Biology subjects

Marsilia, C.

Publications and source records attributed to Marsilia, C..

3 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

The Crk4-Cyc4 complex regulates G2 phase of apicomplexan endodyogeny

Division of apicomplexan parasites differs drastically from the division of their host cells. A fraction of apicomplexans divides in the traditional binary mode, such as Toxoplasma gondii in asexual stages, whereas the vast majority instead divide in a multinuclear fashion. Such variety of replication modes and a dearth of conserved conventional regulators have hindered the progress of apicomplexan cell cycle studies. We previously identified five Cdk-related kinases (Crk) involved in endodyogenic division of T. gondii tachyzoites. The current study investigates the roles of a novel essential cell cycle kinase TgCrk4. We identified this kinase cyclin partner and demonstrated that TgCrk4 regulates processes carried out during conventional G2 phase, such as repression of chromosome rereplication and centrosome re-duplication. Accumulation of TgCyc4 in the nucleus and on the centrosomes supported the role of TgCrk4-TgCyc4 complex as a coordinator of chromosome and centrosome cycles in T. gondii. Examination of the TgCrk4-deficient tachyzoites confirmed a cell cycle stop prior to the TgCrk6-regulated spindle assembly checkpoint. Furthermore, we identified an ortholog of the DNA replication licensing factor Cdt1 that was a dominant interactor of the TgCrk4-TgCyc4 complex. T. gondii Cdt1 is highly divergent but preserved critical signature domains and appeared to play a minimal or no role in licensing DNA replication in G1 phase. Functional analyses indicated the primary role of TgCdt1 is in controlling chromosome rereplication and centrosome reduplication. Global phosphoproteome analyses identified immediate TgCrk4 substrates, such as DNA replication licensing factor TgORC4, component of the anaphase-promoting complex TgCdc20, {gamma}-tubulin nucleation factor TgGCP2, and the catalytic subunit of cell cycle phosphatase TgPP2ACA. Importantly, our phylogenetic and structural analyses revealed that the functional TgCrk4-TgCyc4 complex was encoded in the limited group of apicomplexans dividing in a binary fashion. Together with the minimal representation of binary division in Apicomplexa phylum, our findings support the novel view of apicomplexans acquiring binary division to repress ancestral multinuclear mechanisms.

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↗