Overexpression of CAF1 encoding a novel Ca2+-binding protein stimulates the transition of Dictyostelium cells from growth to differentiation

Among the expressed genes associated with the switch-over of Dictyostelium cells from cell proliferation to differentiation, the Calfumirin-1 ( CAF1 ) gene has been shown to be preferentially expressed at the initial step of differentiation, encoding a novel Ca²⁺-binding protein (Abe & Maeda 1995). To analyze precisely the function of CAF1 , transformants overexpressing the CAF1 mRNA at the vegetative growth phase and also CAF1 -null mutants were prepared, and their developmental features were compared with those of parental wild-type cells. As a result, the CAF1 -overexpression was found to promote cell differentiation, possibly through prompt induction of the cAMP receptor 1 ( CAR1 ) gene expression. In addition, the CAF1 -overexpressing cells were able to differentiate even under low external Ca²⁺ ([Ca²⁺]_e) conditions around 10⁻⁶mol/L at which non-transformed wild-type cells never differentiated. Unexpectedly, however, the CAF1 -null mutant produced by homologous recombination exhibited apparently normal development to form fruiting bodies on non-nutrient agar. These results seem to indicate that CAF1 -overexpression has a stimulatory effect on differentiation, but that the CAF1 protein is not necessarily required for the phase-shift of cells from growth to differentiation.     

Cell-cycle progression during the development of Dictyostelium discoideum and its relation to the subsequent cell-sorting in the multicellular structures

Previous studies have shown that the cell-cycle phase at the onset of starvation is a naturally occurring variable that is closely involved in the subsequent sorting and differentiation of cells during Dictyostelium development. Here the cell-cycle progression during the development of D. discoideum Ax-2 cells and its relation to the subsequent cell-sorting were analyzed in detail using synchronized cells and their pulse-labeling by 5'-bromodeoxyuridine (BrdU). Measurements of cell number and nuclearity provided evidence that about 80% of cells progressed their cell-cycle after formation of multicellular structures (mounds). Many cells (T7 cells) starved at mid–late G2-phase (just before the PS-point from which cells initiate development when starved) progressed to the cell-cycle after mound formation. In contrast, a less amount of cells (T1 cells) starved at late G2-phase (just after the PS-point) progressed through the cell-cycle after mound formation. The significance of cell-cycle progression presented here is discussed, with reference to cell differentiation and pattern formation.     

Possible involvements of 101 kDa, 90 kDa, and 32 kDa phosphoproteins in the phase-shift of Dictyostelium cells from growth to differentiation

Abstract. As demonstrated previously, the transition of starving Dictyostelium cells from growth to differentiation phase occurs at a particular position (putative shift point; PS-point) in G₂-phase of the cell cycle of Dictyostelium discoideum Ax-2. In this study we examined what proteins are phosphorylated or dephosphorylated at the onset of starvation, with special emphasis on changes of phosphoproteins near the PS-point. When AX-2 cells at any particular phase of the cell cycle were pulse-labeled with inorganic ³²P (³²P_i) in the presence or absence of nutrients, it was found that 101 kDa and 90 kDa phosphoproteins exhibit specific changes around the PS-point. From the chase-experiments of ³²P-labeled cells, the 101 kDa and 90 kDa proteins were found to fail to be phosphorylated at the PS-point under starvation conditions. The protein phosphatase inhibitors such as okadaic acid and calyculin A inhibited completely entry of starving Ax-2 cells to differentiation, and also blocked perfectly dephosphorylation of 32 kDa protein. Taken together it is likely that dephosphorylation of 32 kDa protein as well as low phosphorylation levels of 101 kDa and 90 kDa proteins may be required for the phase-shift of Ax-2 cells from growth to differentiation. Subcellular fractionation showed the 101 kDa phosphoprotein to be located in cytoplasm, while parts, at least, of the 90 kDa and 32 kDa phosproproteins were in the nucleus. In addition, the results of cellulose thin-layer electrophoresis of digested 101 kDa and 90 kDa phosphoproteins show that in both proteins only serine residues are phosphorylated. The significance of phosphorylation states of 101 kDa, 90 kDa, and 32 kDa proteins is discussed in relation to a breakaway of cells from proliferation to differentiation.