Entamoeba invadens: dynamics of DNA synthesis during differentiation from trophozoite to cyst.

The DNA dynamics which mediate conversion of uni-nucleate trophozoite into quadrinucleate cyst in Entamoeba histolytica is not well understood. Here, we have addressed this question in Entamoeba invadens (a model system for encystation) through a detailed time course study of the differentiation process. We combined flow cytometric analysis with the change in rate of thymidine incorporation and the number of nuclei per cell. Our data shows that during encystment the cell population passes through three phases: (1) Early phase (0-8h); of rapid DNA synthesis which may correspond to completion of ongoing DNA replication. Bi-nucleated cells increase with concomitant drop in uni-nucleated cells. (2) Commitment phase (8-24h); in which DNA synthesis rate slows down. Possibly new rounds of replication are initiated which proceed slowly, followed by mitosis at 20 h. After this the number of bi- and uni-nucleated cells gradually decline and the tri- and tetra-nucleated cells begin to increase. (3) Consolidation phase (24-72 h); in which the rate of DNA synthesis shows a small increase till 32 h and then begins to decline. The G2/M peak reappears at 48 h, showing that more rounds of DNA replication may be getting completed, followed by nuclear division. By 72 h the encystment is virtually complete. The bi-nucleated stage could be an intermediate both in the conversion of trophozoite to cyst and back. Our study provides a comprehensive view of DNA dynamics during encystation and excystation of E. invadens.