Chemoattractant-elicited translocation of myosin in motile Dictyostelium

The distribution of myosin was studied in amebae of the Ax-3 and NC-4 strains of Dictyostelium migrating at room temperature, using indirect immunofluorescence of aggregation-competent amebae and the agar-overlay technique. Amebae were fixed in methanol-formaldehyde or absolute acetone at -15 degrees C before or after stimulation with micromolar cyclic AMP at room temperature (20-25 degrees C). Myosin was detected by monoclonal antibodies to Dictyostelium myosin heavy chain followed by a fluorescent secondary antibody that had been preabsorbed to remove nonspecific staining. In both strains there was a striking increase in intensity of anti-myosin immunofluorescence in the cortex where it appeared as a continuous ring 30 seconds after addition of cyclic AMP. This correlated with a rounding up of the cell body. Sixty seconds after stimulation there was a clear reduction of cytoplasmic myosin rods in conjunction with the increased cortical localization. At this time extensions of largely hyaline cytoplasm were observed that extended beyond the cortical shell of myosin. Two minutes after the stimulus the immunofluorescence remained as a distinct line at the cortex, but the cells began to resume in elongated shape. By 3 minutes (NC-4 strain) or 5 minutes (Ax-3 strain) the amebae had largely returned to the control shape, and myosin had returned to its control distribution. Counts of the treated cells at different time points substantiated the observations of individual cells. The time course of translocation of myosin in the Ax-3 strain parallels the time course of myosin phosphorylation reported in previous studies. The results are interpreted in terms of a working hypothesis for the mechanism of translocation.