Open Biology

BackgroundA hallmark of the eukaryotic cell is the regulated transport between the nucleus and cytoplasm, which is mediated by a multi-subunit protein assembly called the nuclear pore complex (NPC). While its overall architecture has been preserved across eukaryotes, the NPC structure varies in different organisms, which appears to have tuned its function. Outside of a handful of model systems, the NPC has not been comprehensively studied. This is particularly true of species that are not closely related to well-studied models, such as apicomplexan parasites. Indeed, the evolutionary divergence of Apicomplexa has complicated facile prediction of NPC proteins in these organisms. Because of this, the NPC components remain largely unidentified, and therefore NPC cellular function in Apicomplexa is poorly understood. Principal FindingsHere we identified, experimentally validated, and functionally characterized protein components of the NPC in the apicomplexan parasite Toxoplasma gondii. By combining proximity biotinylation with careful bioinformatic analysis, we identified 16 previously uncharacterized proteins that localize to the Toxoplasma NPC. We demonstrated 8 of these proteins are essential to parasite replication. Importantly, we defined components of the mRNA export machinery, as well as Nups required for the stability and/or assembly of specific NPC subcomplexes. Consistent with the evolutionary distance between Toxoplasma and well-studied models, the majority of our newly validated NPC components show no clear homology to NPC proteins in yeast, animals, or plants. Moreover, we demonstrated that the Toxoplasma mRNA export machinery has a distinct composition from other well-established systems. Intriguingly, Sus1, a well-defined protein of the TREX-2 and SAGA complexes, is missing from the Toxoplasma genome. In contrast, others, such as Centrin-3, have been conserved in Toxoplasma, but are not required for mRNA export in the parasite. ConclusionOur work highlights the distinct composition of multiple subcomplexes of the Toxoplasma NPC and paves the way for future studies to provide high-resolution structural information on the parasites unusual NPC architecture.

Despite glutamates widespread role as the dominant excitatory transmitter in vertebrate brains, the early evolution of glutamate and its recruitment into neural signaling remain largely unknown. The major limitation is the lack of information on its distribution in early-branching basal metazoans, such as ctenophores (comb jellies). Here, using glutamate immunoreactivity (IR) in two ctenophore species with distinct ecologies (Pleurobrachia bachei and Beroe abyssicola), we show that glutamate IR is present in subpopulations of neurons within the subepithelial neural network and in small groups of mesogleal neuron-like cells, and that it differentially labels some muscle fibers. Remarkably, we also observed an enriched glutamate-ir signal within the nuclei of subepithelial neurons in Beroe. However, glutamate expression levels are species-specific, suggesting a tight coupling of glutamate recruitment for neural communication with energetic demands.

The timing of DNA replication and centrosome duplication is tightly regulated with cell cycle progression to ensure the faithful duplication of the genome during cell division. Both DNA and centrosomes are licensed for replication in late telophase/early G1, replicated in S phase and segregated during mitosis; yet how defects in DNA replication licensing are coupled to centrosome homeostasis remains poorly understood. Here, we show that depletion of the replication licensing inhibitor Geminin in proliferating mouse embryonic fibroblasts induces robust centrosome amplification together with impaired primary cilium assembly. Rather than promoting whole-genome reduplication, knockdown of Geminin triggers a replication stress response, characterized by DNA damage accumulation throughout the cycle, and activation of an ATR-dependent DNA damage response. Mechanistically, Geminin depletion-induced replication stress activates the ATR-Chk1-Wee1 checkpoint axis prolonging G2 and leading to premature centriole disengagement and centrosome amplification. These findings identify replication stress as the signaling module that couples defective DNA replication licensing to centrosome amplification.

Diplonemids are highly abundant unicellular flagellates that are evolutionarily divergent from traditional model eukaryotes. Diplonema papillatum is a model diplonemid for which several molecular tools have recently been established. However, no method for conditional protein depletion was available. Here, we have adapted an auxin-inducible degron (AID) system in D. papillatum. Depletion of the KIN-A motor protein (a glycomonad-specific component of the chromosomal passenger complex) causes defects in chromosome segregation and cell proliferation. The AID system also successfully depleted a divergent Mad1 protein that localizes at nuclear pores, and a flagellar component, CFAP20. Our study demonstrates the power of the AID system to perform loss-of-function studies in D. papillatum.

Metabolic alterations commonly accompany neurodevelopmental disorders and may contribute to their pathophysiology. Neurofibromatosis type 1 (OMIM 162200) is a genetic disorder that results from mutations in the NF1 gene and its encoded neurofibromin protein (Nf1). The disorder is multisystemic, affecting multiple aspects of development, physiology, and brain function. In addition, recent evidence suggests that Nf1 deficiency alters metabolic function in both humans and animal models. Whether the metabolic alterations result from changes in neurodevelopment is not known. Here we approach this question in Drosophila melanogaster, which expresses a conserved NF1 gene, exhibits phenotypes reminiscent of the human disease, and shares key developmental mechanisms with humans. Flies with nf1 mutations or RNAi-mediated knockdown exhibit altered metabolism in adulthood. Conditional Nf1 inactivation revealed that the adult metabolic phenotype resulted from loss of Nf1 in neurons during a developmental critical period (third instar larva/pupa), which corresponded to the period of nervous system maturation. Prior to the developmental critical period, nf1 mutants did not exhibit metabolic difference - rather, the metabolic alterations appeared only after the critical period. This suggests that the adult phenotype results from the onset of the developmental alteration during the critical period. High-resolution respirometry on adult mitochondria revealed no differences in complex I/II function or fatty acid oxidation between nf1 mutants and controls, suggesting that the metabolic alterations localize upstream of the electron transport chain at the cellular level. Overall, these data suggest that loss of Nf1 alters adult metabolism via effects during a critical period of nervous system development.

Lamins are nuclear intermediate filament proteins that maintain nuclear architecture through interactions with the chromatin. The A- and B-type lamins tether the genome at the peripheral lamina underlying the inner nuclear membrane in the form of heterochromatic lamina-associated domains (LADs). However, LADs associated with A-type lamins are confined not only to lamina but also to the nuclear core, thereby pointing to their distinct and multifarious roles in chromatin organisation and regulation. Previous studies involving lamin B1 depletion depicted the detachment of LADs from the nuclear periphery, accompanied by alteration of chromatin distribution while preserving the topologically associating domains (TADs) in structurally intact form. In this piece of work, we have shown, for the first time in HGSOC, the effects of lamin A/C knockdown on chromatin organisation, which was characterised by significant detethering of gene-poor chromatin from the periphery along with active (A) to inactive (B) compartment switching. This was associated with a rewiring of oncogenic super-enhancer elements corroborated by the differential gene expression profile. Overall analysis tipped the scale in favour of reduced cellular proliferation upon lamin A/C knockdown. This finding was strengthened by the observed proliferative potential of spheroids ex vivo and tumours in a mouse xenograft model.

Heterozygous pathogenic variants in CSNK2A1 or CSNK2B encoding the Casein Kinase 2 (CK2) protein complex, lead to pediatric neurodevelopmental disorders, Okur-Chung Neurodevelopmental Syndrome (OCNDS) and Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS). OCNDS and POBINDS are characterized by a range of symptoms, including developmental delay, intellectual disability, facial dysmorphism, and seizures. Despite over 250 reported cases of OCNDS and POBINDS, we do not fully understand how specific alterations in CK2 relate to the heterogeneity observed in patients. To investigate this, we used the fruit fly, Drosophila melanogaster, as a model system. To assess variant impact, we co-expressed human CSNK2A1 and CSNK2B reference or disease-causing variants in flies. In parallel, we determined the role of Drosophila CkII in the developing and mature nervous system, specifically in neurons and glia. We found that 12/13 variants tested act as full or partial loss-of-function with one CSNK2A1 variant showing gain-of-function. Phospho-proteomic studies in neurons revealed separate signatures for loss- and gain-of-function variants. We found that neuronal and glial CkII is critical for organismal development. Reduction of neuronal CkII in the adult nervous system causes motor and seizure-like phenotypes. Finally, given the known role of CK2 in potentiating Wnt/{beta}-catenin signalling, we show that Wnt agonists partially rescue phenotypes associated with adult-specific neuronal reduction of CkII. This work generates Drosophila models of CSNK2A1 and CSNK2B expression to functionally assess variant impact, as well as an adult-specific neuronal loss-of-function model for drug screening and mechanistic studies.

Spatiotemporal allocation and dynamic redistribution of centrosomal proteins are critical for coordinating the structural and molecular events that ensure successful cell division. While depletion of the multifunctional protein Optineurin (OPTN) impairs cell-cycle progression, our previous report of the presence of OPTN at key cell-division assemblies raised the question of how its trafficking is coordinated during cell division. In this study, using cell-cycle synchronisation coupled with flow cytometric analysis and confocal microscopy, we report that OPTN progressively dissociates from centrosomes during mitosis, redistributes to cytokinetic structures, accumulates at the spindle midzone, and then concentrates at the midbody during cytokinesis. Moreover, following this transition, OPTN progressively re-emerges at centrosomes during G1 of the subsequent cell cycle. Further, microtubule perturbation and regrowth assays provide evidence that OPTN recruitment and retention at the pericentriolar matrix (PCM) can occur independently of microtubules, whereas its exit is microtubule-dependent. Collectively, this study depicts, for the first time, a detailed movement trail of OPTN, highlighting its centrosome-linked redistribution and microtubule dependency. This context-dependent localisation and trafficking of OPTN across the centrosome-midbody axis establish a definitive spatiotemporal roadmap that can be strategically utilised to study its functional role in cell division.

Background. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative route for electron transfer, contributing to respiratory flexibility and redox homeostasis under stress conditions. In trypanosomatids, AOX displays a highly uneven distribution associated with extensive diversification of mitochondrial electron transport chains (ETCs). In addition to canonical AOX, many trypanosomatids encode an enigmatic AOX-like protein (AOX-L), whose evolutionary origin and functional significance remain unresolved. Results. Here, we investigated the evolutionary distribution, phylogenetic relationships, structural conservation, and functional divergence of AOX and AOX-L across euglenozoans. Comparative genomic analyses revealed a patchy distribution of both proteins among kinetoplastids, with multiple independent losses during evolution. Phylogenetic analyses demonstrated that AOX and AOX-L represent distinct evolutionary lineages, indicating independent origins rather than duplication-derived divergence. While AOX showed a conserved eukaryotic origin, AOX-L occupied a separate phylogenetic position and lacked key residues required for ubiquinol oxidation. Structural modelling revealed that AOX-L retained the characteristic AOX fold, predicted membrane association, and dimeric organization, but lacked the catalytic architecture necessary for enzymatic activity. Transcriptomic and biochemical analyses of four AOX-containing trypanosomatid species showed that AOX expression and activity correlated with mitochondrial ETC organization. Species lacking cytochrome-dependent complexes III and IV displayed substantially higher AOX expression and enzymatic activity, consistent with AOX functioning as the primary terminal oxidase in these lineages. Conversely, species retaining a canonical ETC exhibited lower AOX activity, suggesting a role in metabolic flexibility and redox regulation. Conclusions. Our study provides a comprehensive evolutionary and functional framework for alternative oxidases in trypanosomatids. We demonstrate that AOX and AOX-L are evolutionarily distinct proteins with different predicted functions: AOX maintains respiratory electron flow according to lineage-specific mitochondrial requirements, whereas AOX-L represents a structurally conserved but catalytically inactive protein family that may have acquired an alternative regulatory role. These findings highlight how mitochondrial respiratory components diversify during eukaryotic evolution and provide a basis for future investigations into the biological function of AOX-L.

The highly conserved body plan of nematodes makes members of this phylum excellent models to study cell type evolution. Early branching nematode lineages, mostly occupying aquatic habitats, usually contain caudal glands deployed for underwater attachment to a substrate, but have been thought to lack phasmid sensory organs, resulting in their historical classification as "Aphasmidia". With the transition to a terrestrial environment, nematodes lost caudal glands and gained phasmid sensory neurons. The supposed mutually exclusive existence of caudal glands and phasmids has led to the suggestion that phasmid neurons may have evolved from caudal glands. Here, we rule out this possibility through light and electron microscopical analysis of Mononchus aquaticus, a member of the early branching Dorylaimia lineage, showing that phasmid sensory neurons and caudal glands do coexist. This observation not only argues against a proposed cell type evolution scenario accompanying aquatic-to-terrestrial transitions but also indicates that the presence of phasmid sensory organs may have been an ancestral trait of the nematode phylum.

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Orc6 is the smallest and least evolutionarily conserved among all ORC subunits. In Drosophila, Orc6 binds tightly with the core ORC(1-5) and is required for DNA binding and replication initiation, whereas in Xenopus and human systems Orc6 loosely associates with the rest of the complex resulting in some differences for replication-associated activities. Despite these variations, Orc6 remains essential for viability in all species. In current study we analyzed specific residues within the C-terminal 11 helix that is critical for stable association of Orc6 with the ORC complex in Drosophila. Human Orc6 lacks these residues, however it possesses a strong nuclear localization signal (NLS) that is absent in Drosophilidae. We propose that this NLS drives human protein to the nucleus and compensates for weaker Orc6-ORC(1-5) interactions by increasing the nuclear concentration of Orc6 and shifting the equilibrium toward formation of the fully assembled ORC complex at the DNA.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

PTEN-induced kinase 1 (PINK1) is a mitochondrial serine/threonine kinase that initiates ubiquitin-dependent mitophagy and is mutated in early-onset Parkinson's disease. Despite extensive characterization of insect PINK1 orthologues, obtaining soluble and catalytically active recombinant Homo sapiens PINK1 (HsPINK1) has remained challenging, limiting its biochemical and structural investigation. Here, we identify an evolutionary divergence in the mechanisms supporting PINK1 stability and activity. Comparative analysis of 48 metazoan PINK1 orthologues revealed that predicted binding to the mitochondrial import receptor TOM20 is predominantly a vertebrate feature and coincides with divergence of the PINK1 N- and C-terminal extensions (NTE and CTE). Consistent with this distinction, invertebrate PINK1 orthologues were purified as active kinases, whereas vertebrate orthologues lacked detectable ubiquitin kinase activity. NMR spectroscopy demonstrated that humanization of ten residues within the Tribolium castaneum (Tc)PINK1 NTE-CTE interface is sufficient to confer direct binding to human TOM20, while co-expression with TOM20 increased the activity of recombinant HsPINK1. Guided by these evolutionary differences, we engineered a chimeric PINK1 containing a predominantly human kinase domain supported by Tc-derived NTE-CTE elements. The resulting kinase was catalytically active and required compatible interactions between the NTE-CTE and kinase C-lobe for activity. The chimera enabled characterization of PINK1 variants and revealed R152W as a likely pathogenic variant. Together, our findings support a model in which the NTE and CTE act as intramolecular determinants of PINK1 kinase stability and suggest that vertebrate PINK1 has evolved an increased dependence on the mitochondrial import machinery to maintain a functional state.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Abstract The neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) controls behaviours that are essential for the parasitic lifestyle of plant parasitic nematodes (PPNs). Here, we used serotonin immunochemistry and in situ hybridization for the serotonin synthesis genes, Gpa-tph-1 and Gpa-bas-1, to map its signalling pathways in Globodera pallida J2 infective stages. We identified fourteen serotonin-immunoreactive cell bodies around the pharyngeal median bulb and revealed that eleven of them co-express Gpa-tph-1 and Gpa-bas-1, confirming these are serotonin-synthesising neurons. Serotonin immunoreactivity was also localised in structures consistent with labelling of amphids, and in sixteen non-neuronal cells located in the middle and posterior region of the nematode. Depletion of serotonin with reserpine reduced the serotonin immunoreactivity in all fourteen cell bodies confirming their neuronal identity, but not in non-neuronal tissue. We identified additional, but infrequent expression of Gpa-tph-1 in two cells and expression of Gpa-bas-1 in one pair of symmetrically bilateral cells anterior to the median bulb, that lacked serotonin immunoreactivity. The results reveal distinct pools of serotonin distribution in G. pallida and indicate serotonin signalling is more complex than in other parasitic nematodes and C. elegans, highlighting evolutionary divergence in neurotransmitter identity.

Pannexin 1 (PANX1) is a channel-forming glycoprotein that has been implicated in regulating melanoma cell properties such as growth, migration, and invasion. Here we report that the CRISPR-Cas9 mediated ablation of PANX1 or channel inhibition with probenecid shifted A375-P cell cycle distribution towards G2/M, while channel inhibition with spironolactone stalled cell cycle progression at the G1/S checkpoint regardless of PANX1 expression. Bulk RNA sequencing revealed cell cycle related gene expression was downregulated with PANX1 targeting strategies and this was exacerbated when PANX1 deficient cells were treated with spironolactone. When grown as spheroids, we found the deletion and inhibition of PANX1 disrupted spheroid compaction, with spheroids displaying poorly defined borders and reduced circularity. The deletion of PANX1 increased spheroid fragmentation, and this phenotype was intensified when spheroids devoid of PANX1 were treated with spironolactone, highlighting a potential synthetic lethal effect. Using liquid chromatography mass spectrometry, we sought to determine whether PANX1 had binding partners that may act to stabilize spheroid integrity and found that PANX1 co-immunoprecipitated various cell adhesion molecules such as {beta}-actin and F-actin-capping protein subunit -1, suggesting PANX1 and its interacting partners may stabilize cell-cell contacts. When control and PANX1 ablated cells were grown in chick chorioallantoic membrane xenografts to assess their tumour growth characteristics, we discovered that the deletion of PANX1 reduced tumour surface area and viability. Our findings indicate that PANX1 regulates cell cycle progression and the structural architecture of A375-P melanoma tumours, highlighting the therapeutic utility of targeting PANX1 in melanoma.

Trisomy 21 (T21) is the most common genetic cause of intellectual disability, yet the molecular mechanisms underlying maternal meiosis I errors--responsible for ~70% of free T21 cases--remain poorly understood. In this preliminary study, we used long-read sequencing and genome assembly to investigate the DNA sequence and epigenetic features of chromosome 21 (chr21) centromeres in a family with recurrent free T21 due to maternal meiosis I errors. The mother, who had two affected and three unaffected children, showed no mosaicism or structural rearrangements. One of her two chr21 centromeres lacked a pronounced centromere dip region (CDR), displaying instead a diffuse hypomethylation pattern (dCDR) with much higher methylated CpG levels (55%) compared to its homologue (36%). This dCDR was transmitted to an unaffected child and the affected proband analyzed, suggesting it was present in one of the maternal chr21 since she was at least 32 years of age. Chr21 dCDRs were not observed in seven young mothers with children with T21 or previously described in the literature in 108 population haplotypes. We hypothesize that dCDRs may weaken kinetochore function, increasing nondisjunction risk, and propose two models linking such epigenetic variation to maternal age-related T21 risk. These findings highlight the value of complete centromere characterization in families with children with T21 and suggest centromere methylation status of chr21 as a potential T21 risk factor for future investigation.

The tunicate Ciona robusta offers a tractable non-vertebrate chordate model for probing gene function via tissue-specific, CRISPR/Cas9-mediated mutagenesis in F0. Building on Arcadia Sciences Zoogle platform, which identifies and ranks orthologs of human genes from various non-traditional model organisms, we carried out a pilot project to probe the developmental roles of three notochord- and endoderm-expressed candidate orthologs of human disease genes (Fcho, Pgm3, and Nckap1) alongside a fourth gene (Plastin) implicated in papilla cell elongation. This preprint compiles and updates a series of research project milestones previously posted episodically on Zenodo. Here we summarize the full results and our conclusion about this pilot project. Using CRISPR/Cas9, we found that tissue-specific knockout of Pgm3 and, to a lesser extent, Fcho caused significant defects in larval tail elongation. Separately, CRISPR knockout of Plastin, an actin-bundling gene expressed throughout the sensory-adhesive papillae of the larva, caused a subtle reduction in papilla cell elongation when combined as a duoble knockout with another actin-bundling protein-encoding gene, Villin. These results identify Pgm3 as the most promising candidate for further development as a Ciona-based model of human disease and demonstrate the utility of tissue-specific CRISPR screening for prioritizing candidate disease gene orthologs identified through comparative genomics platforms like Zoogle.

Cyclic episodes of chromosome compaction and de-condensation are features of eukaryotic cell division that aid mitotic segregation and help prevent aneuploidy. While biophysical data on mitotic chromosome structure has been previously obtained, heterogeneity within samples can confound analyses and precludes direct like-for-like comparisons. To circumvent this, we employed advanced flow cytometry to purify specific metaphase chromosomes with biotinylated telomeres from stably engineered mouse cells. We show that ESC-derived metaphase chromosomes 3 and 19 display distinct properties but share a conserved force-dependent mechanical response. In contrast, chromosome equivalents isolated from NSCs and preB cells show markedly different force-dependent responses, reflecting progressive differentiation stages. Covalent crosslinking of ESC-derived chromosomes alters biomechanical properties to mimic equivalents from more differentiated cells. Collectively, these results highlight the need to isolate specific, homogeneous metaphase chromosome samples to accurately decipher their complex behaviours.

Background: Nervous system regeneration requires creation of new neurons and their integration into new and existing networks. How new neurons find their targets and reconnect in the environment of regenerating tissue remains a major gap in knowledge. The planarian Schmidtea mediterranea is well suited to the study of neural regeneration due to the remarkable ability of planarians to regenerate their entire body, including the nervous system. However, we have not yet been able to explore synaptogenesis in regeneration using planarians due to a lack of tools to study synapses. Here we investigate potential players in planarian synapse, focusing on the conserved syntaxin protein family, which functions in neurotransmitter vesicle fusion at the synapse. Results: We found that reduction of either syntaxin-1 (stx1) or syntaxin-binding protein 1-1 (stxbp1-1) decreased locomotion and impeded brain regeneration, specifically in synapse-rich regions. Conclusions: Our observations support STX-1 and STXBP1-1 as bona fide synapse components, providing a starting point for studies neuronal connectivity during homeostatic remodeling and regeneration.