TARGETED DISRUPTION OF THE DICTYOSTELIUM MYOSIN ESSENTIAL LIGHT-CHAINGENE PRODUCES CELLS DEFECTIVE IN CYTOKINESIS AND MORPHOGENESIS

We have previously demonstrated that the myosin essential light chain (ELC) is required for myosin function in a Dictyostelium cell line, 7- 11, in which the expression of ELC was inhibited by antisense RNA over expression. We have now disrupted the gene encoding the ELC (mlcE) in Dictyostelium by gene targeting, The mlcE(-) mutants provide a clean g enetic background for phenotypic analysis and biochemical characteriza tion by removing complications arising from the residual ELC present i n 7-11 cells, as well as the possibility of mutations due to insertion of the antisense construct at multiple sites in the genome, The mlcE( -) mutants, when grown in suspension, exhibited the typical multinucle ate phenotype observed in both myosin heavy chain mutants and 7-11 cel ls, This phenotype was rescued by introducing a construct that express ed the wildtype Dictyostelium ELC cDNA, Myosin purified from the mlcE( -) cells exhibited significant calcium ATPase activity, but the actin- activated ATPase activity was greatly reduced, The results obtained fr om the mlcE(-) mutants strengthen our previous conclusion based on the antisense cell line 7-11 that ELC is critical for myosin function, Th e proper localization of myosin in mlcE(-) cells suggests that its phe notypic defects primarily arise from defective contractile function of myosin rather than its mislocalization, The enzymatic defect of myosi n in mlcE(-) cells also suggests a possible mechanism for the observed chemotactic defect of mlcE(-) cells, We have shown that while mlcE(-) cells were able to respond to chemoattractant with proper directional ity, their rate of movement was reduced, During chemotaxis, proper dir ectionality toward chemoattractant may depend primarily on proper loca lization of myosin, while efficient motility requires contractile func tion, In addition, we have analyzed the morphogenetic events during th e development of mlcE(-) cells using lacZ reporter constructs expresse d from cell type specific promoters, By analyzing the morphogenetic pa tterns of the two major cell types arising during Dictyostelium develo pment, prespore and prestalk cells, we have shown that the localizatio n of prespore cells is more susceptible to the loss of ELC than presta lk cells, although localization of both cell types is abnormal when de veloped in chimeras formed by mixing equal numbers of wild-type and mu tant cells, These results suggest that the morphogenetic events during Dictyostelium development have different requirements for myosin func tion.