Cellular responses to internal and external stimuli are orchestrated by intricate intracellular signaling pathways. To ensure an efficient and specific information flow, cells employ scaffold proteins as critical signaling organizers. These proteins have the ability to bind multiple signaling molecules, sequester signaling components within specific subcellular domains and modulate the efficiency of signal transduction. Scaffold proteins also serve as regulatory targets, finetuning the output of signaling pathways. In our research, we have identified that the substrate specificity and activity of the GLYCOGEN SYNTHASE KINASE 3-like kinase, BRASSINOSTEROID-INSENSITIVE 2 (BIN2), in the stomatal lineage is regulated by the scaffold protein POLAR and its close homolog POLAR-LIKE1 (PL1). These scaffold proteins are specifically expressed in stomatal precursors and are polarly localized before the asymmetric cell division. Preliminary data suggest that BIN2, POLAR and PL1 form a complex. Additionally, PL1 has been observed to undergo phase separation in an ATP-dependent manner, and putative ATP-binding domains have been identified in both POLAR and PL1. In this project, we will combine cryo-electron microscopy with biochemical, genetic and high-resolution live-cell imaging approaches to obtain structural information about the BIN2-POLAR-PL1 complex and to unravel the molecular mechanisms underlying BIN2 activity regulation by the scaffold proteins in stomatal lineage.