Cytokinesis, the physical division of one cell into two, is accomplished by a transient organelle called the contractile ring. The PI is focused on the molecular and biophysical mechanisms of contractile ring function. Ongoing work in the PI's laboratory has yielded an explanation of asymmetric (non-concentric) ring closure, which is seen throughout metazoa. To explain this asymmetry, a biomechanical feedback loop was proposed, among cytoskeletal filament alignment, filament sliding, and membrane curvature. An in silico model based on this feedback can recapitulate ring closure asymmetry, as well as the kinetics of closure initiation and duration in the C. elegans zygote, the primary animal model for this work. To expand and strengthen this model, the proposed work aims to define the molecular and physical mechanisms of each component of the feedback loop. Specifically, the conserved proteins that contribute to alignment of cytoskeletal filaments with each other and with the membrane will be defined. The existence of myosin in the form of bipolar minifilaments in the contractile ring will be defined. Last, the shape of the cell throughout cytokinesis will be described and correlated with local protein enrichment and organization. The proposal centers on the use of three dimensional live-cell (time-lapse) microscopy and quantitative image analysis. Several novel quantitative assays for contractile ring assembly, organization and function will be used. These include ways to measure F-actin alignment, kinetics and position of ring closure throughout cytokinesis, the number of molecules in macromolecular cortical complexes, and the three-dimensional shape of the cell during the course of division. The C. elegans zygote serves as an ideal model system for these studies due to its reproducible size, shape, and the kinetics of cell division events, the ease of thorough depletion of essential proteins, the ability to examine the first cell division attempted following protein depletion, and the availability of strains stably expressing fluorescent fusion proteins that serve as markers for various subcellular components and compartments. Importantly, cell cycle regulatory and structural proteins are conserved among C. elegans and mammals. The long-term goal of this work is to aid the development of anti-cancer chemotherapeutics that block cytokinesis. Targeting proteins that act specifically in the contractile ring should avoid the side effects on non-dividing cells of many popular drugs. In addition, because currently used anti-mitotics also have limited success against some tumor types, development of cytokinesis drugs will be a welcome expansion and diversification of our arsenal against cancers.