PROJECT SUMMARY Generating diverse cell types from a limited number of progenitor cells in development is a significant challenge in multicellular organisms. Through asymmetric cell division (ACD), progenitor cells divide to renew themselves and create new cell types. A key process of ACD is the polarization of progenitor cells. Our research aims to elucidate design principles that govern cell polarization, divisional asymmetry, and the coordination of cell division and cell-fate determination in plant ACD. Stomata are microscopic pores in a plant's epidermis. In the model plant Arabidopsis, stomatal development and patterning provide an excellent platform for studying the molecular mechanisms underlying cell polarity- driven ACD in plants. The PI identified BASL (Breaking of Asymmetry in the Stomatal Lineages) as the first intrinsic polarity protein in Arabidopsis that controls stomatal ACD. BASL functions as a paralog of the conserved PAR proteins in metazoans. Our research, supported by the NIH since 2014, has made significant contributions to the current understanding of the molecular mechanisms underlying stomatal ACD. Specifically, we revealed that BASL functions as a scaffold protein that dynamically assembles specific signaling components required for each step of stomatal ACD. Before ACD, the BIN2 GSK3 (Glycogen synthase kinase)-like kinases are polarized to maintain a high cell-division potential. After ACD, a YODA MAPK (Mitogen-activated Protein Kinase) signaling cascade associated with the polarity module promotes cell-fate differentiation. Furthermore, we identified the BSL1 (bri1 Suppressor 1-like 1) phosphatase that, upon mitosis, becomes polarized to dissociate BIN2 while activating YODA at the plasma membrane. Thus, the BSL1 molecular switch enables the transition from cell division to cell-fate differentiation during stomatal ACD. Additionally, our group has determined that polarization of the peripheral membrane protein BASL requires vesicle trafficking regulated by the PRAF (PH, RCC1, and FYVE) endosomal proteins. PRAFs interact with GNOM, an activator of Arf GTPases that plays pivotal roles in endocytic recycling in plants. The discoveries of BSL1 and PRAF represent major milestones in our recent research and offer profound insights into the current understanding of molecular mechanisms underlying cell polarization and plant ACD. In the next five years, by employing a comprehensive approach combining genetics, biochemistry, advanced cell biology, and proteomics/phosphoproteomics, we aim to (1) understand how regulators of membrane trafficking control targeted vesicle delivery to establish the polarity domain at the plasma membrane, and (2) determine how cell-cycle regulation coordinates with the dynamic assembly of polarity proteins in stomatal ACD. Both directions emerge from our recent discoveries and represent significant areas of inquiry. In addition, we will implement and optimize the usage of TurboID and its variants to...