PROJECT SUMMARY The overall goal of NIGMS-funded research in my lab is to define the molecular and cellular mechanisms that control cell size and shape. Defects in cell size and shape are associated with human diseases including cancer, so defining the underlying mechanisms can identify future therapeutic targets. We use the fission yeast S. pombe as a model system to study these fundamental processes. These rod-shaped eukaryotic cells grow by linear extension due to polarized secretion at growing cell tips, and enter mitosis at a highly reproducible size due to regulated activation of the ubiquitous cyclin-dependent kinase Cdk1. Decades of genetic screens have identified an extensive “parts list” for regulation of cell size and shape. Our current challenge is to assemble these parts into defined signaling networks that spatially control cell growth and activate Cdk1 in a size-dependent manner. For this work, we take a multidisciplinary approach that combines genetics, quantitative live-cell microscopy, phosphoproteomics, and biochemical reconstitution. In this proposal, we will address four key unanswered questions. First, how do cortical multiprotein clusters called “nodes” control fission yeast cell size at division? We discovered that nodes contain conserved cell cycle regulators including the protein kinases Cdr2, Cdr1, and Wee1, but we do not know the mechanisms of assembly or signal transduction within nodes. Second, what is the role of multiple cell cycle pathways in monitoring aspects of cell size such as volume and surface area? We will focus on the mitotic inducers Cdc25 and Cdc13/cyclin, with the goal of generating systems-level knowledge supported by mathematical modeling. Third, how do cell polarity mechanisms that function far away from the growing cell tips contribute to cell shape? We will exploit our recent discoveries that implicate RNA granules and SNARE protein clusters as novel “at-a-distance” regulators of cell polarity and shape. Fourth, how do cell size and shape influence spatial patterning of nodes in cells? We have identified cell tips, cortical anchors, and the nucleus as critical regulators of node positioning. We will combine genetic mutants with quantitative fluorescence microscopy and particle-based simulations to define the underlying design principles of this system. Based on extensive conservation of these pathways and processes between yeast and mammals, we fully expect that discoveries from our work will impact and guide future work in other organisms and biological systems.