Deciphering cell cycle organization of Toxoplasma endodyogeny

Area of Science:

• Cell Biology
• Parasitology
• Molecular Biology

Background:

• Apicomplexan parasites like Toxoplasma gondii have unique cell division mechanisms.
• Understanding the parasite cell cycle is crucial for disease control but remains poorly understood due to atypical regulation.
• Previous research indicated differences from host cell cycles, but event order was unestablished.

Purpose of the Study:

• To investigate the atypical cell cycle organization of Toxoplasma gondii tachyzoites.
• To develop and utilize novel Fluorescent Ubiquitination-based Cell Cycle Indicator (ToxoFUCCI) probes for visualizing the parasite cell cycle.
• To determine the order and duration of cell cycle events in T. gondii.

Main Methods:

• Development of ToxoFUCCI probes (ToxoFUCCI^S and ToxoFUCCI^SC) using tagged DNA replication (TgPCNA1) and budding (TgIMC3) factors.
• Quantitative live and immunofluorescence microscopy analyses.
• Utilizing auxin-inducible degradation systems for parasite manipulation.

Main Results:

• TgPCNA1 confirmed as a suitable FUCCI probe, showing bell-shaped dynamics and cell cycle asynchrony.
• DNA replication initiates near centromeric regions.
• An unusual composite cell cycle phase was identified, incorporating overlapping S, G2, mitosis, and cytokinesis stages.
• Five distinct intervals within the composite phase were quantified, with durations ranging from <1% to 19%.

Conclusions:

• Toxoplasma gondii possesses a highly complex and unusual cell cycle with significant phase overlaps.
• The ToxoFUCCI probes provide valuable tools for studying apicomplexan cell cycle dynamics.
• Findings offer insights into parasite survival strategies and potential therapeutic targets.