Regulation of apicomplexan mitosis coupled to budding - Abstract Apicomplexan parasites contribute significantly to the human disease burden, including Plasmodium spp. infection causing ~400,000 deaths per year, and Toxoplasma gondii permanently infecting about ~1/3 of the human populations. Existing treatments are limited, which fuels studies that aim to uncover new vulnerable processes to control such infections. Our project investigates the core survival mechanism that controls parasite division taking place in its host, the cell cycle. Apicomplexan cell division differs vastly from that of their host cells, and represent a remarkably versatile, novel, and poorly understood biological process. Some apicomplexan species replicate their genome once per division cycle (binary division) and produce two progeny, while the majority of species instead replicate their genome multiple times (multinuclear division) and produce thousands of daughter cells in a single round of division. The variety of replication modes, dearth of conserved conventional regulators, and the complexity of internal structures have hindered the progress of apicomplexan cell cycle studies. Our project is built on an innovative concept of apicomplexan cell cycle organization, where instead of following the traditional G1-S-G2-M/C sequence, the G1 phase of T. gondii cell cycle is followed by a composite S/G2/M/C cell cycle phase that lacks clear phase transitions. The new view of apicomplexan cell cycle may explain why, until now, the apicomplexan G2 phase was considered missing, why tachyzoites require multiple cell cycle Cdk-related kinases (Crks) and why the complete sequence of cell cycle events had never been established. Our main hypothesis is that parasite-specific Crk-Cyclin complexes engage novel protein networks to coordinate spatially segregated, but concurrent events during an overlap of G2 phase, mitosis, and budding. We will examine how T. gondii controls progression throughout this composite cell cycle phase while maintaining the overall order of cell cycle events. To elevate the studies of apicomplexan cell cycles, we have designed new tools: a ToxoFUCCISC cell cycle probe, and two checkpoint synchronization models. To test our hypothesis, we will characterize the protein networks acting in G2 (Aim 1) and at the spindle assembly checkpoint (SAC) (Aim 2) and explore each checkpoint mechanism in detail. Using our new checkpoint-based synchronization approach, we will perform global profiling of the entry (TgCrk4-Cyc4-regulated G2/M) and exit from mitosis (TgCrk6-Cyc1-regulated SAC) to generate the landscape of the composite S/G2/M/C cell cycle period (Aim 3). Our study will address outstanding questions of apicomplexan parasite biology regarding how parasites regulate their amplification in host cells. Our results will have an impact on studies across the Apicomplexa phylum and lay the groundwork to uncover the cell cycle strategies that produce multiple progenies per cycle (e.g., Plasmodium spp. and Cryptosporidium spp.).
