A temperature-sensitive adenylyl cyclase mutant of Dictyostelium

Dictyostelium development starts with the chemotactic aggregation of up to 10(6) amoebae in response to propagating cAMP waves. cAMP is produced by th e aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Temperature-sensitive mutants of ACA were selected from a population of aca null cells transformed with a library of ACA genes, a m ajor segment of which had been amplified by error-prone PCR, One mutant (ts aca2) that can complement the aggregation null phenotype of aca null cells at 22 degrees C but not at 28 degrees C was characterized in detail The bas al catalytic activity of the enzyme in this mutant was rapidly and reversib ly inactivated at 28 degrees C. Using this mutant strain we show that cell movement in aggregates and mounds is organized by propagating waves of cAMP , Synergy experiments between wild-type and tsaca2 cells, shifted to the re strictive temperature at various stages of development, showed that ACA pla ys an important role in the control of cell sorting and tip formation.