Using a permeabilized cell system and cell physiology to understand cytokinetic actomyosin ring constriction (part of programme Cytokinetic actomyosin ring: from understanding the ultrastructure and assembly to (360G-Wellcome-101885_C_13_Z)

Cell division in many eukaryotes requires an actomyosin-based contractile ring, whose constriction bisects a mother cell into two. Although this F-actinand myosin II motor-containing ring has been described for > 40 years, we know very little about how it constricts and generates force. Our goals are to understand the mechanism of actomyosin ring constriction and its regulation inthe cell cycle. We will use Schizosaccharomyces pombe for our studies, since it divides using an actomyosin-based co ntractile ring, and is amenable to themethods of genetics, biochemistry, and cytology. We have developed a permeabilized cell system in which addition of adenosine triphosphate leads tocomplete contraction of intact actomyosin rings. We will use this system to 1.estimate the force generated by the constricting ring and 2. perform contractility assays on permeabilized cells generated from mutants in actomyosin ring proteins, to identify ring components important for its constriction. We wil l use proteomic approaches to uncover the molecular differences between actomyosin rings before and during constriction and to understand the cell cycle regulation of ring constriction. We will use cross-linking amino acid mutagenesis of actin to identify changes in actin filaments during cytokinesis and employ a new approach to identify protein conformational changes during cytokinesis. Finally, we will investigate if overlapping mechanisms regulate ring constriction in vivo. The combination of in vitro contractility, in vivo studies, and screens based on emerging technologies should provide tremendous insight into actomyosin ring constriction mechanisms and can lead the field in new directions.