A FUCCI sensor reveals complex cell cycle organization of Toxoplasma endodyogeny
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In this study, we report the application of the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) in a unicellular eukaryote model. Toxoplasma gondii is an Apicomplexan parasite and opportunistic pathogen that can infect a wide range of warm-blooded hosts, including humans. The remarkably flexible cell division of T. gondii and 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 the T. gondii tachyzoite, we have created Toxo FUCCI S and Toxo FUCCI SC probes. 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 suitable T. gondii FUCCI probe. The Toxo FUCCI S analysis 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 of Toxo FUCCI S and its derivatives co-expressing epitope-tagged cell cycle markers have revealed an unusual composite cell cycle phase that incorporates overlapping S, G 2 , mitosis and cytokinesis (budding). We identified five intervals of the composite phase and their approximate duration: S (19%), S/G 2 /C (3%), S/M/C (9%), M/C (18%) and C/G 1 (<1%). The Toxo FUCCI S probe efficiently detected G 2 /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.
