3D analysis of cell movement during normal and myosin-II-null cell morphogenesis in dictyostelium

To gain insights into the possible guidance mechanisms used by Dictyostelium cells as they undergo morphogenesis, we have used time-lapse computational optical-sectioning microscopy to visualize and quantify the three-dimensional (3D) trajectories of both normal (Ax2) and myosin-II-null cells. To accomplish this, we typically collected 30-60 time-lapse 3D images every 2-3 min at the earliest multicellular stage, the mound. These time-lapse data were used to generate 3D movies of morphogenesis and to construct 3D trajectories for individual cells. In contrast to previous 2D time-lapse cinematography studies which revealed predominantly spiral trajectories of Ax2 cells in the mound, we have found a complex assortment of motile behaviors: some cells jiggled in place; others appeared to follow either linear or spiral trajectories; some cells reversed their directions; and others apparently converted from one motile behavior to another. These results suggest that a number of different, potentially competing cell-guidance mechanisms are operative in the mound. To assess one molecular mechanism underlying this assortment of motile behaviors, we have examined cell locomotion in a mutant, namely, in myosin-II-null cells which never develop beyond the mound. Previous studies had shown that these cells can crawl, albeit somewhat slowly, on a 2D substrate. We also found, at the earliest stages of myosin-II-null mound formation, some directed cell locomotion. But later, as the mound condensed into a tightly packed cell conglomerate, extended cell trajectories disappeared, and instead virtually all of the cells jiggled in place. Thus, our results suggest that myosin-II is absolutely essential for normal 3D ameboid locomotion.