The contractile ring is the array of microfilaments located beneath the plasma membrane of the cleavage furrow of dividing cells and represents the intracellular machinery responsible for cytokinesis. Because it is composed of cytoskeletal proteins it has been characterized superficially as a muscle-like organelle. The contractile ring has defied detailed experimental or compositional analysis because it is too small, transient, and labile for conventional isolation. This joint research project will exploit a promising new method of preparing contractile rings in large numbers that will allow the first detailed analysis of the macromolecular composition and organization of this important cytoskeletal structure and its association with the plasma membrane. The cell fraction enriched in contractile rings will be prepared from synchronously dividing sea urchin eggs. In one set of studies, the morphological organization of the actin-myosin-membrane complex of the contractile ring will be explored by immunofluorescence and electron microscopy, utilizing available antiactin and antimyosin antibodies already known to be effective. In another set of studies, new monoclonal and polyclonal antibodies will be generated in order to discover and identify as yet unknown regulatory and stabilizing molecules, using combined microscopic and immunochemical techniques. A final set of experiments is designed to ascertain the extent of myosin phosphorylation in the contractile ring and to evaluate its role in myosin organization. The results of this collaborative research should elucidate the macromolecular machinery responsible for the separation of two daughter cells during cell division.
