Cytokinesis is essential for development and survival of all organisms. Defects in cytokinesis cause aneuploidy and genomic instability, and thereby contribute to serious diseases such as cancer, neuronal disorders, and anemia. Thus, mechanistic study of cytokinesis is important not only for understanding the basic principles of a fundamental process but also for designing new strategies to treat human diseases. Cytokinesis in animal and fungal cells requires concerted functions of a contractile actomyosin ring (AMR), targeted vesicle fusion, and localized extracellular matrix (ECM) remodeling. Despite extensive studies of cytokinesis over a century, the basic architecture of the AMR remains unknown in any system. It is also unclear how myosin-II localization and filament assembly are regulated during cytokinesis. Increasing evidence suggests that ECM remodeling is critical for cytokinesis not only for yeast cells but also for animal cells. However, the underlying mechanisms remain poorly understood. We propose to address these key questions in cytokinesis using both budding yeast and mammalian cells as our experimental models. In Aim 1, we will determine the AMR structure in budding yeast and then develop a quantitative model for it. This model will open new avenues to address broad questions in cytokinesis, e.g. those regarding the mechanism of ring constriction as well as those concerning the assembly, regulation, and function of the ring. We will also examine the AMR in mammalian cells to establish the degree of conservation in this core cytokinetic structure. In Aim 2, we will test our hypothesis that IQGAP functions as a dual regulator of myosin localization and filament assembly during cytokinesis in both budding yeast and mammalian cells. In Aim 3, we will test our hypothesis that the AMR-associated protein Inn1 interacts with SNAREs via its C2 domain to facilitate vesicle fusion at the division site, thereby increasing the local concentration of the cargo enzyme Chs2 (chitin synthase-II), which is subsequently activated by Cyk3 via its transglutaminase-like domain to promote septum formation (equivalent of ECM remodeling in animal cells) during cytokinesis. The proposed research is innovative, as diverse and cutting-edge technologies will be applied to generate new information and concepts regarding the core mechanisms of cytokinesis.