A FUCCI sensor reveals complex cell cycle organization ofToxoplasmaendodyogeny

In this study, we report the application of the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) in a unicellular eukaryote model.Toxoplasma gondiiis an Apicomplexan parasite and opportunistic pathogen that can infect a wide range of warm-blooded hosts, including humans. The remarkably flexible cell division ofT. gondiiand other apicomplexan parasites differs considerably from the cell division modes employed by other model eukaryotes. Additionally, there is a lack of recognizable cell cycle regulators, which have contributed to the difficulties in deciphering the order of events in the Apicomplexan cell cycle. To aid in studies of the cell cycle organization of theT. gondiitachyzoite, we have createdToxoFUCCI^SandToxoFUCCI^SCprobes. We introduced a DNA replication factor TgPCNA1 tagged with NeonGreen that can be used alone or in conjunction with an mCherry-tagged budding indicator TgIMC3 in an RH auxin-induced degradation (AID) parental strain. The varied localization and dynamic cell cycle oscillation have confirmed TgPCNA1 to be a suitableT. gondiiFUCCI probe. TheToxoFUCCI^Sanalysis showed that tachyzoite DNA replication starts at or near centromeric regions, has a bell-shaped dynamic and a significant degree of the cell cycle asynchrony within the vacuoles. Quantitative live and immunofluorescence microscopy analyses ofToxoFUCCI^Sand its derivatives co-expressing epitope-tagged cell cycle markers have revealed an unusual composite cell cycle phase that incorporates overlapping S, G₂, mitosis and cytokinesis (budding). We identified five intervals of the composite phase and their approximate duration: S (19%), S/G₂/C (3%), S/M/C (9%), M/C (18%) and C/G₁(<1%). TheToxoFUCCI^Sprobe efficiently detected G₂/M and Spindle Assembly Checkpoints, as well as the SB505124-induced TgMAPK1 dependent block. Altogether, our findings showed an unprecedented complexity of the cell cycle in apicomplexan parasites.