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Centrosomes are the major microtubule organising centres of animal cells. Deregulation in their number occurs in cancer and was shown to trigger tumorigenesis in mice. However, the incidence, consequence and origins of this abnormality are poorly understood. Here, we screened the NCI-60 panel of human cancer cell lines to systematically analyse centriole number and structure. Our screen shows that centriole amplification is widespread in cancer cell lines and highly prevalent in aggressive breast carcinomas. Moreover, we identify another recurrent feature of cancer cells: centriole size deregulation. Further experiments demonstrate that severe centriole over-elongation can promote amplification through both centriole fragmentation and ectopic procentriole formation. Furthermore, we show that overly long centrioles form over-active centrosomes that nucleate more microtubules, a known cause of invasiveness, and perturb chromosome segregation. Our screen establishes centriole amplification and size deregulation as recurrent features of cancer cells and identifies novel causes and consequences of those abnormalities. Cancer cells are characterised by abnormalities in the number of centrosomes and this phenotype is linked with tumorigenesis. Here the authors report centriole length deregulation in a subset of cancer cell lines and suggest a link with subsequent alterations in centriole numbers and chromosomal instability.

Toxoplasmosis is a disease of worldwide distribution, causing high morbidity and mortality in humans, as well as heavily impacting animal health and the economy. Toxoplasma gondii, the causative agent, is an intracellular parasite with a complex life cycle whose completion entails asexual, pre-sexual, and sexual stage conversions. Pre-sexual and sexual differentiation take place only within the intestinal epithelium of felines. Recently, several transcriptional factors and epigenetic components crucial to trigger parasite stage transitions within the cat have been identified, allowing, through precise genetic manipulation, obtaining pre-sexual stages known as merozoites in vitro. Through conditional depletion of two pre-sexual stage-specific gene silencing transcription factors, AP2XII-1 and AP2XII-2, we have characterized the interplay between cell division and the sequence of events leading up to differentiation of tachyzoites into merozoites. We explored genome duplication, assembly of daughter cells, karyokinesis, and cytokinesis, characterizing the differential cell division modes and kinetics undergone by critical structures along the differentiation axis. Building onto the pre-existing body of knowledge, primarily describing the underpinnings of these forms of division by transmission electron microscopy, our work contributes previously unexplored temporal and spatial resolution to the transitions between endodyogeny and endopolygeny, providing a conceptual framework for understanding and exploring T. gondii’s route of sexual differentiation.IMPORTANCESexual development in Toxoplasma gondii is essential for transmission, but remains poorly understood, largely because pre-sexual stages are restricted to the feline intestine and have only recently become experimentally accessible. Here, we leverage an in vitro differentiation system to resolve how parasites transition toward merozoite formation at the cellular level. By combining expansion microscopy, stage-specific markers, and quantitative analyses, we define the temporal sequence of nuclear division and daughter cell assembly during merogony, addressing longstanding ambiguity regarding division modes in these stages. Our findings reveal that parasites can adopt alternative division strategies emerging from a polyploid intermediate, highlighting an unexpected degree of flexibility in how cell division is executed during differentiation. Beyond refining this developmental framework, this work establishes a foundation for future mechanistic studies of pre-sexual biology and provides broader insight into the diversity of eukaryotic cell division strategies.

Toxoplasma gondii is a ubiquitous apicomplexan parasite that can infect virtually any warm-blooded animal. Acquired infection during pregnancy and the placental breach, is at the core of the most devastating consequences of toxoplasmosis. T. gondii can severely impact the pregnancy’s outcome causing miscarriages, stillbirths, premature births, babies with hydrocephalus, microcephaly or intellectual disability, and other later onset neurological, ophthalmological or auditory diseases. To tackle T. gondii’s vertical transmission, it is important to understand the mechanisms underlying host-parasite interactions at the maternal-fetal interface. Nonetheless, the complexity of the human placenta and the ethical concerns associated with its study, have narrowed the modeling of parasite vertical transmission to animal models, encompassing several unavoidable experimental limitations. Some of these difficulties have been overcome by the development of different human cell lines and a variety of primary cultures obtained from human placentas. These cellular models, though extremely valuable, have limited ability to recreate what happens in vivo . During the last decades, the development of new biomaterials and the increase in stem cell knowledge have led to the generation of more physiologically relevant in vitro models. These cell cultures incorporate new dimensions and cellular diversity, emerging as promising tools for unraveling the poorly understood T. gondii ´s infection mechanisms during pregnancy. Herein, we review the state of the art of 2D and 3D cultures to approach the biology of T. gondii pertaining to vertical transmission, highlighting the challenges and experimental opportunities of these up-and-coming experimental platforms.

Toxoplasma gondii parasites cause toxoplasmosis, arguably the most widespread and prevalent parasitosis of humans and animals. During the clinically relevant stage of its life cycle, the parasites divide by endodyogeny. Centrosomes are the main microtubule-organizing center of the cell. They are normally formed by two centrioles, embedded in a cloud of proteins known as pericentriolar material (PCM). The PCM ascribes centrioles with their microtubule nucleation capacity. Toxoplasma gondii , the causative agent of toxoplasmosis, divides by endodyogeny. Successful cell division is critical for pathogenesis. The centrosome, one of the microtubule organizing centers of the cell, plays central roles in orchestrating the temporal and physical coordination of major organelle segregation and daughter cell formation during endodyogeny. The Toxoplasma centrosome is constituted by multiple domains: an outer core, distal from the nucleus; a middle core; and an inner core, proximal to the nucleus. This modular organization has been proposed to underlie T. gondii ’s cell division plasticity. However, the role of the inner core remains undeciphered. Here, we focus on understanding the function of the inner core by finely studying the localization and role of its only known molecular marker; TgCep250L1. We show that upon conditional degradation of TgCep250L1 parasites are unable to survive. Mutants exhibit severe nuclear segregation defects. In addition, the rest of the centrosome, defined by the position of the centrioles, disconnects from the nucleus. We explore the structural defects underlying these phenotypes by ultrastructure expansion microscopy. We show that TgCep250L1’s location changes with respect to other markers, and these changes encompass the formation of the mitotic spindle. Moreover, we show that in the absence of TgCep250L1, the microtubule binding protein TgEB1, fails to localize at the mitotic spindle, while unsegregated nuclei accumulate at the residual body. Overall, our data support a model in which the inner core of the T. gondii centrosome critically participates in cell division by directly impacting the formation or stability of the mitotic spindle. IMPORTANCE Toxoplasma gondii parasites cause toxoplasmosis, arguably the most widespread and prevalent parasitosis of humans and animals. During the clinically relevant stage of its life cycle, the parasites divide by endodyogeny. In this mode of division, the nucleus, containing loosely packed chromatin and a virtually intact nuclear envelope, parcels into two daughter cells generated within a common mother cell cytoplasm. The centrosome is a microtubule-organizing center critical for orchestrating the multiple simultaneously occurring events of endodyogeny. It is organized in two distinct domains: the outer and inner cores. We demonstrate here that the inner core protein TgCEP250L1 is required for replication of T. gondii . Lack of TgCEP250L1 renders parasites able to form daughter cells, while unable to segregate their nuclei. We determine that, in the absence of TgCEP250L1, the mitotic spindle, which is responsible for karyokinesis, does not assemble. Our results support a role for the inner core in nucleation or stabilization of the mitotic spindle in T. gondii .

Toxoplasmosis, caused by the obligate intracellular parasite Toxoplasma gondii , is one of the most prevalent zoonotic parasitic infections worldwide. When acquired during pregnancy, T. gondii can be transmitted to the fetus, with clinical outcomes influenced by gestational age at time of infection and the parasite’s genotype. Prenatal screening enables the detection of maternal seroconversion and offers a critical window for intervention. In Uruguay, despite mandatory serological screening during pregnancy, national data on T. gondii seroprevalence and maternal seroconversion have not been updated in two decades. In addition, the genetic diversity of local strains remains poorly characterized. In this study, we analysed publicly available serological data from pregnant individuals attending Uruguay’s largest public maternity hospital between 2019 and 2023. We found that seroprevalence has modestly declined from 50% (reported in 1998) to 45.5%, with a congenital transmission rate of 0.58%. Clinical analysis of affected newborns revealed chorioretinitis as the predominant manifestation. To investigate parasite diversity, we performed genotyping of T. gondii strains using in silico PCR-RFLP following molecular detection. Our findings revealed substantial genetic diversity, including novel allele combinations not previously described in the region. These results highlight both the continued public health burden and the evolving genetic landscape of T. gondii in Uruguay. Our findings underscore the need to strengthen surveillance and prevention strategies for congenital toxoplasmosis in South America.

Key Points Apicomplexa are eukaryotic parasites that cause important human and veterinary diseases, such as malaria, toxoplasmosis and cryptosporidiosis. Apicomplexa replicate within the cells of their hosts by highly flexible and adaptable mechanisms, which can generate thousands of progeny to spread the infection. The cell division of Apicomplexa occurs by closed mitosis of the nucleus and budding of daughter cells. The regulation of apicomplexan cell cycle progression occurs at a global level throughout the cytoplasm and at a local level for each individual nucleus. Control is exerted by the activity of regulatory kinases, by modulating transcription, translation and protein stability and by the presence of physical tethers. The apicomplexan nucleus is highly structured into defined nuclear territories. Centromeres and telomeres occupy defined positions, which are closely connected to the position of the centrosome to ensure genome and epigenome integrity during division. Daughter cells are assembled in a stepwise and highly ordered process that is temporally and spatially guided by cytoskeletal self-organization and that is physically linked to the centrosome. The centrosome emerges as a central regulatory location for the progression and completion of cell division in Apicomplexa. Apicomplexan parasites have a complex life cycle that relies on highly regulated development inside host cells. Here, Francia and Striepen give an overview of apicomplexan cell cycle progression and cell division and the ways in which these processes are spatially and temporally guided by the centrosome in these pathogenic parasites. Toxoplasma gondii and Plasmodium falciparum are important human pathogens. These parasites and many of their apicomplexan relatives undergo a complex developmental process in the cells of their hosts, which includes genome replication, cell division and the assembly of new invasive stages. Apicomplexan cell cycle progression is both globally and locally regulated. Global regulation is carried out throughout the cytoplasm by diffusible factors that include cell cycle-specific kinases, cyclins and transcription factors. Local regulation acts on individual nuclei and daughter cells that are developing inside the mother cell. We propose that the centrosome is a master regulator that physically tethers cellular components and that provides spatial and temporal control of apicomplexan cell division.

Microtubule organizing centers (MTOCs) perform critical cellular tasks by nucleating, stabilizing, and anchoring microtubule’s minus ends. These capacities impact tremendously a wide array of cellular functions ranging from ascribing cell shape to orchestrating cell division and generating motile structures, among others. The phylum Apicomplexa comprises over 6000 single-celled obligate intracellular parasitic species. Many of the apicomplexan are well known pathogens such as Toxoplasma gondii and the Plasmodium species, causative agents of toxoplasmosis and malaria, respectively. Microtubule organization in these parasites is critical for organizing the cortical cytoskeleton, enabling host cell penetration and the positioning of large organelles, driving cell division and directing the formation of flagella in sexual life stages. Apicomplexans are a prime example of MTOC diversity displaying multiple functional and structural MTOCs combinations within a single species. This diversity can only be fully understood in light of each organism’s specific MT nucleation requirements and their evolutionary history. Insight into apicomplexan MTOCs had traditionally been limited to classical ultrastructural work by transmission electron microscopy. However, in the past few years, a large body of molecular insight has emerged. In this work we describe the latest insights into nuclear MTOC biology in two major human and animal disease causing Apicomplexans: Toxoplasma gondii and Plasmodium spp.

Apicomplexa are intracellular parasites that cause important human diseases including malaria and toxoplasmosis. During host cell infection new parasites are formed through a budding process that parcels out nuclei and organelles into multiple daughters. Budding is remarkably flexible in output and can produce two to thousands of progeny cells. How genomes and daughters are counted and coordinated is unknown. Apicomplexa evolved from single celled flagellated algae, but with the exception of the gametes, lack flagella. Here we demonstrate that a structure that in the algal ancestor served as the rootlet of the flagellar basal bodies is required for parasite cell division. Parasite striated fiber assemblins (SFA) polymerize into a dynamic fiber that emerges from the centrosomes immediately after their duplication. The fiber grows in a polarized fashion and daughter cells form at its distal tip. As the daughter cell is further elaborated it remains physically tethered at its apical end, the conoid and polar ring. Genetic experiments in Toxoplasma gondii demonstrate two essential components of the fiber, TgSFA2 and 3. In the absence of either of these proteins cytokinesis is blocked at its earliest point, the initiation of the daughter microtubule organizing center (MTOC). Mitosis remains unimpeded and mutant cells accumulate numerous nuclei but fail to form daughter cells. The SFA fiber provides a robust spatial and temporal organizer of parasite cell division, a process that appears hard-wired to the centrosome by multiple tethers. Our findings have broader evolutionary implications. We propose that Apicomplexa abandoned flagella for most stages yet retained the organizing principle of the flagellar MTOC. Instead of ensuring appropriate numbers of flagella, the system now positions the apical invasion complexes. This suggests that elements of the invasion apparatus may be derived from flagella or flagellum associated structures.

While infection with the parasite Toxplasma gondii is largely asymptomatic in healthy adults, severe disease and death can result in immunocompromised individuals and in those infected congenitally. With minimal treatments for toxoplasmosis available, it is crucial to study parasite-specific processes to identify new drug targets. This study investigated the protein TgGSK, uncovering its essentiality for parasite proper division and survival. We performed an in-depth study of the functional role of this kinase. Importantly, TgGSK was shown to bear higher homology to plant proteins than its mammalian counterparts, which may allow for specific targeting of this protein.