MUTANTS LACKING MYOSIN-II CANNOT RESIST FORCES GENERATED DURING MULTICELLULAR MORPHOGENESIS

We have used fluorescent labeling, confocal microscopy and computer-as sisted motion analysis to observe and quantify individual wild-type an d myosin II mutant cell behavior during early multicellular developmen t in Dictyostelium discoideum. When cultured with an excess of unlabel ed wild-type cells, labeled control cells are randomly distributed wit hin aggregation streams, while myosin II mutant cells are found primar ily at the lateral edges of streams, Wild-type cells move at average r ates of 8.5+/-4.9 mu m/min within aggregation streams and can exhibit regular periodic movement at 3.5 minute intervals; half as long as the 7 minute period reported previously for isolated cells, Myosin II mut ants under the same conditions move at 5.0+/-4.8 mu m/min, twice as fa st as reported previously for isolated myosin II mutant cells, and fai l to display regular periodic movement, When removed from aggregation streams myosin II mutant cells move at only 2.5+/-2.0 mu m/min, while wild-type cells under these conditions move at 5.9+/-4.5 mu m/min. Ana lysis of cell morphology further reveals that myosin II mutant cells a re grossly and dynamically deformed within wild-type aggregation strea ms but not when removed from streams and examined in isolation. These data reveal that the loss of myosin II has dramatic consequences for c ells undergoing multicellular development, The segregation of mutant c ells to aggregation stream edges demonstrates that myosin II mutants a re unable to penetrate a multicellular mass of wild-type cells, while the observed distortion of myosin II mutant cells suggests that the co rtex of such cells is too flacid to resist forces generated during mov ement, The increased rate of mutant cell movement and distortion of mu tant cell morphology seen within wild-type aggregation streams further argues both that movement of wild-type cells within a multicellular m ass can generate traction forces on neighboring cells and that mutant cell morphology and behavior can be altered by these forces, In additi on, the distortion of myosin II mutant cells within wild-type aggregat ion streams indicates that myosin is not required for the formation of cell-cell contacts, Finally, the consequences of the loss of myosin I I for cells during multicellular development are much more severe than has been previously revealed for isolated cells, The techniques used here to analyze the behavior of individual cells within multicellular aggregates provide a more sensitive assay of mutant cell phenotype tha n has been previously available and will be generally applicable to th e study of motility and cytoskeletal mutants in Dictyostelium.