DICTYOSTELIUM-DISCOIDEUM CELLS LACKING THE 34,000-DALTON ACTIN-BINDING PROTEIN CAN GROW, LOCOMOTE, AND DEVELOP, BUT EXHIBIT DEFECTS IN REGULATION OF CELL STRUCTURE AND MOVEMENT - A CASE OF PARTIAL REDUNDANCY

Cells lacking the Dictyostelium 34,000-D actin-bundling protein, a cal cium-regulated actin cross linking protein, were created to probe the function of this polypeptide in living cells. Gene replacement vectors were constructed by inserting either the UMP synthase or hygromycin r esistance cassette into cloned 4-kb genomic DNA containing sequences e ncoding the 34-kD protein. After transformation and growth under appro priate selection, cells lacking the protein were analyzed by PCR analy ses on genomic DNA, Northern blotting, and Western blotting. Cells lac king the 34-kD protein were obtained in strains derived from AX2 and A X3. Growth, pinocytosis, morphogenesis, and expression of developmenta lly regulated genes is normal in cells lacking the 34-kD protein. In c hemotaxis studies, 34-kD(-) cells were able to locomote and orient nor mally, but showed an increased persistence of motility. The 34-kD(-) c ells also lost bits of cytoplasm during locomotion. The 34-kD(-) cells exhibited either an excessive number of long and branched filopodia, or a decrease in filopodial length and an increase in the total number of filopodia per cell depending on the strain. Reexpression of the 34 -kD protein in the AX2-derived strain led to a ''rescue'' of the defec t in the persistence of motility and of the excess numbers of long and branched filopodia, demonstrating that these defects result fi om the absence of the 34-kD protein. We explain the results through a model of partial functional redundancy, Numerous other actin cross-linking p roteins in Dictyostelium may be able to substitute for some functions of the 34-kD protein in the 34-kD(-) cells. The observed phenotype is presumed to result from functions that cannot be adequately supplanted by a substitution of another actin cross-linking protein. We conclude that the 34-kD actin-bundling protein is not essential for growth, bu t plays an important role in dynamic control of cell shape and cytopla smic structure.