DICTYOSTELIUM MUTANTS LACKING MULTIPLE CLASSIC MYOSIN-I ISOFORMS REVEAL COMBINATIONS OF SHARED AND DISTINCT FUNCTIONS

Dicryostelium cells that lack the myoB isoform were previously shown t o exhibit reduced efficiencies of phagocytosis and chemotactic aggrega tion (''streaming'') and to crawl at about half the speed of wild-type cells. Of the four other Dictyostelium myosin I isoforms identified t o date, myoC and myoD are the most similar to myoB in terms of tail do main sequence. Furthermore, we show here that myoC, like myoB and myoD , is concentrated in actin-rich cortical regions like the leading edge of migrating cells. To look for evidence of functional overlap betwee n these isoforms, we analyzed myoB, myoC, and myoD single mutants, myo B/myoD double mutants, and myoB/myoC/myoD triple mutants, which were c reated using a combination of gene targeting techniques and constituti ve expression of antisense RNA. With regard to the speed of locomoting , aggregation-stage cells, of the three single mutants, only the myoB mutant was significantly slower. Moreover, double and triple mutants w ere only slightly slower than the myoB single mutant. Consistent with this, the protein level of myoB alone rises dramatically during early development, suggesting that a special demand is placed on this one is oform when cells become highly motile. We also found, however, that th e absolute amount of myoB protein in aggregation-stage cells is much h igher than that for myoC and myoD, suggesting that what appears to be a case of nonoverlapping function could be the result of large differe nces in the amounts of functionally overlapping isoforms. Streaming as says also suggest that myoC plays a significant role in some aspect of motility other than cell speed. With regard to phagocytosis, both myo B and myoC single mutants exhibited significant reductions in initial rate, suggesting that these two isoforms perform nonredundant roles in supporting the phagocytic process. In triple mutants these defects we re not additive, however. Finally, because double and triple mutants e xhibited significant and progressive decreases in doubling times, we a lso measured the kinetics of fluid phase endocytic flux (uptake, trans it time, efflux). Not only do all three isoforms contribute to this pr ocess, but their contributions are synergistic. While these results, w hen taken together, refute the simple notion that these three ''classi c'' myosin I isoforms perform exclusively identical functions, they do reveal that all three share in supporting at least one cellular proce ss (endocytosis), and they identify several other processes (motility, streaming, and phagocytosis) that are supported to a significant exte nt by either individual isoforms or various combinations of them.