The mechanism by which animal cells divide their cytoplasm during cell division (cytokinesis) is poorly understood. Although a band of actin-like microfilaments is found at the base of the constriction of dividing cells, the mechanisms by which the microfilaments arrive there are exert contractile force are unknown. Recent, carefully-performed immunocytochemical studies with antibodies to myosin, Alpha-actinin, and filamine have not definitively proven an association or lack of association of these proteins with cleavage furrows. Moreover, no consistent changes in ionic activities have been noted in associateion with cytokinesis. We propose to study the proteins associated with cleavage furrows and the local ionic changes that may accompany their progress. Specifically, we will concentrate on assessing the degree to which myosin is involved with cytokinesis and determining the roles that changes in pCa and pH may play. Cytokinesis will be studied in rat lymphoma cells, as well as in the fertilized egges of marine mollusks (Ilyanassa, Mytilus) that also undergo a change in cell shape representing a long-lived mimic of cytokinesis, polar lobe formation. Mouse hybridomas will be used to produce monoclonal antibodies against cytoplasmic myosins from rat lymphoma cells and fertilized Mytilus eggs, against myosin heavy chains, as well as light chains (phosphorylated and dephosphorylated). Degree and patterns of myosin antibody binding in cleavage furrows of fixed and permeabilized cells will be assessed by immunofluorescence and electron microscopy, using monoclonal antibodies directly coupled to fluorochromes or ferritin, respectively. Degree of myosin light chain phosphorylation will be assessed by autoradiography of SDS-polyacrylamide gels after 3 2P labeling of cells before and during cytokinesis. Microelectrodes will be used to record changes in intracellular ionic activities (concentrations of free ions), as well as to inject putative perturbants of contractile activity. Determining the regulatory steps in cytokinesis of normal cells is required before the bases of abnormal patterns of cell division can be understood.
