Assessing the <em>Toxoplasma</em> Tachyzoite Cell Cycle Phases Using Fluorescent Ubiquitination-Based Cell Cycle Indicator

<i>Toxoplasma gondii</i> is an apicomplexan parasite that infects a wide variety of eukaryotic hosts and causes toxoplasmosis. The cell cycle of <i>T. gondii</i> 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 <i>Toxo</i>FUCCI^S 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 <i>Toxoplasma</i> tachyzoite cell cycle. • Quantitative immunofluorescence analysis and real-time microscopy enable the measurement of each cell cycle phase. • The <i>Toxo</i>FUCCI^S probe helps to gain new insights into the highly flexible, overlapping nature of cell cycle organization in apicomplexan parasites.