question to definitively test the Rb dilution and alternative models, we will perform a series of in vivo experiments. This is important because recent studies in cell culture have reported conflicting results about how animal cells control their size. To determine how animal cell growth triggers cell division in vivo, we propose to examine the mouse epidermis because it has a large population of proliferating stem cells whose division dynamics can be assayed using live- cell imaging. We will measure changes in keratinocyte cell size in a series of mouse lines in which the Rb family of genes has been conditionally deleted or over-expressed in the mouse epidermis. This will test our central hypothesis that Rb1 is crucial for cell size control in vivo. We will also use mouse genetics to test the alternative hypothesis that the p38 stress activated protein kinase controls cell size. Taken togethe...