A wide variety of cell types delay cell cycle transitions until they reach a critical size threshold, but the mechanisms that measure size and transmit this information to the core cell cycle machinery are largely unknown. Conserved cell cycle regulators form two discrete populations of large, multi-protein structures called “nodes” at the cortex of fission yeast cells. One population of nodes contains the protein kinases Cdr2, Cdr1, and Wee1, which function in a linear, genetically defined pathway to regulate mitotic entry. We discovered a second population of nodes that contain Skb1, which inhibits mitotic entry by binding to Cdr1 and Wee1. Node assembly is required for control of cell size at division, but we do not know the mechanisms of assembly or signal transduction within nodes. We will address key open questions using powerful genetic, biochemical, and quantitative imaging approaches. We will focus on the fundamental process of cell cycle regulation, but our work has broad implications for spatial control of signal transduction because higher-order clusters and node-like structures are emerging as critical sites of signal transduction throughout cell biology. The specific aims of this grant are to: (1) define the molecular mechanism by which Cdr2 organizes nodes and transmits cell size signals, (2) test the hypothesis that node function and organization respond to environmental changes, and (3) determine how Skb1 nodes inhibit mitotic entry by signaling to Cdr2-Cdr1-Wee1 nodes. Successful completion of these goals will advance scientific knowledge by identifying how protein clustering in nodes coordinates cell growth and division. Moreover, the signaling mechanisms that we uncover will provide insights for how size controls the activity of other biological systems.