A Close View of the Production of Bioactive Fungal Metabolites Mediated by Chromatin Modifiers.

Secondary metabolites produced by fungi are well known for their biological properties, which play important roles in medicine. These metabolites aid in managing infections and treating chronic illnesses, thereby contributing substantially to human health improvement. Despite this extensive knowledge, the vast biodiversity and biosynthetic potential of fungi is still largely unexplored, highlighting the need for further research in natural products. In this review, several secondary metabolites of fungal origin are described, emphasizing novel structures and skeletons. The detection and characterization of these metabolites have been significantly facilitated by advancements in analytical systems, particularly modern hyphenated liquid chromatography/mass spectrometry. These improvements have primarily enhanced sensitivity, resolution, and analysis flow velocity. Since the in vitro production of novel metabolites is often lower than the re-isolation of known metabolites, understanding chromatin-based alterations in fungal gene expression can elucidate potential pathways for discovering new metabolites. Several protocols for inducing metabolite production from different strains are discussed, demonstrating the need for uniformity in experimental procedures to achieve consistent biosynthetic activation.

Sepsis Impairs IFN-γ Production in CD8 T Cells through Changes in Local Chromatin Landscape.

Sepsis is a complex condition of inflammatory and immune dysregulation, triggered by severe infection. In survivors, chronic inflammation and immune dysregulation linger, facilitating the emergence of infections. CD8 dysfunction contributes to immunosuppression in sepsis survivors. We devised an animal model that enabled us to identify and analyze CD8-intrinsic defects induced by sepsis. We adoptively transferred CD45.1 CD8 OT-I T cells into CD45.2 congenic mice and subjected them to cecal ligature and puncture, to induce abdominal sepsis. One month later, we isolated the transferred CD8 cells. Surface marker expression confirmed they had not been activated through the TCR. CD8 OT-I T cells isolated from septic (or sham-operated) mice were transferred to second recipients, which were challenged with OVA-expressing Listeria monocytogenes. We compared effector capacities between OT-I cells exposed to sepsis and control cells. Naive mice that received OT-I cells exposed to sepsis had higher bacterial burden and a shorter survival when challenged with OVA-expressing L. monocytogenes. OT-I cells isolated from septic mice produced less IFN-γ but had conserved activation, expansion potential, and cytotoxic function. We observed lower transcript levels of IFN-γ and of the long noncoding RNA Ifng-as1, a local regulator of the epigenetic landscape, in cells exposed to sepsis. Accordingly, local abundance of a histone modification characteristic of active promoter regions was reduced in sepsis-exposed CD8 T cells. Our results identify a mechanism through which inflammation in the context of sepsis affects CD8 T cell function intrinsically.

Non-B DNA in plant genomes: prediction, mapping, and emerging roles.

Regulating gene expression in plant development and environmental responses is vital for mitigating the effects of climate change on crop growth and productivity. The eukaryotic genome largely shows the canonical B-DNA structure that is organized into nucleosomes with histone modifications shaping the epigenome. Nuclear proteins and RNA interactions influence chromatin conformations and dynamically modulate gene activity. Non-B DNA conformations and their transitions introduce novel aspects to gene expression modulation, particularly in response to environmental shifts. We explore the current understanding of non-B DNA structures in plant genomes, their interplay with epigenomics and gene expression, and advances in methods for their mapping and characterization. The exploration of so far uncharacterized non-B DNA structures remains an intriguing area in plant chromatin research and offers insights into their potential role in gene regulation.

Class I TCP transcription factors TCP14 and TCP15 promote axillary branching in Arabidopsis by counteracting the action of Class II TCP BRANCHED1.

Shoot branching is determined by a balance between factors that promote axillary bud dormancy and factors that release buds from the quiescent state. The TCP family of transcription factors is classified into two classes, Class I and Class II, which usually play different roles. While the role of the Class II TCP BRANCHED1 (BRC1) in suppressing axillary bud development in Arabidopsis thaliana has been widely explored, the function of Class I TCPs in this process remains unknown. We analyzed the role of Class I TCP14 and TCP15 in axillary branch development in Arabidopsis through a series of genetic and molecular studies. In contrast to the increased branch number shown by brc1 mutants, tcp14 tcp15 plants exhibit a reduced number of branches compared with wild-type. Our findings provide evidence that TCP14 and TCP15 act by counteracting BRC1 function through two distinct mechanisms. First, they indirectly reduce BRC1 expression levels. Additionally, TCP15 directly interacts with BRC1 decoying it from chromatin and thereby preventing the transcriptional activation of a set of BRC1-dependent genes. We describe a molecular mechanism by which Class I TCPs physically antagonize the action of the Class II TCP BRC1, aligning with their opposite roles in axillary bud development.

Transitions in chromatin conformation shaped by fatty acids and the circadian clock underlie hepatic transcriptional reorganization in obese mice.

The circadian clock system coordinates metabolic, physiological, and behavioral functions across a 24-h cycle, crucial for adapting to environmental changes. Disruptions in circadian rhythms contribute to major metabolic pathologies like obesity and Type 2 diabetes. Understanding the regulatory mechanisms governing circadian control is vital for identifying therapeutic targets. It is well characterized that chromatin remodeling and 3D structure at genome regulatory elements contributes to circadian transcriptional cycles; yet the impact of rhythmic chromatin topology in metabolic disease is largely unexplored. In this study, we explore how the spatial configuration of the genome adapts to diet, rewiring circadian transcription and contributing to dysfunctional metabolism. We describe daily fluctuations in chromatin contacts between distal regulatory elements of metabolic control genes in livers from lean and obese mice and identify specific lipid-responsive regions recruiting the clock molecular machinery. Interestingly, under high-fat feeding, a distinct interactome for the clock-controlled gene Dbp strategically promotes the expression of distal metabolic genes including Fgf21. Alongside, new chromatin loops between regulatory elements from genes involved in lipid metabolism control contribute to their transcriptional activation. These enhancers are responsive to lipids through CEBPß, counteracting the circadian repressor REVERBa. Our findings highlight the intricate coupling of circadian gene expression to a dynamic nuclear environment under high-fat feeding, supporting a temporally regulated program of gene expression and transcriptional adaptation to diet.

Novel putative causal mutations associated with fat traits in Nellore cattle uncovered by eQTLs located in open chromatin regions.

Intramuscular fat (IMF) and backfat thickness (BFT) are critical economic traits impacting meat quality. However, the genetic variants controlling these traits need to be better understood. To advance knowledge in this area, we integrated RNA-seq and single nucleotide polymorphisms (SNPs) identified in genomic and transcriptomic data to generate a linkage disequilibrium filtered panel of 553,581 variants. Expression quantitative trait loci (eQTL) analysis revealed 36,916 cis-eQTLs and 14,408 trans-eQTLs. Association analysis resulted in three eQTLs associated with BFT and 24 with IMF. Functional enrichment analysis of genes regulated by these 27 eQTLs revealed noteworthy pathways that can play a fundamental role in lipid metabolism and fat deposition, such as immune response, cytoskeleton remodeling, iron transport, and phospholipid metabolism. We next used ATAC-Seq assay to identify and overlap eQTL and open chromatin regions. Six eQTLs were in regulatory regions, four in predicted insulators and possible CCCTC-binding factor DNA binding sites, one in an active enhancer region, and the last in a low signal region. Our results provided novel insights into the transcriptional regulation of IMF and BFT, unraveling putative regulatory variants.

Exploring the epigenetic profile of ID4 in breast cancer: bioinformatic insights into methylation patterns and chromatin accessibility dynamics.

PURPOSE: Inhibitor of differentiation 4 (ID4) is a dominant-negative regulator of basic helix-loop-helix (bHLH) transcription factors. The expression of ID4 is dysregulated in various breast cancer subtypes, indicating a potential role for ID4 in subtype-specific breast cancer development. This study aims to elucidate the epigenetic regulation of ID4 within breast cancer subtypes, with a particular focus on DNA methylation and chromatin accessibility. METHODS: Bioinformatic analyses were conducted to assess DNA methylation and chromatin accessibility in ID4 regulatory regions across breast cancer subtypes. Gene Set Enrichment Analysis (GSEA) was conducted to identify related gene sets. Transcription factor binding within ID4 enhancer and promoter regions was explored. In vitro experiments involved ER+ breast cancer cell lines treated with estradiol (E2) and Tamoxifen. RESULTS: Distinct epigenetic profiles of ID4 were observed, revealing increased methylation and reduced chromatin accessibility in luminal subtypes compared to the basal subtype. Gene Set Enrichment Analysis (GSEA) implicated estrogen-related pathways, suggesting a potential link between estrogen signaling and the regulation of ID4 expression. Transcription factor analysis identified ER and FOXA1 as regulators of ID4 enhancer regions. In vitro experiments confirmed the role of ER, demonstrating reduced ID4 expression and increased methylation with estradiol treatment. Conversely, Tamoxifen treatment increased ID4 expression, indicating the potential involvement of ER signaling through ERα in the epigenetic regulation of ID4 in breast cancer cells. CONCLUSION: This study shows the intricate epigenetic regulation of ID4 in breast cancer, highlighting subtype-specific differences in DNA methylation and chromatin accessibility.

Chromatin structure and dynamics: one nucleosome at a time.

Eukaryotic genomes store information on many levels, including their linear DNA sequence, the posttranslational modifications of its constituents (epigenetic modifications), and its three-dimensional folding. Understanding how this information is stored and read requires multidisciplinary collaborations from many branches of science beyond biology, including physics, chemistry, and computer science. Concurrent recent developments in all these areas have enabled researchers to image the genome with unprecedented spatial and temporal resolution. In this review, we focus on what single-molecule imaging and tracking of individual proteins in live cells have taught us about chromatin structure and dynamics. Starting with the basics of single-molecule tracking (SMT), we describe some advantages over in situ imaging techniques and its current limitations. Next, we focus on single-nucleosome studies and what they have added to our current understanding of the relationship between chromatin dynamics and transcription. In celebration of Robert Feulgen's ground-breaking discovery that allowed us to start seeing the genome, we discuss current models of chromatin structure and future challenges ahead.

Cannabidiol modulates hippocampal genes involved in mitochondrial function, ribosome biogenesis, synapse organization, and chromatin modifications.

BACKGROUND: Cannabidiol (CBD) is one of the main cannabinoids present in Cannabis sativa female flowers. Previous investigation has already provided insights into the CBD molecular mechanism; however, there is no transcriptome data for CBD effects on hippocampal subfields. Here, we investigate transcriptomic changes in dorsal and ventral CA1 of adult mice hippocampus after 100 mg/kg of CBD administration (i.p.) for one or seven consecutive days. METHODS: C57BL/6JUnib mice were treated with either vehicle or CBD for 1 or 7 days. The collected brains were sectioned, and the hippocampal sub-regions were laser microdissected for RNA-Seq analysis. RESULTS: The transcriptome analysis following 7 days of CBD administration indicates the differential expression of 1559 genes in dCA1 and 2924 genes in vCA1. Furthermore, GO/KEGG analysis identified 88 significantly enriched biological process and 26 significantly enriched pathways for dCBD7, whereas vCBD7 revealed 128 enriched BPs and 24 pathways. CONCLUSION: This dataset indicates a widespread decrease of electron transport chain and ribosome biogenesis transcripts in CA1, while chromatin modifications and synapse organization transcripts were increased following CBD administration for 7 days.

Bioinspired peptides induce different cell death mechanisms against opportunistic yeasts.

The management of fungal diseases imposes an urgent need for the development of effective antifungal drugs. Among new drug candidates are the antimicrobial peptides, and especially their derivatives. Here, we investigated the molecular mechanism of action of three bioinspired peptides against the opportunistic yeasts Candida tropicalis and Candida albicans. We assessed morphological changes, mitochondrial functionality, chromatin condensation, ROS production, activation of metacaspases, and the occurrence of cell death. Our results indicated that the peptides induced sharply contrasting death kinetics, of 6 h for RR and 3 h for D-RR to C. tropicalis and 1 h for WR to C. albicans. Both peptide-treated yeasts exhibited increased ROS levels, mitochondrial hyperpolarization, cell size reduction, and chromatin condensation. RR and WR induced necrosis in C. tropicalis and C. albicans, but not D-RR in C. tropicalis. The antioxidant ascorbic acid reverted the toxic effect of RR and D-RR, but not WR, suggesting that instead of ROS there is a second signal triggered that leads to yeast death. Our data suggest that RR induced a regulated accidental cell death in C. tropicalis, D-RR induced a programmed cell death metacaspase-independent in C. tropicalis, while WR induced an accidental cell death in C. albicans. Our results were obtained with the LD100 and within the time that the peptides induce the yeast death. Within this temporal frame, our results allow us to gain clarity on the events triggered by the peptide-cell interaction and their temporal order, providing a better understanding of the death process induced by them.

Long noncoding RNA-mediated epigenetic regulation of auxin-related genes controls shade avoidance syndrome in Arabidopsis.

The long noncoding RNA (lncRNA) AUXIN-REGULATED PROMOTER LOOP (APOLO) recognizes a subset of target loci across the Arabidopsis thaliana genome by forming RNA-DNA hybrids (R-loops) and modulating local three-dimensional chromatin conformation. Here, we show that APOLO regulates shade avoidance syndrome by dynamically modulating expression of key factors. In response to far-red (FR) light, expression of APOLO anti-correlates with that of its target BRANCHED1 (BRC1), a master regulator of shoot branching in Arabidopsis thaliana. APOLO deregulation results in BRC1 transcriptional repression and an increase in the number of branches. Accumulation of APOLO transcription fine-tunes the formation of a repressive chromatin loop encompassing the BRC1 promoter, which normally occurs only in leaves and in a late response to far-red light treatment in axillary buds. In addition, our data reveal that APOLO participates in leaf hyponasty, in agreement with its previously reported role in the control of auxin homeostasis through direct modulation of auxin synthesis gene YUCCA2, and auxin efflux genes PID and WAG2. We show that direct application of APOLO RNA to leaves results in a rapid increase in auxin signaling that is associated with changes in the plant response to far-red light. Collectively, our data support the view that lncRNAs coordinate shade avoidance syndrome in A. thaliana, and reveal their potential as exogenous bioactive molecules. Deploying exogenous RNAs that modulate plant-environment interactions may therefore become a new tool for sustainable agriculture.

The antiproliferative effect of FGF2 in K-Ras-driven tumor cells involves modulation of rRNA and the nucleolus.

The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm.

BACKGROUND: In vitro incubation of epididymal and vas deferens sperm with Mn2+ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DNAses and thus far only described in epididymal mouse sperm, can be triggered in ejaculated sperm is yet to be elucidated. The current study aimed to determine if exposure of pig ejaculated sperm to divalent ions (Mn2+ and Mg2+) activates SCF, and whether this has any impact on sperm function and survival. For this purpose, sperm DNA integrity was evaluated through the Comet assay and Pulsed Field Gel Electrophoresis (PFGE); sperm motility and agglutination were assessed with computer assisted sperm analysis (CASA); and sperm viability and levels of total reactive oxygen species (ROS) and superoxides were determined through flow cytometry. RESULTS: Incubation with Mn2+/Ca2+ activated SCF in a dose-dependent (P < 0.05) albeit not time-dependent manner (P > 0.05); in contrast, Mg2+/Ca2+ only triggered SCF at high concentrations (50 mM). The PFGE revealed that, when activated by Mn2+/Ca2+ or Mg2+/Ca2+, SCF generated DNA fragments of 33-194 Kb, compatible with the size of one or multiple toroids. Besides, Mn2+/Ca2+ affected sperm motility in a dose-dependent manner (P < 0.05), whereas Mg2+/Ca2+ only impaired this variable at high concentrations (P < 0.05). While this effect on motility was concomitant with an increase of agglutination, neither viability nor ROS levels were affected by Mn2+/Ca2+ or Mg2+/Ca2+ treatments. CONCLUSION: Mn2+/Ca2+ and Mn2+/Ca2+ were observed to induce SCF in ejaculated sperm, resulting in DNA cleavage at TLRs. The activation of this mechanism by an intracellular, non-oxidative factor sheds light on the events taking place during sperm cell death.

Citrullination of actin-ligand and nuclear structural proteins, cytoskeleton reorganization and protein redistribution across cellular fractions are early events in ionomycin-induced NETosis.

Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release.

Genome-wide analysis of the chromatin sites targeted by HEX 70a storage protein in the honeybee brain and fat body.

Hexamerins, the proteins massively stored in the larval haemolymph of insects, are gradually used throughout metamorphosis as a source of raw material and energy for the development of adult tissues. Such behaviour defined hexamerins as storage proteins. Immunofluorescence experiments coupled with confocal microscopy show a hexamerin, HEX 70a, in the nucleus of the brain and fat body cells from honeybee workers, an unexpected localization for a storage protein. HEX 70a colocalizes with fibrillarin, a nucleolar-specific protein and H3 histone, thus suggesting a potential role as a chromatin-binding protein. This was investigated through chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq). The significant HEX 70a-DNA binding sites were mainly localized at the intergenic, promoter and intronic regions. HEX 70a targeted DNA stretches mapped to the genomic regions encompassing genes with relevant functional attributes. Several HEX 70a targeted genes were associated with H3K27ac or/and H3K27me3, known as active and repressive histone marks. Brain and fat body tissues shared a fraction of the HEX 70 targeted genes, and tissue-specific targets were also detected. The presence of overrepresented DNA motifs in the binding sites is consistent with specific HEX 70a-chromatin association. In addition, a search for HEX 70a targets in RNA-seq public libraries of fat bodies from nurses and foragers revealed differentially expressed targets displaying hex 70a-correlated developmental expression, thus supporting a regulatory activity for HEX 70a. Our results support the premise that HEX 70a is a moonlighting protein that binds chromatin and has roles in the brain and fat body cell nuclei, apart from its canonical role as a storage protein.

Chromatin Structure from Development to Ageing.

Nuclear structure influences genome architecture, which contributes to determine patterns of gene expression. Global changes in chromatin dynamics are essential during development and differentiation, and are one of the hallmarks of ageing. This chapter describes the molecular dynamics of chromatin structure that occur during development and ageing. In the first part, we introduce general information about the nuclear lamina, the chromatin structure, and the 3D organization of the genome. Next, we detail the molecular hallmarks found during development and ageing, including the role of DNA and histone modifications, 3D genome dynamics, and changes in the nuclear lamina. Within the chapter we discuss the implications that genome structure has on the mechanisms that drive development and ageing, and the physiological consequences when these mechanisms fail.

Differential chromatin binding preference is the result of the neo-functionalization of the TB1 clade of TCP transcription factors in grasses.

The understanding of neo-functionalization of plant transcription factors (TFs) after gene duplication has been extensively focused on changes in protein-protein interactions, the expression pattern of TFs, or the variation of cis-elements bound by TFs. Yet, the main molecular role of a TF, that is, its specific chromatin binding for the direct regulation of target gene expression, continues to be mostly overlooked. Here, we studied the TB1 clade of the TEOSINTE BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTORS (TCP) TF family within the grasses (Poaceae). We identified an Asp/Gly amino acid replacement within the TCP domain, originated within a paralog TIG1 clade exclusive for grasses. The heterologous expression of Zea mays TB1 and its two paralogs BAD1 and TIG1 in Arabidopsis mutant plants lacking the TB1 ortholog BRC1 revealed distinct functions in plant development. Notably, the Gly acquired in the TIG1 clade does not impair TF homodimerization and heterodimerization, while it modulates chromatin binding preferences. We found that in vivo TF recognition of target promoters depends on this Asp/Gly mutation and directly impacts downstream gene expression and subsequent plant development. These results provided new insights into how natural selection fine-tunes gene expression regulation after duplication of TFs to define plant architecture.

Chromatin Dispersion Test to Asses DNA Damage in Cervical Epithelial Cells.

The chromatin dispersion test (CDT) is based on the removal of nuclear proteins under the assumption that cells with fragmented DNA produce a typical halo of circular DNA loops, which is absent in cells with non-fragmented DNA. This method represents a simple, rapid, accurate, highly reproducible, and inexpensive technique to assess nuclear DNA damage in somatic cells. The visualization of DNA damage and the capacity of the test to provide a threshold value to discriminate between high and low levels of cervical lesions would aid in determining the malignant transformation. All of these advantages associated with the CDT protocol could promote this technique as a tool for the quick and reliable diagnosis of cervical epithelial disorders, even at primary-care centers.

Trypanosoma cruzi infection changes the chromatin proteome profile of infected human cells.

Chagas disease is endemic in 22 Latin American countries, with approximately 8 million individuals infected worldwide and 10,000 deaths yearly. Trypanosoma cruzi presents an intracellular life cycle in mammalian hosts to sustain infection. Parasite infection activates host cell responses, promoting an unbalance in reactive oxygen species (ROS) in the intracellular environment inducing genomic DNA lesions in the host cell during infection. To further understand changes in host cell chromatin induced by parasite infection, we investigated alterations in chromatin caused by infection by performing quantitative proteomic analysis. DNA Damage Repair proteins, such as Poly-ADP-ribose Polymerase 1 (PARP-1) and X-Ray Repair Cross Complementing 6 (XRRC6), were recruited to the chromatin during infection. Also, changes in chromatin remodeling enzymes suggest that parasite infection may shape the epigenome of the host cells. Interestingly, the abundance of oxidative phosphorylation mitochondrial and vesicle-mediated transport proteins increased in the host chromatin at the final stages of infection. In addition, Apoptosis-inducing Factor (AIF) is translocated to the host cell nucleus upon infection, suggesting that cells enter parthanatos type of death. Altogether, this study reveals how parasites interfere with the host cells' responses at the chromatin level leading to significant crosstalk that support and disseminate infection. SIGNIFICANCE: The present study provides novel insights into the effects of Trypanosoma cruzi on the chromatin from the host cell. This manuscript investigated proteomic alterations in chromatin caused by parasite infection at early and late infection phases by performing a quantitative proteomic analysis. In this study, we revealed that parasites interfere with DNA metabolism in the early and late stages of infection. We identified that proteins related to DNA damage repair, oxidative phosphorylation, and vesicle-mediated transport have increased abundance at the host chromatin. Additionally, we have observed that Apoptosis-inducing Factor is translocated to the host cell nucleus upon infection, suggesting that the parasites could lead the cells to enter Parthanatos as a form of programmed cell death. The findings improve our understanding on how the parasites modulate the host cell chromatin to disseminate infection. In this study, we suggest a mechanistic parasite action towards host nucleus that could be used to indicate targets for future treatments.

Separate To Operate: the Centriole-Free Inner Core of the Centrosome Regulates the Assembly of the Intranuclear Spindle in Toxoplasma gondii.

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.