Project Summary/Abstract: Cytokinesis is fundamental to life, and the septins form cytoskeletal scaffolds or diffusion barriers of distinct architectures to impact diverse cellular functions including cytokinesis, cell migration, ciliogenesis, dendritic spine morphogenesis, spermiogenesis, and bacterial infection. Defects in cytokinesis or septins cause lethality or serious diseases including cancer, infertility, hereditary neuropathy, and neurodegenerative diseases such as Alzheimer's and Parkinson's. Thus, understanding cytokinesis and the septin cytoskeleton is critically important not only for basic science but also for public health. In this application, we will address the core issues regarding cytokinesis including the architecture and constriction mechanism of the actomyosin ring (AMR) as well as the mechanism of vesicle fusion at the division site. We will also address the key questions in the septin field including how a high-order structure such as the septin ring or hourglass is assembled and then remodeled into a distinct architecture for a distinct function. These questions will be addressed chiefly using the budding yeast model due to the stereotyped behaviors of cellular and molecular events involved in cytokinesis and septin architecture and remodeling during its cell cycle. Some of the key questions such as the architecture of the AMR, especially the organization of distinct myosin-II isoforms in the contractile ring, will also be investigated in fission yeast and mammalian cells to illustrate the mechanistic conservation and divergence during evolution. All questions will be addressed using an integrative approach that combines precise genetic editing, cell synchronization, AlphaFold2-based structural predictions, biochemical analysis, and cutting-edge imaging technologies including super-resolution microscopy, platinum-replica EM, and cryo-FIB-SEM/cryo-ET. We have made major contributions to the understanding of cytokinesis and the septin cytoskeleton. The proposed project is based on rigorous published and unpublished data, which sets the stage for testing specific hypotheses and exploring new directions. The completion of this project is expected to generate deep architectural and mechanistic insights into cytokinesis and septin assembly and remodeling.