Assessing the Toxoplasma Tachyzoite Cell Cycle Phases Using Fluorescent Ubiquitination-Based Cell Cycle Indicator.

Toxoplasma gondii is an apicomplexan parasite that infects a wide variety of eukaryotic hosts and causes toxoplasmosis. The cell cycle of T. gondii exhibits a distinct architecture and regulation that differ significantly from those observed in well-studied eukaryotic models. To better understand the tachyzoite cell cycle, we developed a fluorescent ubiquitination-based cell cycle indicator (FUCCI) system that enables real-time visualization and quantitative assessment of the different cell cycle phases via immunofluorescence microscopy. Quantitative immunofluorescence and live-cell imaging of the ToxoFUCCIS probe with specific cell cycle markers revealed substantial overlap between cell cycle phases S, G2, mitosis, and cytokinesis, further confirming the intricacy of the apicomplexan cell cycle. Key features • This protocol describes the development of the transgenic lines capable of detecting individual cell cycle phases and processes of the Toxoplasma tachyzoite cell cycle. • Quantitative immunofluorescence analysis and real-time microscopy enable the measurement of each cell cycle phase. • The ToxoFUCCIS probe helps to gain new insights into the highly flexible, overlapping nature of cell cycle organization in apicomplexan parasites.