The apicomplexan centrosome has a unique bipartite structure comprising an inner and outer core responsible for the nuclear cycle (mitosis) and budding cycles (cytokinesis), respectively. Although these two cores are always associated, they function independently to facilitate polyploid intermediates in the production of many progeny per replication round. Here, we describe the function of a large coiled-coil protein in Toxoplasma gondii, TgCep250, in connecting the two centrosomal cores and promoting their structural integrity. Throughout the cell cycle, TgCep250 localizes to the inner core but, associated with proteolytic processing, is also present on the outer core during the onset of cell division. In the absence of TgCep250, stray centrosome inner and outer core foci were observed. The detachment between centrosomal inner and outer cores was found in only one of the centrosomes during cell division, indicating distinct states of mother and daughter centrosomes. In mammals, Cep250 processing is required for centrosomal splitting and is mediated by Nek phopsphorylation. However, we show that neither the nonoverlapping spatiotemporal localization of TgNek1 and TgCep250 nor the distinct phenotypes upon their respective depletion support conservation of this mechanism in Toxoplasma. In conclusion, TgCep250 has a tethering function tailored to the unique bipartite centrosome in the Apicomplexa.

A unique aspect of apicomplexan biology is the requirement for egress from and invasion into host red blood cells (RBCs). The cellular mechanisms and molecular mediators of RBC egress and invasion remain poorly characterized in Babesia spp., a group of parasites of veterinary importance and emerging cause of zoonotic disease. Through the use of video microscopy, transcriptomics, and chemical genetics we have implicated signaling, proteases and gliding motility in egress and/or invasion by Babesia divergens. We developed CRISPR/Cas9 and two inducible knockdown systems to perform a genetic screen of putative mediators of egress. We found that proteases ASP2 and ASP3 are required for invasion, and the latter is also required for egress. Strikingly, parasites continue to replicate intracellularly in the absence of the protein kinases, PKG or CDPK4, indicating that they are required for exit from the replication cycle and egress. These essential molecules present druggable targets for Babesia spp. All together we have established a molecular framework for the spread of infection through host RBCs, with egress of B. divergens more closely resembling T. gondii than the more evolutionarily related Plasmodium spp.

The phylum Apicomplexa encompasses deadly pathogens such as malaria and Cryptosporidium. Apicomplexa cell division is mechanistically divergent from that of their mammalian host, potentially representing an attractive source of drug targets. Depending on the species, apicomplexan parasites can modulate the output of cell division, producing two to thousands of daughter cells at once. The inherent flexibility of their cell division mechanisms allows these parasites to adapt to different niches, facilitating their dissemination. Toxoplasma gondii tachyzoites divide using a unique form of cell division called endodyogeny. This process involves a single round of DNA replication, closed nuclear mitosis, and assembly of two daughter cells within a mother. In higher Eukaryotes, the four-subunit chromosomal passenger complex (CPC) (Aurora kinase B (ARKB)/INCENP/Borealin/Survivin) promotes chromosome bi-orientation by detaching incorrect kinetochore-microtubule attachments, playing an essential role in controlling cell division fidelity. Herein, we report the characterization of the Toxoplasma CPC (Aurora kinase 1 (Ark1)/INCENP1/INCENP2). We show that the CPC exhibits dynamic localization in a cell cycle-dependent manner. TgArk1 interacts with both TgINCENPs, with TgINCENP2 being essential for its translocation to the nucleus. While TgINCENP1 appears to be dispensable, interfering with TgArk1 or TgINCENP2 results in pronounced division and growth defects. Significant anti-cancer drug development efforts have focused on targeting human ARKB. Parasite treatment with low doses of hesperadin, a known inhibitor of human ARKB at higher concentrations, phenocopies the TgArk1 and TgINCENP2 mutants. Overall, our study provides new insights into the mechanisms underpinning cell cycle control in Apicomplexa, and highlights TgArk1 as potential drug target.

The Apicomplexa are famously named for their apical complex, a constellation of organelles at their apical end dedicated to invasion of their host cells. In contrast, at the other end of the cell, the basal complex (BC) has been overshadowed since it is much less prominent and specific functions were not immediately obvious. However, in the past decade a staggering array of functions have been associated with the BC and strides have been made in understanding its structure. Here, these collective insights are supplemented with new data to provide an overview of the understanding of the BC in Toxoplasma gondii. The emerging picture is that the BC is a dynamic and multifunctional complex, with a series of (putative) functions. The BC has multiple roles in cell division: it is the site where building blocks are added to the cytoskeleton scaffold; it exerts a two-step stretch and constriction mechanism as contractile ring; and it is key in organelle division. Furthermore, the BC has numerous putative roles in ‘import’, such as the recycling of mother cell remnants, the acquisition of host-derived vesicles, possibly the uptake of lipids derived from the extracellular medium, and the endocytosis of micronemal proteins. The latter process ties the BC to motility, whereas an additional role in motility is conferred by Myosin C. Furthermore, the BC acts on the assembly and/or function of the intravacuolar network, which may directly or indirectly contribute to the establishment of chronic tissue cysts. Here we provide experimental support for molecules acting in several of these processes and identify several new BC proteins critical to maintaining the cytoplasmic bridge between divided parasites. However, the dispensable nature of many BC components leaves many questions unanswered regarding its function. In conclusion, the BC in T. gondii is a dynamic and multifunctional structure at the posterior end of the parasite.

23Invasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for 24 their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility 25 and invasion-related proteins from microneme organelles is regulated by oscillations of 26 intracellular Ca 2+ . Later stages of invasion are considered Ca 2+ -independent, including 27 the secretion of proteins required for host cell entry and remodeling from the parasite's 28 rhoptries. We identified a family of three Toxoplasma proteins with homology to the 29 ferlin family of double C2 domain-containing Ca 2+ sensors. In humans and model 30 organisms such Ca 2+ sensors orchestrate Ca 2+ -dependent exocytic membrane fusion with 31 the plasma membrane. One ferlin that is conserved across the Apicomplexa, TgFER2, 32 localizes to the parasite's cortical membrane skeleton, apical end, and rhoptries. 33Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes 34 normally and were completely motile. However, these parasites were unable to invade 35 host cells and were therefore not viable. Specifically, knockdown of TgFER2 prevented 36 rhoptry secretion and these parasites failed to form the moving junction on the parasite-37 host interface necessary for host cell invasion. Collectively, these data demonstrate that 38 the putative Ca 2+ sensor TgFER2 is required for the secretion of rhoptries. These findings 39 provide the first regulatory and mechanistic insights into this critical yet poorly 40 understood aspect of apicomplexan host cell invasion. 41 42 3 Graphical abstract 43 44 45