In vivo observations of myosin II dynamics support a role in rear retraction

To investigate myosin II function in cell movement within a cell mass, we i maged green fluorescent protein-myosin heavy chain (GFP-MHC) cells moving w ithin the tight mound of Dictyostelium discoideum. In the posterior cortex of cells undergoing rotational motion around the center of the mound, GFP-M HC cyclically formed a "C," which converted to a spot as the cell retracted its rear. Consistent with an important role for myosin in rotation, cells failed to rotate when they lacked the myosin II heavy chain (MHC-) or when they contained predominantly monomeric myosin II (3xAsp). Ln cells lacking the myosin II regulatory light chain (RLC-), rotation was impaired and even tually ceased. These rotational defects reflect a mechanical problem in the 3xAsp and RLC- cells, because these mutants exhibited proper rotational gu idance cues. MHC- cells exhibited disorganized and erratic rotational guida nce cues, suggesting a requirement for the MHC in organizing these signals. However, the MHC- cells also exhibited mechanical defects in rotation, bec ause they still moved aberrantly when seeded into wild-type mounds with pro per rotational guidance cues. The mechanical defects in rotation may be med iated by the C-to-spot, because RLC- cells exhibited a defective C-to-spot, including a slower C-to-spot transition, consistent with this mutant's slo wer rotational velocity.