TheToxoplasmacentrocone 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, essential for chromosome replication 1 (ECR1), that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures. ECR1 was discovered by complementation of a temperature sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40 o C similar to a ts-mutant carrying a defect in topoisomerase. ECR1 is a 52kDa protein containing divergent RING and TRAF-Sina like zinc-binding domains that is dynamically expressed in the tachyzoite cell cycle.ECR1 first appears in the centrocone compartment 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 daughters resolve from the mother, ECR1 is down regulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitinmediated 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 CDK-related kinase, TgCrk5, which shares a similar cell cycle expression and localization during tachyzoite replication. Altogether, the results of this study suggest ECR1 may be a unique E3 ligase that regulates DNA licensing and other mitotic processes. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle. IMPORTANCEParasites of the apicomplexan family are important causes of human disease including malaria, toxoplasmosis, and cryptosporidiosis. Parasite growth is the underlying cause of pathogenesis, yet despite this importance the molecular basis for parasite replication is poorly understood. Filling this knowledge gap cannot be accomplished by mining recent whole genome sequencing because apicomplexan cell cycles differ substantially and lack many of the key regulatory factors of wellstudied yeast and mammalian cell division models. We have utilized forward genetics to discover essential factors that regulate cell division in these parasites using the Toxoplasma gondii model.

Apicomplexa Cell Cycles: Something Old, Borrowed, Lost, and New

“…Cell division in Toxoplasma tachyzoites is founded on a number of ancestral mechanisms, especially those processes for copying (S phase) and segregating (mitosis) chromosomes ( Figure 2 ). Present apicomplexans possess the core DNA synthetic machinery conserved across eukaryote domains, including all subunits of the MCM helicase complex and functionally specialized DNA polymerases [ 9 , 10 ] ( Figure 3 and Box 2 ). Key factors that direct the DNA synthetic machinery to specific initiation sites in the chromosomes are also present [ 9 ].…”

Toxoplasma gondii UBL‐UBA shuttle proteins contribute to the degradation of ubiquitinylated proteins and are important for synchronous cell division and virulence

“…38 Disruption of TgOTUD3A results in dysregulation of cell cycle progression, suggesting the important roles of ubiquitin-mediated proteins degradation in cell cycle regulation. 39 In addition, studies about ECR1 and FBXO1, two putative E3 ligase which are important factors for Toxoplasma replication, have elucidated partial molecular basis for Toxoplasma endodyogeny.. 4,40 All these results show that ubiquitinylation plays key roles in Toxoplasma cell cycle regulation. Our present works that deletion of UBL-UBA shuttle proteins results in K48-linked polyubiquitinylated proteins accumulation and cell division asynchronous also confirmed this conclusion, however, the precise mechanisms are unknown.…”

Loss of the conserved Alveolate kinase MAPK2 decouplesToxoplasmacell growth from the cell cycle

“…A number of kinases with orthologs in model organisms have been identified as critical to the Toxoplasma cell cycle. These include kinases in the CDK (5,16), Aurora (17,42), and NEK (8) families. Progression through G1/S is a critical control point throughout eukaryota, and parasite kinases appear to perform similar functions to their metazoan orthologs in regulating this checkpoint.…”

“…Organelles are then partitioned between these elongating scaffolds (14). A growing number of the regulatory components of the parasite cell cycle have been identified, including homologs of scaffolds (4,15) and kinases (5,8,16,17) that are broadly conserved in other organisms.…”