The Toxoplasma Centrocone Houses Cell Cycle Regulatory Factors

Abstract

Our knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures, and we named this factor ECR1 for essential for chromosome replication 1. ECR1 was discovered by complementation of a temperature-sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40°C similar to a ts mutant carrying a defect in topoisomerase. ECR1 is a 52-kDa protein containing divergent RING and TRAF-Sina-like zinc binding domains that are dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the unique spindle compartment of the Apicomplexa (centrocone) of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughter parasites separate from the mother parasite, ECR1 is downregulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex, and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the cyclin-dependent kinase (CDK)-related kinase, T. gondii Crk5 (TgCrk5), which displays a similar cell cycle expression and localization during tachyzoite replication. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates that this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.

Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Toxoplasma gondii

“…The most critical function of the centrosome is to anchor the mitotic spindle at the kinetochore to drive mitosis and karyokinesis. In light of mitosis in Toxoplasma being closed (the nuclear envelope does not disintegrate as the chromosomes separate), a specialized structure termed the centrocone ( 42 – 44 ) is required to establish access for the cytoplasmically located centrosomes to the chromosomes in the nucleus. The presence, therefore, of multiple centrosomes in multiply gravid TgOTUD3A-KO parasites would necessitate the formation of matched centrocones to ensure the correct engagement of the respective genome equivalents to the centrosomes, ensuring the fidelity of inheritance.…”

“…Recent studies show that the bipartite centrosome in T. gondii plays a major regulatory role in determining the ploidy and, by extension, the number of progeny ( 35 , 48 ). Cell cycle control is mediated to a significant degree by the activity of atypical cyclins and cyclin-dependent kinase (Cdk)-related kinases (Crks), serving as checkpoints in the progression of the T. gondii cell cycle ( 44 , 49 ).…”

“…Among ubiquitinated targets, histones, histone-modifying enzymes, DNA licensing factors, and enzymes regulating epigenetic processes point to the centrality of ubiquitin-mediated regulation in ensuring the fidelity of replication ( 19 ). A recent functional study further shows that one Crk family protein, Crk5, and its interacting partner ECR1 ( e ssential for c hromosome r eplication 1), regulating DNA licensing at the origin of replication, are regulated by ubiquitin-mediated mechanisms ( 44 ).…”

“…The other deviation from the norm is the balanced reduplication wherein both the centrosomes reduplicate either before or after the first round of karyokinesis is complete and that can be explained by signaling events similar to those described above. Of interest is a recent finding of a role for the ECR1/TgCrk complex in centrocone duplication and the ubiquitin-mediated turnover of ECR1, which is related to the geminins in higher eukaryotes ( 44 ). Geminins play a critical role in preventing relicensing of DNA synthesis, chromosome duplication, and spindle formation ( 53 , 54 ).…”

Immunogenicity and Protective Effect of a Virus-Like Particle Containing the SAG1 Antigen of Toxoplasma gondii as a Potential Vaccine Candidate for Toxoplasmosis

“…Moreover, it is known that T. gondii blocks or inhibits the signal pathways of cell cycle initiators or the apoptotic stage in host cells. T. gondii promotes its proliferation by rapidly forming PVM or regulating cell cycle factors of the host for survival after invasion, which finally increases the viability of T. gondii in the host [34,35]. Recently the subcellular organelles of T. gondii, such as dense granules (GRAs), the rhoptry, the microneme, and the inner membrane complex, have been used as major recombinant proteins and/or antigens for vaccine candidates, as well as major targets for inhibiting T. gondii infection.…”