Open Biology

Protein phosphorylation is a central post-translational modification that regulates signaling pathways across all living organisms. Through the antagonistic activities of protein kinases and phosphatases, phosphorylation modulates protein function by inducing conformational changes that affect enzyme activity, protein-protein interactions, stability, and subcellular localization. These molecular events regulate diverse cellular processes, including cell cycle progression, differentiation, gene expression, and metabolism. In unicellular parasites such as Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., specialized signaling pathways have evolved to enable adaptation to the fluctuating environments of insect vectors and mammalian hosts. In many eukaryotes, phosphorylation of histone H3 at serine 10 (H3Ser10p) is essential for proper chromosome condensation during mitosis and is catalyzed by Aurora kinase B. Although trypanosomatids possess an Aurora kinase B homolog and a conserved serine residue at position 10 of histone H3, this modification had not been previously detected in these organisms. Here, using a stage-specific approach, we report the first detection of H3Ser10p in T. cruzi and explore its association with cell cycle progression. Western blot analyses using a specific antibody revealed H3Ser10p in exponentially growing epimastigotes, both in total protein extracts and nucleosome-enriched fractions, indicating its incorporation into chromatin. Fluorescence microscopy showed that this histone mark is restricted to the nuclei of dividing cells. Furthermore, H3Ser10p was detected exclusively in replicative stages of the parasite. Analysis of cell cycle-associated structures and flow cytometry demonstrated that H3Ser10 phosphorylation is dynamically regulated, peaking in the G2/M phase. These findings identify H3Ser10p as a novel epigenetic mark in T. cruzi that is tightly regulated during the cell cycle.

All cells of a multicellular organism usually share an identical genome, faithfully transmitted through successive divisions. Yet, a number of animal species deviate from this dogma, as parts of their DNA are systematically eliminated in all their somatic nuclei, in a process called Programmed DNA Elimination (PDE). PDE leads to the unexpected reorganisation of the genome at every generation in all somatic cells but its molecular mechanism, evolutionary origins, and functional significance remain unknown. This lack of understanding partially stems from limitations in genetically tractable model species. PDE can target an entire chromosome, or involve chromosome fragmentation followed by selective fragment retention and elimination, raising further questions on genome stability, genome integrity and mechanisms of DNA repair. PDE by chromosome fragmentation has been described in parasitic nematodes in the family Ascarididae, copepods in the genus Cyclops and unicellular ciliates. More recently, PDE has been discovered in three non-parasitic, lab-tractable nematode species from the Rhabditidae family, opening new perspectives. In this study, we used cytological approaches to screen 25 new Rhabditidae species for PDE. We found evidence of PDE in 17 species. Our work reveals that PDE is present in 12 out of 17 tested genera, demonstrating its widespread presence in Rhabditidae nematodes, with the notable exception of C. elegans. Genetic tools have already been established for some species. This work provides a collection of lab-tractable species that can be used to test many aspects of somatic Programmed DNA Elimination by chromosome fragmentation in animals.

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and preliminary structure-functional analyses of three crystal structures of TvPPi-PFK are presented. All three structures organize as tetramers with the conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. Comparative analysis with structural neighbors from other organisms demonstrated that despite sharing <29% sequence identity, TvPPi-PFKs protomer shares overall topology with both PPi- and ATP-dependent PFKs. Mass photometry confirmed that TvPPi-PFK formed tetramers under near-physiological conditions. Unexpectedly, TvPPi-PFK crystals dephosphorylate ATP to AMP during soaking. In all three structures, either ATP or AMP is bound at the enzymes dimer interface, typical of ATP-PFKs, but a novel finding for PPi-PFKs. Furthermore, a sugar phosphate binding site was observed in proximity to the ATP-binding site. Thus, the three reported TvPPi-PFK structures validate its established PPi-dependent activity while revealing previously unreported ATP and sugar phosphate binding. This study also lays a foundation for future research into putative ATP-dependent activity of TvPPi-PFK and for evaluating known phosphofructokinase inhibitors as potential therapeutics for trichomoniasis. These findings expand our understanding of PFK superfamily diversity and support the continued exploration of TvPPi-PFK as a drug target for trichomoniasis. SynopsisThe production, crystallization, and three crystal structures of a pyrophosphate-dependent phosphofructokinase from Trichomonas vaginalis (TvPPi-PFK) reveal ATP binding and structural similarity to both ATP-dependent and pyrophosphate-dependent phosphofructokinases. TvPPi-PFK dephosphorylates ATP and has a novel ATP-PFK-like ATP-binding cavity.

African trypanosomes employ specialised mechanisms of membrane trafficking as a key strategy to persist in both the mammalian host and insect vector. Their survival and pathogenicity rely on the continuous synthesis and surface delivery of extremely abundant surface coat proteins, imposing an extraordinary biosynthetic burden on the secretory pathway. Despite this, the luminal proteome of the T. brucei endoplasmic reticulum (ER) and Golgi apparatus remains incompletely characterised. Here, we exploit TurboID proximity biotinylation, using the abundant ER chaperone BiP (Binding-immunoglobulin protein) as luminal bait to map the ER proteome in bloodstream and procyclic lifecycle stages of Trypanosoma brucei. Comparison with BiPN, a truncated secretory form of BiP that transits the Golgi, provides differential compartmental labelling, together identifying 366 (BiP) and 428 (BiPN) proximity partners respectively and encompassing established ER quality control machinery, secretory cargo, and Golgi proteins. Quantitative ranking of BiP labelling intensity identifies a cohort of candidate BiP interactors: the most strongly enriched is Tb927.5.1160, a protein sharing structural homology with the mammalian BiP nucleotide exchange inhibitor MANF (mesencephalic astrocyte-derived neurotrophic factor). Endogenous mNeonGreen tagging confirms ER localisation of TbMANF in both life cycle stages, and reciprocal manipulation of its abundance by RNAi and inducible expression produces opposing shifts in cellular sensitivity to ER stress. These data are consistent with a role in regulating BiP ATPase cycling in an organism that, unlike yeast and mammals, lacks a canonical unfolded protein response, making TbMANF the first candidate regulator of BiP activity identified in kinetoplastids. Finally, TurboID proximity labelling anchored at the inner face of the nuclear pore via NUP65 extends our endomembrane map to the inner nuclear membrane, identifying candidate proteins of this specialised ER-continuous domain. AUTHOR SUMMARYAfrican sleeping sickness is caused by Trypanosoma brucei, a parasite that survives in the mammalian bloodstream by constantly renewing its protective protein coat. To synthesise and export this surface coat, the parasite relies on two intracellular compartments, the endoplasmic reticulum (ER) and the Golgi apparatus, which function as a quality control and sorting factory for proteins entering the secretory pathway. However, the identity of the proteins that populate these compartments in blood-stage parasites, and that maintain functioning under stress conditions, has remained poorly mapped. Here, we used an enzyme-based proximity labelling strategy that identifies neighbouring proteins in live cells without disturbing their targeting signals, generating a comprehensive protein inventory of both compartments across the two main T. brucei lifecycle stages. Among the most strongly labelled proteins was Tb927.5.1160, a protein structurally related to a mammalian regulator of the master ER chaperone BiP. Reducing or increasing the abundance of Tb927.5.1160 in parasites produced opposite changes in ER stress tolerance, identifying it as a candidate modulator of ER homeostasis in a lineage that regulates protein quality control through mechanisms distinct from those operating in yeast or human cells. Together, our findings provide a new molecular resource for understanding how T. brucei sustains secretory pathway function under the biosynthetic demands of mammalian and insect host infection.

Echinoderms possess a complex immune system, primarily relying on coelomocytes - immune cells circulating in coelomic fluids. Over the last few decades, various coelomocytes have been described based on morphological features, with holothuroids exhibiting the highest diversity of cell morphotypes among the different echinoderm classes. However, while the overall immune function of these cells is broadly accepted, their respective functions remain unclear, and molecular data specific to the different cell types are still limited in the literature. In this study, we address this gap in functional information and molecular data by using single-cell RNA sequencing (scRNA-seq) on coelomocytes from the perivisceral fluid of Holothuria forskali. We identified 10 distinct clusters, each assumed to correspond to a distinct transcriptional coelomocyte population. Among these, cluster 0 occupies a central position relative to the others, suggesting it may represent "progenitor cells", whereas cluster 6 is markedly divergent from all other clusters. Functional enrichment analyses revealed that some clusters ensure key immune functions, including pathogen recognition, phagocytosis, complement activation and redox balance regulation. In addition, examination of the processed samples under a microscope confirms the presence of a small proportion of recently discovered carotenocytes (7.0%) in the perivisceral fluid, a cell type rich in carotenoids. By using transcriptomics data previously obtained for this cell type by bulk RNA sequencing (bRNA-seq), it was possible to confidently identify cluster 6 as carotenocytes and provide further insights into their gene expression. While further analyses are needed to link other clusters to the different morphotypes previously described in the literature, this pioneer study presents preliminary data on the functional diversity of holothuroid coelomocytes, which could be of broad interest for a better understanding of holothuroid immunity as well as for the study of immune cell lineage evolution across deuterostomes.

Sponges are widely recognized as important model organisms for studying animal evolution, due to their phylogenetic position at the base of the animal tree of life, as well as similarities to the nearest animal relatives, the choanoflagellates. A critical aspect of animal evolution concerns the origin of germ layers, the embryonic structures which give rise to all tissues and organs of animal bodies. Haeckels hypothesis suggested a germ layer homology between sponges and corals, and thus all eumetazoans (complex animals including cnidarians and bilaterians). According to this hypothesis, sponge choanoderm (composed of the feeding cells, choanocytes) and sponge pinacoderm (the outer epithelium) would be homologous to eumetazoan endoderm (from which the digestive system originates) and the ectoderm (giving rise to the epidermis), respectively. We addressed this hypothesis comparing tissue-specific transcriptomes derived from single-cell transcriptome datasets of sponges and cnidarians. We have sequenced single cell transcriptomes of Australian calcareous sponge, Sycon capricorn, and identified its cell types using a combination of in silico annotation of the cell clusters and in situ hybridization with marker genes. Single-cell transcriptome datasets for two demosponge species and two cnidarian species were extracted from recent literature. Homology was assessed using the SAMap algorithm, which has been designed to identify homologous cell types across vast evolutionary distances by detection of shared expression profiles. Our results are fully consistent with Haeckels hypothesis, supporting homology between the innermost layers of sponges and cnidarians (choanoderm and endoderm/gastrodermis) as well as the outermost layers of sponges and cnidarians (pinacoderm and ectoderm/epidermis). Thus, sponge body plan appears to represent an intermediate step between single cell protists (choanoflagellates) and complex animals, rather than being independent experiment in animal multicellularity as suggested by alternative hypotheses.

In tunicates, larvaceans represent a fascinating case of evolution, where the chordate body plan has been maintained despite a rapidly evolving genome characterized by strong In contrast to other tunicates, larvaceans keep the chordate body plan during their entire life. They have acquired a highly specialized epithelium in charge of producing the "house", a complex extracellular apparatus used for filter feeding in the plankton. To what extent the house and this epithelium represent true molecular innovations withing chordates is a question for which thorough transcriptomics can bring novel insights. We conducted a developmental profiling of gene expression at the single-cell level in the larvacean Oikopleura dioica. We provide detailed descriptions of cellular transcriptomes associated with the house-synthesizing organ, which permits to define the molecular specifics of epithelial cell territories. We followed their emergence during development, and we identified genes that represent key candidate molecules for regulating the morphogenesis of the house-producing organ. Dynamic changes in gene expression and cell identities during major developmental transitions of the lifecycle illustrate that our dataset effectively allows access to the diversity of O. dioicas cell types in embryos and in adults. The resources presented here constitute critical assets to investigate larvacean biology and evolution for mechanistic and comparative goals.

Left-right asymmetry of the brain is well recognized in various animals including C. elegans, drosophila and zebrafish. In primates, most of the brain studies describe side of the brain. However, in spite of huge amounts of accumulating rodent studies on neuroscience, most of rodent studies do not distinguish the brain side. The pig brain is considered to occupy an intermediate position between primates and rodents in terms of structural complexity and brain function. Moreover, the numbers of studies using genetic manipulation of pigs are drastically increasing. So, we investigated microminipig (MMP) brain mesoscopic anatomy focusing on left-right differences of its morphology. Here, we show the anterior cingulate cortex, perirhinal cortex, and cerebellum of male and female MMPs, are structurally asymmetrical. The cerebellar vermis, paravermis is tilted from the midline and the consequently the cerebellar cortex exhibits asymmetrical morphology. The anterior cingulate gurus exhibited protrusion and invagination toward the midline on the right and left side, respectively. The left perirhinal lobe exhibited distinct patterns of cortical gyration between left and right side. These data demonstrate that MMPs are one of the suitable model animals for investigating cerebral and cerebellar asymmetry.

FAM122A regulates cell cycle progression through inhibition of the PP2A-B55 phosphoprotein phosphatase. Recent structural work has uncovered helical elements in the N-terminus of FAM122A as binding determinants for PP2A-B55 but whether FAM122A inhibition towards PP2A-B55 is regulated is presently unclear. To address this we performed a systematic analysis of the PP2A-B55 interaction with FAM122A in cells uncovering a novel region in the C-terminus of FAM122A, spanning residues 150-170, required for binding. This C-terminal region and the N-terminal helices are both required for efficient binding to PP2A-B55 suggesting a bipartite binding mechanism. We perform amino acid resolution scans of FAM122A 150-170 uncovering several residues in this region contributing to binding including the conserved Ser158, a reported phosphorylation site. We show that Ser158 is important for PP2A-B55 inhibition in human cells as well as efficient stimulation of mitotic entry in Xenopus laevis egg extracts. In human cells and in Xenopus laevis Ser158 phosphorylation is regulated with increased occupancy correlating with cell cycle stages requiring PP2A-B55 inhibition. Collectively our work uncovers novel aspects of FAM122A interaction with PP2A-B55 and provides a possible mechanism for how the inhibitory activity of FAM122A can be regulated during the cell cycle.

Coordination of metabolism, cell growth and cell division is essential to life. Recent single-cell measurements in S. cerevisiae have shown that metabolic processes and the cellular redox state are dynamic along the cell cycle. However, it is unknown whether similar metabolic oscillations also occur in other organisms. Until now, the dynamics of metabolism in other eukaryotes have predominantly been studied in cell cycle synchronised populations. Since cell cycle synchronisation methods can perturb metabolism, they may also introduce artefacts in the recorded dynamics. Here, we performed time-lapse microscopy analyses of exponentially growing single cells of the budding yeast S. cerevisiae, the fission yeast S. pombe and murine leukaemia L1210 cells. Measuring the NAD(P)H autofluorescence and the cell surface area growth rate in unsynchronised cells, we discovered oscillations along the cell cycle of the cellular redox state and lipid metabolism, respectively. Thus, our work shows that metabolism is dynamic along the cell cycle of these three evolutionarily distant eukaryotic organisms. This finding suggests that such metabolic oscillations could be a conserved characteristic among eukaryotes.

Horizontal gene transfer (HGT) is one of the fundamental processes in the evolution of prokaryotic genomes, while its importance in eukaryotes is still debated. Some of the hallmark eukaryotic organelles, such as mitochondria and chloroplasts, are of an ancient endosymbiotic origin. The process of acquiring (and losing) new endosymbionts is dynamic and still ongoing in many lineages. Endosymbiotic gene transfer (EGT) between symbionts and their hosts has been considered as one of the major sources of overall HGTs in eukaryotes. Thanks to recent advances in genomics and microscopy, more and more endosymbionts are discovered in protists offering suitable models for the study of EGT and its impact on the host. Recently, the presence of holosporacean and chlamydiacean symbionts in the novel strains of marine euglenozoan flagellates of genus Rhynchopus has been discovered. Here, we present an analysis of the genomes and transcriptomes of five Rhynchopus strains and examine the extent of EGT/HGT and the role of endosymbiosis in shaping the nuclear genome of symbiont-bearing hosts. Our results have shown that there is no evidence of a recent EGT from either Holosporales or Chlamydiales symbionts. The absence of such transfers suggests that EGT or at least a stable retention of EGT genes is not a requisite for a successful endosymbiosis. Furthermore, our results show striking differences between patterns of detected HGTs among the Rhynchopus lineages pointing to a dynamic and largely neutral evolution of horizontally-acquired genes.

Schistosoma mansoni is a parasitic flatworm that has two, genetically determined, sexes. We used aggregated data of 8 posttranslational histone modifications (ChIP-Seq), chromatin accessibility (ATAC-Seq), transcription (RNA-Seq) and genome feature annotations to decipher the histone code of genes involved in the differentiation of schistosome gonads (i.e. female ovaries and male testes). We show schistosome gonads express at least two classes of protein coding genes: H3K4me3-positive genes that display canonical features of eukaryotic protein-coding genes such as peaks of H3K4me3 at the transcription start sites (TSS) and increases in histone acetylation marks towards the transcription end site (TES), but also a non-canonical H3K9/27me3 plateau just upstream of the TSS. H3K4me3 enrichment at the TSS is highly predictive for transcription strength in these genes compared to a second class of protein coding genes (H3K4me3-negative genes) that do not display this pattern and is characterised by absence of the investigated histone marks at TSS and TES. This is indicative of the existence of hitherto unknown, potentially schistosome-specific histone marks in these genes. The absence of H3K4me3 at the TSS is not associated with inducible or stable gene expression in the gonads. Instead, gene ontology analysis indicates that H3K4me3-positive genes are related to functions which typically govern processes such as metabolism or signal transduction while H3K4me3-negative genes are dedicated to cell communication or immune responses. Second, individual histone modifications and their combinations are associated with functional features of the schistosome genome, known as "chromatin colours". In H3K4me3-positive genes, there is clear co-linearity of 3 colours, which strongly suggests a functional role for histone modifications in the control of transcription pre-initiation, promotor release, and transcription termination. Third, there are striking chromatin structure changes during maturation of the gonads in all genomic features including protein-coding and non-protein coding genes as well as repetitive sequences. The nature of these changes is different in both sexes. H3K36me3 and H3K9me3, as well as H3K23ac and H3K9ac show the strongest variations. Last, we show that pharmacological inhibition of histone demethylation activity by IOX1 leads to a concentration-dependent separation ("divorce") of schistosome couples confirming the importance of H3K36/H3K9 methylation for pairing maintenance and indicating histone demethylases as a potential drug target family. Collectively, our findings offer unprecedented insights into histone codes and chromatin dynamics governing the reproductive development of S. mansoni gonads.

Colorectal cancer across sub-Saharan Africa presents a growing global health burden, with increasing cases and mortality linked to late diagnosis, limited healthcare access and lack of effective treatments. African patients typically present with aggressive disease marked by distinct genomic signatures, indicating the need for targeted treatment approaches. We integrated genetic modelling, phenotypic scoring, imaging and biochemical analysis to explore how mutations found in individual Nigerian colorectal cancer patients influence drug responsiveness. We used the data from Cancer Genome Atlas to identify mutation profiles specific to Nigerian patients. We then generated ten stable Drosophila melanogaster personalised patient avatar lines designed to model patient genomic profiles. This study focused on three lines; each line included oncogenic RAS plus targeting patient-specific variants. These models exhibited various phenotypes including altered larval size, gut size and reduced survival. Two of the three avatar lines showed improved survival, reduced hindgut proliferation zone expansion and restored redox balance after treatment with regorafenib and trametinib. Mirroring clinical patient responses, we found that response to therapy is dependent on the specific genetic profile of the tumour. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/714433v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@110518aorg.highwire.dtl.DTLVardef@5965a0org.highwire.dtl.DTLVardef@11f16a3org.highwire.dtl.DTLVardef@744a1_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIAfrican colorectal cancer showed distinct mutation patterns that contribute to tumour heterogeneity. C_LIO_LIPatient-derived Drosophila avatars were engineered using tumour-specific genetic mutations with key features of human colorectal cancer. C_LIO_LITreatment with targeted therapies showed responses patterned by tumour genotype. C_LIO_LIResponse patterns indicated the need for personalised for colorectal cancer therapies among diverse populations. C_LI

Dendritic spines are highly dynamic structures whose morphology and lifespan are modified in response to synaptic efficacy changes. Structural modifications following activity support the long-term encoding of information and could allow for the remodeling of neural circuits. Long-term depression (LTD) is a key mechanism for synaptic weight regulation, yet its structural correlates -- particularly for long-lasting, protein synthesis dependent forms -- remain poorly understood. Furthermore, in humans, this type of plasticity is often disrupted in neurodevelopmental disorders, correlating with cognitive dysfunction and structural abnormalities. Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and is characterized by excessive metabotropic receptor-mediated synaptic depression, excessive protein synthesis, and spine abnormalities. Here, we investigate the relationship between long lasting synaptic depression and structural plasticity, as well as the role of protein availability in determining how many spines can simultaneously undergo structural changes during LTD in both healthy and FXS mutant neurons. Using high resolution optical methods, we developed and tested a method for inducing metabotropic glutamate receptor (mGluR)-dependent depression at single spines via glutamate uncaging in mouse hippocampal neurons. We found that this form of activity leads to robust spine shrinkage, which requires new protein synthesis. However, when we induced this depression at multiple spines, they competed for structural changes and only one spine shrank. We hypothesized that this was due to limited resources, in the form of newly made proteins, and therefore, we decided to test if spine competition would be altered in the mouse model of FXS, where protein levels are abnormally elevated. Indeed, we found that competition was absent in FXS mutant neurons, and all of the stimulated spines underwent shrinkage following LTD induction. Importantly, we found that single spine structural plasticity in FXS was expressed to the same degree as in WT controls. Taken together, these findings suggest that the hallmark phenotype of excess mGluR LTD in FXS may result from a greater number of inputs undergoing synaptic depression, rather than excessive LTD at individual synapses. By probing plasticity at the level of individual inputs, our findings highlight the importance of evaluating activity across groups of synapses, in order to uncover plasticity interactions that are critical for learning. Understanding how these mechanisms are disrupted in neurodevelopmental disorders such as FXS can inform the development of effective therapeutic strategies.

Protein subcellular localisation is informative for understanding potential protein function, particularly in highly structured unicellular eukaryotes. Microscopy is especially powerful for interrogating localisation, providing high resolution single cell data about where a protein resides. We previously generated the TrypTag dataset - a genome-wide protein localisation resource for the human unicellular parasite Trypanosoma brucei using fluorescent protein tagging. This is a puissant dataset due to its scale: Originally captured with high content image analysis in mind, it is a formidable resource for machine learning or artificial intelligence tool development and testing. Here, we describe a Python module for programmatic access to this data rich resource. Images of each tagged cell line, together with segmented cell masks, can be accessed arbitrarily by gene ID and tagging terminus, the database can be searched by protein localisation, and tools are provided to assist foundational image analysis of individual T. brucei cell cycle stage and morphology. We stress-tested this tool by using it to examine a key feature of T. brucei morphogenesis during division: The old and newly formed flagellum and associated organelles tend to have different protein compositions, and using the TrypTag toolkit we show that there is extensive age-based differential content of these organelles while the daughter nuclei completely lack such asymmetry.

Unraveling how adult neurons reshape their architecture is key to understanding post-developmental plasticity. Drosophila clock neurons, which remodel their terminals on a daily basis, offer a unique model to examine the mechanisms underlying structural plasticity. In this study, we examine the impact of the experimental design on the remodeling process. We established a simple fixation protocol that preserves tissue integrity and prevents its deformation while enabling the fixation of a larger number of individuals within the appropriate time window. We show that intrinsic (i.e., targeting fluorescent reporters to the membrane) or extrinsic (i.e., temperature) variables may influence this dynamic process. Examining ex vivo preparations, we found that the s-LNv terminals display numerous thin filopodia extending from their synaptic boutons. However, these fine membrane protrusions are lost upon fixation, as they could only be accurately visualized ex vivo. Finally, we present MorphoScope, a Python-based interface that eliminates observer bias in complexity measurements. Altogether, we present a powerful and robust model to investigate the principles of adult neuronal plasticity, with implications extending beyond circadian biology.

Presynaptic homeostatic plasticity (PHP) is a potent form of homeostatic plasticity that has been documented at synapses as diverse as the glutamatergic Drosophila neuromuscular junction (NMJ), cholinergic mammalian NMJ (including human), and glutamatergic synapses in the mammalian brain. Published experimental evidence in favor of PHP in adult hippocampus and cerebellum includes patch-clamp electrophysiology, presynaptic capacitance measurement, calcium imaging, optical reporters of vesicle release and correlated three-dimensional electron microscopy. These studies are grounded in newly optimized experimental protocols that differ substantively from those typically used to study activity-dependent plasticity in neonatal and juvenile slice preparations. Here, we elaborate and extend our assays and methodologies for the study of PHP in the adult mammalian brain. Our assays are designed to optimize synapse, cell and tissue health and minimize the incorporation of unintended adverse experimental conditions that may interfere with the induction and/or expression of PHP. In addition, we provide benchmark criteria for assessment of cell health, necessary for analysis of PHP and, in so doing, advance our understanding of postsynaptic conditions necessary for PHP induction in the adult brain. Our data underscore why PHP may have been previously overlooked, inclusive of a recent manuscript challenging the robust expression of PHP in the mammalian brain (Dou et al., 2026 BioRxiv [preprint]).

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, impacting hundreds of millions of people and animals globally. Disease pathology primarily originates from host immune responses to parasite eggs, which are produced only when female schistosomes are continuously paired with males. Past research focused on pairing-dependent female sexual maturation, while scarce data exist for the males reproductive biology. In this study, we characterized the G protein-coupled receptor Smgpcr9 (Smp_244240), an orphan Class A (Rhodopsin-like) GPCR with a testis-preferential and pairing-influenced expression profile in S. mansoni males. Previous bulk RNA-seq analyses of adult worms and their isolated gonads revealed that Smgpcr9 belongs to a subgroup of GPCR genes with abundant testis-preferential and pairing-influenced transcript levels in males but low and extremely low expression in unpaired and paired females, respectively. This male-/unpaired female-biased expression pattern mirrors that of neuropeptide (npp) genes of S. mansoni such as Smnpp26 and Smnpp41. In a deorphanization approach using yeast-two-hybrid analyses, GPCR internalization experiments, bioluminescence resonance energy transfer assays, and by modeling and docking analyses, we provide first evidence that both NPPs can interact with SmGPCR9. Furthermore, we optimized a GPCR RNAi approach and achieved efficient transcript knockdown (> 90%) enabling robust functional characterization of Smgpcr9. Following RNAi, physiological and morphological analyses revealed that SmGPCR9 regulates key aspects of male reproductive biology like testis morphology and spermatogenesis. Remarkably, ovary structure and egg production were also affected in paired females post RNAi. We observed similar phenotypes plus motility constraints and reduced stem-cell proliferation in both sexes upon RNAi of Smnpp26 and Smnpp41. In all cases, RNAi downstream analyses by RT-qPCR of marker genes substantiated the observed phenotypic effects. These results strongly indicate the importance of SmGPCR9, SmNPP26, and SmNPP41 for spermatogenesis and further physiological processes in male and female S. mansoni. Author SummaryResearch of the reproductive biology of schistosomes focused mainly on females so far, which upon pairing sexually mature to produce eggs that are important for the life cycle maintenance but also for the pathogenesis of schistosomiasis, the infectious disease caused by these parasites. We investigated a yet unknown G protein-coupled receptor, Smgpcr9, which showed a testis-preferential and pairing-influenced expression profile in Schistosoma mansoni males. To this end, we optimized an RNA interference (RNAi) approach for knockdown analysis, identified neuropeptides (NPPs) as potential ligands by different biochemical approaches and modeling and docking analyses, and we investigated the roles of SmGPCR9 and two interacting NPPs, SmNPP26 and SmNPP41, by physiological, microscopical, and molecular techniques. Our results strongly suggest that SmGPCR9 and both NPPs regulate spermatogenesis. Furthermore, we detected effects on ovary morphology, egg production, and stem-cell proliferation of paired females post RNAi. Taken together, we deorphanized SmGPCR9 and showed for the first time the essential role of a so far uncharacterized GPCR and two interacting neuropeptides for spermatogenesis. Our results shed first light on spermatogenesis regulatory processes controlled by GPCRs and neuropeptides in male S. mansoni and thus expand our understanding of the roles of GPCR-NPP signaling for schistosome reproductive biology.

Background/ObjectivesRNA polymerase II is a multifunctional complex that is critical for gene regulation and environmental responses. Its POLR2I subunit in human is associated with various pathologies, including cancer chemoresistance. However, much of our understanding of how POLR2I could function indirectly derives from studies of its homologs in yeasts called Rpb9. Here, we endogenously humanized the rpb9 gene of the fission yeast Schizosaccharomyces pombe to examine the functional capabilities of POLR2I. MethodsWe edited the genomic rpb9 locus in S. pombe so that it encodes the human POLR2I protein, and investigated functional and structural conservation. ResultsWith our humanized yeast system, we find widespread functional complementation by human POLR2I of S. pombe rpb9 roles in yeast growth, chronological aging, and stress responses. We also find that POLR2I complements novel roles for yeast rpb9 in facultative heterochromatin assembly, resistance against the chemotherapy 5-fluorouracil, and resistance against the fungicide thiabendazole. In contrast, we find that POLR2I cannot complement the role of rpb9 in resistance against the transcription elongation inhibitor 6-azauracil (6-AU) in our system. Interestingly, POLR2I could complement 6-AU resistance if ectopically expressed. Lastly, we observe extensive structural homology between Rpb9 and POLR2I proteins. ConclusionsOur study establishes an endogenous cross-species gene complementation strategy that uncovers both conserved and rewired functions of fission yeast rpb9 and its human homolog, POLR2I. In addition to validating conserved roles, we also identified conservation of previously unrecognized roles of rpb9 in heterochromatin formation and chemoresistance.

Garra rufa, commonly known as the doctor fish, is a small freshwater cyprinid notable for its exceptional tolerance to high temperatures, surviving even at around the human body temperature of 37 {degrees}C, and has emerging potential as a novel laboratory model for human cancer xenotransplantation and infectious disease research. To establish a foundation for its experimental use, we conducted comprehensive anatomical and histological analyses across major organ systems. The overall body organization and tissue architecture of G. rufa are broadly similar to those of zebrafish (Danio rerio), indicating a conserved cyprinid body plan. However, several organ systems in G. rufa exhibited species-specific differences compared with zebrafish, including a well-developed adhesive disc around the oral region, a long and coiled intestine, and a distinct dark pigmentation of the peritoneum. These species-specific traits may reflect ecological and behavioral adaptations of G. rufa, including benthic scraping in warm, flowing habitats. Physiological assays confirmed that G. rufa maintains high survival rates and normal swimming activity at 37 {degrees}C, whereas zebrafish exhibit significant mortality and reduced locomotion under the same conditions. Collectively, this work provides a comprehensive histo-anatomical atlas of G. rufa, highlighting its unique morphological specializations while establishing an essential reference for the development of this species as a novel experimental fish model.