Biology subjects

Suvorova, E. S.

Publications and source records attributed to Suvorova, E. S..

4 recordsLinked to original sources

bioRxiv2024

A FUCCI sensor reveals complex cell cycle organization of Toxoplasma endodyogeny

In this study, we report the atypical cell cycle organization of the unicellular eukaryotic pathogen Toxoplasma gondii. 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 the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probes, ToxoFUCCIS and ToxoFUCCISC. 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 the 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 ToxoFUCCIS 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 ToxoFUCCIS and its derivatives co-expressing epitope-tagged cell cycle markers have revealed an unusual composite cell cycle phase that incorporates overlapping S, G2, mitosis and cytokinesis (budding). We identified five intervals of the composite phase and their approximate duration: S (19%), S/G2/C (3%), S/M/C (9%), M/C (18%) and C/G1 (<1%). The ToxoFUCCIS probe efficiently detected G2/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.

cell biology↗

bioRxiv2023

Essential role of the Conserved Oligomeric Golgi complex in Toxoplasma gondii

Survival of the apicomplexan parasite Toxoplasma gondii depends on the proper functioning of many glycosylated proteins. Glycosylation is performed in the major membranous organelles ER and Golgi apparatus that constitute a significant portion of the intracellular secretory system. The secretory pathway is bidirectional: cargo is delivered to target organelles in the anterograde direction, while the retrograde flow maintains the membrane balance and proper localization of glycosylation machinery. Despite the vital role of the Golgi in parasite infectivity, little is known about its biogenesis in apicomplexan parasites. In this study we examined T. gondii Conserved Oligomeric Golgi (COG) complex and determined that, contrary to predictions, T. gondii expresses the entire eight-subunit complex and each complex subunit is essential for tachyzoite growth. Deprivation of the COG complex induces a pronounced effect on Golgi and ER membranes, which suggests the T. gondii COG complex has wider role in intracellular membrane trafficking. We demonstrated that besides its conservative role in protein glycosylation and retrograde intra-Golgi trafficking, the COG complex also interacted with anterograde and novel transport machinery. Furthermore, we identified coccidian-specific components of the Golgi transport system: TgUlp1 and TgGlp1. Protein structure and phylogenetic analyses revealed that TgUlp1 is an adaptation of the conservative Golgi tethering factor Uso1/p115, and together with Golgi-localized TgGlp1, TgUlp1 showed dominant interactions with the trafficking machinery that predicted to operate the endosome-to-Golgi recycling. Together, our study showed that T. gondii has expanded function of the conservative Golgi tethering COG complex and evolved additional regulators of the transport likely to serve parasite-specific secretory organelles.

cell biology↗

bioRxiv2017

The Toxoplasma centrocone houses cell cycle regulatory factors

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{degrees}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 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 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.\n\nIMPORTANCEParasites 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 well-studied 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. An example of this approach is described here with the discovery of a putative E3 ligase/protein kinase mechanism involved in regulating chromosome replication and mitotic processes of asexual stage parasites.

microbiology↗

bioRxiv2017

A checkpoint roadmap for the complex cell division of Apicomplexa parasites

The unusual cell cycles of Apicomplexa parasites are remarkably flexible with the ability to complete cytokinesis and karyokinesis coordinately or postpone cytokinesis for several rounds of chromosome replication are well recognized. Despite this surprising biology, the molecular machinery required to achieve this flexibility is largely unknown. In this study, we provide comprehensive experimental evidence that apicomplexan parasites utilize multiple Cdk-related kinases (Crks) to coordinate cell division. We determined that Toxoplasma gondii encodes seven atypical P-, H-, Y- and L- type cyclins and ten Crks to regulate cellular processes. We generated and analyzed conditional tet-OFF mutants for seven TgCrks and four TgCyclins that are expressed in the tachyzoite stage. These experiments demonstrated that TgCrk1, TgCrk2, TgCrk4 and TgCrk6, were required or essential for tachyzoite growth revealing a remarkable number of Crk factors that are necessary for parasite replication. G1 phase arrest resulted from the loss of cytoplasmic TgCrk2 that interacted with a P-type cyclin demonstrating that an atypical mechanism controls half the T. gondii cell cycle. We showed that T. gondii employs at least three TgCrks to complete mitosis. Novel kinases, TgCrk6 and TgCrk4 were required for spindle function and centrosome duplication, respectively, while TgCrk1 and its partner TgCycL were essential for daughter bud assembly. Intriguingly, mitotic kinases TgCrk4 and TgCrk6 did not interact with any cyclin tested and were instead dynamically expressed during mitosis indicating they may not require a cyclin timing mechanism. Altogether, our findings demonstrate that apicomplexan parasites utilize distinctive and complex mechanisms to coordinate their novel replicative cycles.

cell biology↗