This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objective of this project is to acquire 3D structural information about the organization of the actin-myosin-II network in the cleavage furrow cortex of Dictyostelium cells at different stages f furrow ingression. In our work, we have been developing a quantitative framework for cytokinesis in order to understand the molecular mechanisms that govern the dynamics of cell shape change. Myosin-II and actin are central to this process. Many textbook models of cytokinesis invoke a circumferential belt of actin filaments with the myosin-II thick filaments arranged so that the cleavage furrow is constricted like a purse string. While this organization is likely to be correct for some organisms, several lines of evidence challenge this as a general framework. First, myosin-II mutant Dictyostelium cells undergo mitotic cell division nearly as fast as wild-type cells. Second, Dictyostelium myosin-II is kinetically tuned and assembled into thick filaments in such a way as to make it difficult to conceptualize a simple sarcomeric-like contraction mechanism; some mammalian nonmuscle myosin-IIs are similarly tuned. Third, in mammalian and Dictyostelium, cells, a clear ring of actin filaments is not clearly detectable. High resolution structural information would allow us to considerably refine our current models for cytokinesis in Dictyostelium. The initial goal of this project is to determine the orientation of actin filaments and myosin-II thick filaments at various stages of furrow ingression. We have prepared cells by rapidly plunge freezing formvar-coated gold EM grids with attached cells and are imaging these for tomographic reconstruction both as whole mounts in the frozen-hydrated state and as plastic sections after freeze-substitution.