PHOSPHORYLATION OF THE DICTYOSTELIUM MYOSIN-II HEAVY-CHAIN IS NECESSARY FOR MAINTAINING CELLULAR POLARITY AND SUPPRESSING TURNING DURING CHEMOTAXIS

Conversion of the three mapped threonine phosphorylation sites in the myosin II heavy chain tail to alanines results in a mutant (3XALA) in Dictyostelium discoideum, which displays constitutive myosin overassem bly in the cytoskeleton and increased cortical tension. To assess the importance of myosin phosphorylation in cellular translocation and che motaxis, 3XALA mutant cells have been analyzed by 2D and 3D computer-a ssisted methods in buffer, in a spatial gradient of cAMP, and after th e rapid addition of cAMP. 3XALA cells crawling in buffer exhibit disti nct abnormalities in cellular shape, the maintenance of polarity and t he complexity of the pseudopod perimeter. 3XALA cells crawling in buff er also exhibit a decrease in directionality. In a spatial gradient of cAMP, the behavioral defects are accentuated. In a spatial gradient, 3XALA cells exhibit a repeating 1- to 2-min behavior cycle in which th e shape of each cell changes abnormally from elongate to extremely wid e;vith lateral, opposing pseudopods. At the end of each cycle, 3XALA c ells turn 90 degrees into the left or right lateral pseudopod, resulti ng in a dramatic depression in chemotactic efficiency, even though 3XA LA cells are chemotactically responsive to cAMP. These results demonst rate that the phosphorylation of myosin II heavy chain plays a critica l role in the maintenance of cell shape and in persistent translocatio n in a spatial gradient of chemoattractant. (C) 1998 Wiley-Liss, Inc.