ATR-Chkl-Cdc25Signaling Pathways Involved In The Regulation Of Dictyostelium Discoideum During G2/M Cell Cycle

Dictyostelium discoideum is an eukaryotic single cell. It is a social amoeba whose life cycle consists of two distinct phases-growth and differentiation—that are easily controlled by nutritionalconditions. D. discoideum (strainKAx-3) cells grow and multiply by mitosis as long as nutrients are supplied.Upon exhaustion of nutrients, however, starving cells initiate differentiation to acquire aggregation competence. Cell cycle of Dictyostelium discoideum has following traits:(i) a very short period of M-phase (15min or less);(ii) little or noGl-phase;(iii)30min or less of S-phase;and (iv) a long period (about6.5h) of G2-phase,though some studies have claimed the presence of G1-phase. So G2/M checkpoint account for93.10%of the whole cell cycle.Cdc25phosphatase regulate the process of G2/M cell cycle, ATR-Chkl-Cdc25signaling pathway is very important during G2/M transition period. The cell proliferation of wild type KAx-3and mutant type RNAi-allC observed by light microscope and cell counting, RNAi-allc cell was divided8time faste than that of KAx-3cells. To evaluate the reason of cell cycle shortened of RNAi-allC cells, the function of ATR-Chkl-Cdc25signaling pathway was explored by quantitative PCR and western blot techniques. The results showed the differences of ATR, Chkl, Cdc25in mRNA and protein level between KAx-3and RNAi-allC cells, including the expression of ATR and Chkl ratio of RNAi-allC to KAx-3was0.69:1and0.1:1respectively; the expression of Cdc25in RNAi-allC cells was8times that of KAx-3cells. The data suggested though the expression of ATR had little change, Chkl and Cdc25expression changed greatly. Western blotting results were consistent with Q-PCR reports. The Chkl protein contents were significantly less in mutant type RNAi-allC than that in KAx-3cells; the Cdc25protein contents were higher in RNAi-allC cells. The above mentioned results suggested that ATR-Chkl-Cdc25signaling pathway involved in the regulation of the G2/M phase in Dictyostelium discoideum.This provide reference to cell cycle signaling pathways of Dictyostelium discoideum.