Open Biology

Ciliates are a model lineage for studies of genome architecture given their unusual genome structures. All ciliates have both somatic macronuclei (MAC) and germline micronuclei (MIC), both of which develop from a zygotic nucleus following sex (i.e., conjugation). Nuclear developmental stages are not as well explored among non-model ciliate genera, including Chilodonella uncinata (Class-Phyllopharyngea), the focus of our work. Here, we characterize nuclear architecture and genome dynamics in C. uncinata by combining DAPI (4',6-diamidino-2-phenylindole) staining and fluorescence in situ hybridization (FISH) techniques with confocal microscopy. We developed a telomere probe for staining alongside DAPI, which allows for the identification of fragmented somatic chromosomes among the total DNA in the nuclei. We quantify both total DNA and telomere-bound signals to explore changes in DNA content and chromosome maturation across Chilodonellas nuclear life cycle. Specifically, we find that MAC developmental stages in the ciliate C. uncinata are different than the data reported from other ciliate species. These data provide insights into nuclear dynamics during nuclear development and enrich our understanding of genome evolution in non-model ciliates.

Mental retardation autosomal dominant 57 (MRD57) is a rare neurodevelopmental disorder characterised by delayed language and psychomotor development, intellectual disability, hypotonia, gastrointestinal issues and facial dysmorphia. It is linked to genetic mutations in the serine/threonine kinase TLK2, characterised by haploinsufficiency and in some cases, its loss or impaired kinase function. TLK2 is an established cell cycle regulator that has been extensively studied in mitotic cells. It is upregulated in cancers, driving tumour growth, however, the role of TLK2 in postmitotic neurons is not understood. We therefore aimed to gain insight into how TLK2 mutations cause MRD57 by determining where TLK2 is expressed in the brain and its subcellular localisation during neuronal differentiation. Public human and mouse brain transcriptomic data revealed splice variant diversity in the N-terminus of TLK2, which contains its nuclear localisation sequence (NLS). Using splice-specific in situ hybridisation probes, we observed expression of TLK2 isoforms that contain and lack the NLS in the mouse hippocampus and cerebellum. We confirmed these findings in human SH-SY5Y neuroblastoma cells, and found that neuronal differentiation of these cells enhances a cytoplasmic pool of TLK2 by two mechanisms: nuclear export of full length TLK2 and increased expression of TLK2 splice variants lacking the NLS. Finally, acute stimuli that mimic synaptic activity were sufficient to elicit nuclear export of TLK2. Our data highlight the need to establish the neuronal cytoplasmic substrates of TLK2 and determine how the loss of TLK2 activity in MRD57 might impact their function in the developing and mature brain.

The ability to co-ordinate function between multiple cells is a critical requirement for multi-cellularity. This co-ordination is mediated by hormones or growth factors, molecules secreted by one cell type that can convey information to the other cells and influence their behaviour. Hormone-dependent signalling is mediated by second messenger systems;phosphoinositides (PIs) generated by lipid kinase activity are one such key second messenger system. Phosphatidylinositol 5 phosphate 4-kinase (PIP4K) is a lipid kinase that phosphorylates phosphatidylinositol 5-phosphate (PI5P) to generate phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. Following a comprehensive bioinformatics analysis of ca. 23296 proteomes covering the tree of life, we find that PIP4K is a metazoan-specific enzyme, although its homologs are also found in choanoflagellate genomes. To understand their function in early metazoans, we experimentally analysed the biochemical activity and physiological function of PIP4K from several early metazoans. We find that the PIP4K enzyme from an early branching metazoan sponge Amphimedon queenslandica (AqPIP4K), regarded as the earliest evolved metazoan, shows a biochemical activity highly conserved with human PIP4K; AqPIP4K is able to selectively phosphorylate PI5P to generate PI(4,5)P2 just as effectively as the human enzyme. Further, AqPIP4K was able to rescue the reduced cell size, growth and development phenotype in larvae of a null mutant in Drosophila PIP4K. These phenotypes are regulated through activity of the hormone insulin, acting via the cell surface insulin receptor, a member of the receptor tyrosine kinase family, that is unique to metazoans. Together, our findings indicate that in early metazoans, AqPIP4K is likely to function in a signal transduction pathway that is required for receptor tyrosine kinase signalling. Overall, our work defines PIP4K as a signal transduction motif required to regulate receptor tyrosine kinase signalling for intercellular communication in the earliest forms of metazoa.

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.

Mitochondrial DNA of trypanosomatid parasites is organized into a topologically complex structure, named kinetoplast (kDNA). Replication, segregation and expression of kDNA involve an estimated [~]300 proteins, only a fraction of which have been identified and characterized. Here, we report the development of a genetic screen in Trypanosoma brucei to identify novel kDNA maintenance factors. Of the 20 highest-ranked genes identified, six are known kDNA maintenance factors. We selected one hit, Tb927.8.4240, a gene of previously unknown function, for experimental follow-up. Ultrastructure expansion microscopy using a tagged version of the protein reveals a dynamic localization during the cell cycle. RNAi-mediated ablation of Tb927.8.4240 results in the progressive but incomplete loss of kDNA, with only a minor effect on the tripartite attachment complex, suggesting the protein is involved in kDNA replication but not segregation. The growth phenotype of Tb927.8.4240 ablation is fully rescued in a kDNA-independent genetic background, confirming a specific role in kDNA replication. In summary, we describe a functional genetic screen for the identification of kDNA maintenance factors in trypanosomes, validate one hit as a novel kDNA replication factor, and provide a prioritized hit list as a promising starting point for the future identification of additional factors.

The phylogenetic position of chaetognaths has been debated for decades, however recently they have been grouped into the Gnathifera, sister taxon to the Lophotrochozoa. Chaetognaths possess photoreceptor cells that are anatomically unique and arranged remarkably different in the eyes of the various species. Studies investigating eye development and underlying gene regulatory networks are so far missing. In order to gain insights into the development and the molecular toolkit of chaetognath photoreceptors and eyes a new transcriptome of the epibenthic species Spadella cephaloptera was searched for opsins. Our screen revealed single-copies of xenopsin and peropsin and gene expression analyses demonstrated that only xenopsin is expressed in photoreceptor cells of the developing lateral eyes. Adults likewise exhibit two xenopsin+ photoreceptor cells in each of their lateral eyes. Beyond that, a single cryptochrome gene was uncovered and found co-expressed with xenopsin in some photoreceptor cells of the lateral developing eye. In addition, it is co-expressed with peropsin in the cerebral ganglia, a condition reminiscent of a non-visual photoreceptive zone in the apical nervous system of the annelid Platynereis dumerilii that performs circadian entrainment and melatonin release. Cryptochrome expression was also detected in cells of the corona ciliata, a circular organ in the posterior dorsal head region that has been attributed several functions arguing for an involvement of this organ in circadian entrainment. Our study demonstrates the importance to investigate representatives of the Gnathifera, a clade that has been neglected with respect to developmental studies and that might contribute to unravel the evolution of spiralian and bilaterian body plans.

Progression through the cell cycle in eukaryotes is regulated on multiple levels. The main driver of the cell cycle progression is the periodic activity of cyclin-dependent kinase (CDK) complexes. In parallel, transcription during the cell cycle is regulated by a transcriptional program that ensures the just-in-time gene expression. Many core cell cycle regulators are present in all eukaryotes, among them cyclins and CDKs; however, periodic transcriptional programs are divergent between distantly related species. In addition, many otherwise conserved cell cycle regulators have been lost and independently evolved in yeast, a widely used model organism for cell cycle research. To gain insight into the cell cycle regulation in a more representative opisthokont, we investigated the cell cycle regulation at the transcriptional level of Capsaspora owczarzaki, a species closely related to animals. We developed a protocol for cell cycle synchronization in Capsaspora cultures and assessed gene expression over time across the entire cell cycle. We identified a set of 801 periodic genes that grouped into five clusters of expression over time. Comparison with datasets from other eukaryotes revealed that the periodic transcriptional program of Capsaspora is most similar to that of animal cells. We found that orthologues of cyclin A, B and E are expressed at the same cell cycle stages as in human cells and in the same temporal order. However, in contrast to human cells where these cyclins interact with multiple CDKs, Capsaspora cyclins likely interact with a single ancestral CDK1-3. Thus, the Capsaspora cyclin-CDK system could represent an intermediate state in the evolution of animal-like cyclin-CDK regulation. Overall, our results demonstrate that Capsaspora could be a useful unicellular model system for animal cell cycle regulation.\n\nAuthors summaryWhen cells reproduce, proper duplication and splitting of the genetic material is ensured by cell cycle control systems. Many of the regulators in these systems are present across all eukaryotes, such as cyclin and cyclin-dependent kinases (CDK), or the E2F-Rb transcriptional network. Opisthokonts, the group comprising animals, yeasts and their unicellular relatives, represent a puzzling scenario: in contrast to animals, where the cell cycle core machinery seems to be conserved, studies in yeasts have shown that some of these regulators have been lost and independently evolved. For a better understanding of the evolution of the cell cycle regulation in opisthokonts, and ultimately in the lineage leading to animals, we have studied cell cycle regulation in Capsaspora owczarzaki, a unicellular amoeba more closely related to animals than fungi that retains the ancestral cell cycle toolkit. Our findings suggest that, in the ancestor of Capsaspora and animals, cyclins oscillate in the same temporal order as in animals, and that expansion of CDKs occurred later in the lineage that led to animals.

Centrioles are the main components of cilia and centrosomes, which play a central role in cell division and signalling. Their numbers are strictly regulated. Centriole amplification, or the presence of extra centrioles, often occurs in tumours and leads to aneuploidy and altered signalling and has been associated with cancer development and malignancy. Negative selection of cells with extra centrioles prevents numerical errors from expanding in the population, resulting in an overproduction-selection balance. However, how chronic perturbation of key centriolar regulators affects centriole number dynamics is poorly described. PLK4, a key regulator of centriole biogenesis, is often overexpressed in cancer. Here, we studied the long-term dynamics of cell populations exposed to different levels of PLK4 overexpression. We measured absolute and relative fitness in the evolving populations, quantified centriole numbers over time, as well as various aspects of the immediate response to centriole amplification. Our experiments indicated negative selection against cells with extra centrioles and outcompetition of PLK4-overexpressing cells by a cell line carrying a truncated form of PLK4, that does not amplify centrioles. In populations where cells carrying the truncated form of PLK4 were absent, cells overexpressing full-length PLK4 maintained the capacity to amplify centrioles over the course of experimental evolution and, strikingly, converge to the same degree of centriole amplification regardless of the level of PLK4 overexpression. Our results support a population-level response to centrosome amplification to control centriole amplification levels. Future work is necessary to further characterise this response and the mechanisms that allow cell populations to maintain centriole amplification.

In this study, we report the atypical cell cycle organization of the unicellular eukaryotic pathogen Toxoplasma gondii. The remarkably flexible cell division of T. gondii and other apicomplexan parasites differs considerably from the cell division modes employed by other model eukaryotes. Additionally, there is a lack of recognizable cell cycle regulators, which have contributed to the difficulties in deciphering the order of events in the apicomplexan cell cycle. To aid in studies of the cell cycle organization of the T. gondii tachyzoite, we have created the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probes, ToxoFUCCIS and ToxoFUCCISC. We introduced a DNA replication factor TgPCNA1 tagged with NeonGreen that can be used alone or in conjunction with an mCherry-tagged budding indicator TgIMC3 in the auxin-induced degradation (AID) parental strain. The varied localization and dynamic cell cycle oscillation have confirmed TgPCNA1 to be a suitable T. gondii FUCCI probe. The ToxoFUCCIS analysis showed that tachyzoite DNA replication starts at or near centromeric regions, has a bell-shaped dynamic and a significant degree of the cell cycle asynchrony within the vacuoles. Quantitative live and immunofluorescence microscopy analyses of ToxoFUCCIS and its derivatives co-expressing epitope-tagged cell cycle markers have revealed an unusual composite cell cycle phase that incorporates overlapping S, G2, mitosis and cytokinesis (budding). We identified five intervals of the composite phase and their approximate duration: S (19%), S/G2/C (3%), S/M/C (9%), M/C (18%) and C/G1 (<1%). The ToxoFUCCIS probe efficiently detected G2/M and Spindle Assembly Checkpoints, as well as the SB505124-induced TgMAPK1 dependent block. Altogether, our findings showed an unprecedented complexity of the cell cycle in apicomplexan parasites.

Vertebrates show conserved left-right (L-R) asymmetry of internal organs controlled by Nodal-Pitx2/Lefty signaling [1-3]. Modifications in L-R asymmetry occur in mutants [4] and rarely in humans [5], but little is known about natural L-R changes during evolution. Here we describe changes in L-R asymmetry in Astyanax mexicanus, a teleost with ancestral surface (surface fish) and derived cave (cavefish) morphs [6]. In teleosts, Nodal-Pitx2 signaling is activated in the left lateral plate mesoderm (LPM), the cardiac tube jogs to the left and loops to the right (D-looping), and the liver and pancreas form on opposite sides of the midline. Surface fish show conventional L-R patterning, but cavefish can show Nodal-Pitx2 expression in the right LPM or bilaterally, left (L)-looping hearts, and reversed liver and pancreas asymmetry, and these reversals have no effect on survival. The Lefty1 Nodal antagonist is expressed along the surface fish and cavefish midlines, but expression of the Lefty2 antagonist is absent in the LPM of most cavefish embryos, suggesting a role for lefty2 (lft2) in changing organ asymmetry. Although CRISPR-Cas9 lft2 editing affected D-looping in surface fish, the cavefish lft2 gene showed no coding mutations, and was expressed normally during cavefish gastrulation, suggesting downregulation by regulatory changes. Reciprocal hybridization, the fertilization of cavefish eggs with surface fish sperm and vice versa, indicated that the change in cavefish L-R asymmetry is a maternal genetic effect. Our studies reveal natural changes in internal organ asymmetry during evolution and introduce A. mexicanus as a new model to study the underlying mechanisms.

E3 ubiquitin ligases are part of various families of proteins and include hundreds of members, which play key roles in all aspects of cell biology. They generally regulate the half-life of other proteins but can also modulate their cellular localization and functions. The MARCH family of ubiquitin ligases is composed of 11 members and two closely related proteins, MARCH1 and MARCH8, share similar targets, while being active in different cell types. Although they appear to target principally immune cell components, such as MHC class II molecules and the co-stimulatory molecule CD86, the repertory of their targets remains to be fully documented. Here, to further define the MARCH1s interactome, we adapted a proximity-dependent biotin identification (BioID)-based screening approach in live HEK293 cells. We transfected a fusion protein consisting of mouse MARCH1 linked to YFP at its N-terminus and to the biotin ligase of Aquifex aeolicus at its C-terminus. Upon transient overexpression of this construct in the presence of exogenous biotin, we could recover biotinylated proteins that are presumably found within 10nm of MARCH1. To help in the identification of bona fide down-regulated specific targets, we compared MARCH1s interactome with the one obtained using a ubiquitination-deficient MARCH1 mutant (MARCH1W104A). CD98 and CD71, two previously described targets of MARCH1, were identified in this screen. Of 16 other biotinylated proteins identified by semi-quantitative mass spectrometry, 10 were tested directly by flow cytometry to monitor their expression in the presence or absence of transfected MARCH1. The protein levels of five of these endogenous targets, CD29, CD112, NKCC1, CD147 and SNAT2, confirmed their negative regulation by MARCH1 in this system. SNAT2 was particularly sensitive to the presence of MARCH1 and was found to be ubiquitinated on Western blots following immunoprecipitation. Thus, BioID2 is an effective mean of characterizing the interactome of MARCH1 and the identification of SNAT2 suggests a role of this ubiquitin ligase in cellular metabolism.

The study of Nuclear Matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogaster embryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol can be extended to larval tissues like salivary glands and imaginal discs with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant and transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture. SummaryNuclear Matrix is a biochemically defined entity and a basic component of the nuclear architecture. Here we present a protocol to isolate and visualize Nuclear Matrix in situ in the intact embryos and tissues of Drosophila melanogaster and its potential applications.

Platynereis dumerilii, a marine annelid, is a model animal that has gained popularity in various fields such as developmental biology, biological rhythms, nervous system organization and physiology, behaviour, reproductive biology, and epigenetic regulation. The transparency of P. dumerilii tissues at all developmental stages makes it easy to perform live microscopic imaging of all cell types. In addition, the slow-evolving genome of P. dumerilii and its phylogenetic position as a representative of the vast branch of Lophotrochozoans add to its evolutionary significance. Although P. dumerilii is amenable to transgenesis and CRISPR-Cas9 knockouts, its relatively long and indefinite life cycle, as well as its semelparous reproduction have been hindrances to its adoption as a reverse genetics model. To overcome this limitation, an adapted culturing method has been developed allowing much faster life cycling, with median reproductive age at 15 weeks instead of 6-8 months using the traditional protocol. A low worm density in boxes and a strictly controlled feeding regime are important factors for the rapid growth and health of the worms. Moreover, a genetic selection for fast-reproducing individuals has been applied to isolate a "Fast Forward" strain that can be used for egg microinjection. This culture method has several advantages, such as being much more compact, not requiring air bubbling or an artificial moonlight regime for synchronized sexual maturation, and necessitating only limited water change. A full protocol for worm care and handling is provided.

Cryptococcus neoformans (Cn) is an important human pathogen and a model system for basidiomycetes. Here we carry out a dissection of its spindle assembly checkpoint (SAC), focusing on Bub1 and Bub3. In many eukaryotes, including humans, Saccharomyces cerevisiae and Schizosaccharomyces pombe, Bub1 underwent gene duplication, generating paralogues referred to as Bub1 and BubR1 (or Mad3). Bub1 has upstream signalling functions at kinetochores, whilst BubR1/Mad3 is a component of the downstream mitotic checkpoint complex (MCC) that delays anaphase onset until all chromosomes are correctly attached. Here we demonstrate that the single CnBub1 protein carries out all the checkpoint roles of both Bub1 kinase and Mad3/BubR1. Proteomic analysis reveals kinetochore targeting via Spc105KNL1 and interactions with all downstream SAC components and effectors (Cdc20 and the anaphase promoting complex/cyclosome). We demonstrate that CnBub1 kinase activity is required to maintain prolonged checkpoint arrest. Thus CnBub1 acts as a SAC signalling hub and is a future target for anti-mitotic drugs.

Histriobdellidae, the so-called Charlie Chaplin worms, is an enigmatic group of microscopic commensal annelids associated with crustaceans. They crawl by alternately attaching their adhesive anterior appendages and left and right huge lateral feet, and bear a complex jaw apparatus in the ventral muscular pharynx. Although histriobdellids were always thought to be a part of the jaw-bearing clade Eunicida, their exact placement within the annelid tree is still debated due to their highly derived external morphology and long branch attraction artefacts in molecular analyses. In this study we employ morphological and molecular comparative approaches in order to gain new insights into the evolution of Histriobdellidae and its aberrant traits. Our phylogenetic analyses of Eunicida including 52 species and four molecular markers yield further support for Histriobdellidae being the sister group to the eunicid family Dorvilleidae. The detailed morphology of Histriobdella homari Van Beneden, 1858, a commensal of the European lobster, was examined using standard immunohistochemical stainings and subsequent confocal laser scanning microscopy (CLSM) as well as scanning electron microscopy (SEM). Integrative analyses allow us to compare in detail with other eunicidans and unravel extensive anatomical transformations in Histriobdellidae. Neural innervation patterns help verify the presence of antennae and true annelid palps on the histriobdellid prostomium. The arrangement of ganglia and the neuronal scaffold innervating the anterior end supports the presence of a buccal segment (peristomium) in Histriobdella. Additionally, based on our comprehensive investigations we newly propose their adhesive anterior locomotory appendages to be homologous with parapodia, and their posterior-most adhesive locomotory appendages to be homologous with the pygidial lobes of other Annelida. Detailed studies of this highly deviating family of annelids not only exemplify how to reconstruct extreme transformation of canonical annelid characters such as parapodia, but again also highlight the exceptional evolutionary plasticity of the annelid body plan.

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.

Important in many human artistic cultures, checkerboard patterns are rare in nature like many motifs based on squared geometry. Nevertheless, they are expected to be very detectable by the visual systems due to their periodic geometry and contrasted two-tone coloring, therefore potential specific biological functions are suspected. Here, thanks to a biological survey, we draw the first diversity landscape of eukaryotic species bearing checkerboard patterns, confirming their rarity but also their presence in extremely diverse clades. Then, we selected two genera, Sarcophaga flies and Fritillaria flowers, to perform in-depth pattern analyses allowing us to make strong hypotheses on the mechanisms producing these very peculiar patterns, as no morphogenetic process was known to generate checkerboards. Although they share a similar geometry, these two genera appear to produce checkerboards through very different ways, showing a convergence of shape but not of processes. Whereas the Fritillaria analysis points to a geometric constraining of a Turing-like pattern by the parallel network of veins, that of Sarcophaga suggest the reuse of developmental boundaries and right-left symmetry, together by the combination of vertical and horizontal stripes. Furthermore, we present the first description to our knowledge of the striking color-changing nature of Sarcophaga checkerboards, whose light and dark squares can exchange their color depending on the angles of lighting and observation thanks to the planar polarity of the cuticular hairs, the setae. Together, this shows the extent of the processes selected during evolution to generate complex forms and colors, and confirms the importance of studying morphogenesis with in-depth pattern analyses and through species diversity. Finally, by enabling strong hypotheses to be made about the morphogenesis of these patterns, it paves the way for the molecular identification of the morphogenetic processes at work.

Circadian clock drives the 24h rhythm in our behavior and physiology. Previously, we have shown that inner nuclear membrane protein Lamin B receptor (LBR) regulates circadian rhythm in human cells and Drosophila, while the underlying mechanism is unclear (Lin et al., 2014). A very recent study reported that the circadian clock protein PERIOD is organized into discrete foci at the nuclear envelope in fly circadian neurons, which is believed to be important for controlling the subcellular localization of clock genes. Loss of LBR leads to disruption of these foci, but how they are regulated is yet unknown. Here we found that LBR likely facilitates PER foci accumulation by destabilizing the catalytic subunit of protein phosphatase 2A, MICROTUBULE STAR (MTS). MTS is known to dephosphorylate PER and hampers the accumulation of PER foci. On the other hand, the circadian kinase DOUBLETIME (DBT) which phosphorylates PER enhances the accumulation of the foci. These foci are likely phase-separated condensates, the formation of which mediated by intrinsically disordered region in PER. Taken together, here we demonstrate a key role for phosphorylation in promoting the accumulation of PER foci, while LBR modulates this process by impinging on the circadian phosphatase MTS.

Polycomb Repressive Complex 2 (PRC2) is involved in establishing transcriptionally silent chromatin states through its ability to methylate lysine 27 of histone H3 by the catalytic subunit Enhancer of zeste [E(z)]. Polycomb group (PcG) proteins play a crucial role in the maintenance of cell identity and in developmental regulation. Previously, the diversity of PRC2 subunits within some eukaryotic lineages has been reported and its presence in early eukaryotic evolution has been hypothesized. So far however, systematic survey of the presence of PRC2 subunits in species of all eukaryotic lineages is missing. Here, we report the diversity of PRC2 core subunit proteins in different eukaryotic supergroups with emphasis on the early-diverged lineages and explore the molecular evolution of PRC2 subunits by phylogenetics. In detail, we investigate the SET-domain protein sequences and their evolution across the four domains of life and particularly focus on the structural diversity of the SET-domain subfamily containing E(z), the catalytic subunit of PRC2. We show that PRC2 subunits are already present in early eukaryotic lineages, strengthening the support for PRC2 emergence prior to diversification of eukaryotes. We identify a common presence of E(z) and ESC, suggesting that Su(z)12 may have emerged later and/or may be dispensable from the evolutionarily conserved functional core of PRC2. Furthermore, our results broaden our understanding of the E(z) evolution within the SET-domain protein family, suggesting possibilities of function evolution. Through this, we shed light on a possible emerging point of the PRC2 and the evolution of its function in eukaryotes.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the bakers yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen. Author summaryThe study of uncultivable microorganisms has always been a challenge. Such is the case of the human-specific pathogen Pneumocystis jirovecii, the causative agent of pneumocystis pneumonia. P. jirovecii is insensitive to classical antifungal drugs, making options for treatment and prophylaxis limited. In recent years, more and more cases of P. jirovecii infections have become resistant to treatment, highlighting the need to study and understand this pathogens mechanisms of resistance. Here, we use a yeast strain expressing P. jiroveciis DHFR as a reporter for resistance to an antifolate, one of the drug families used to treat infections. We observed that this DHFR was sensitive to methotrexate, a powerful antifolate, in a quantitative manner. Then, by using a large-scale mutational assay, we identified virtually all single mutations that confer this protein resistance to methotrexate. While any of them have also been reported in other eukaryotes, we find new mutations at positions of the protein not previously known to confer resistance or to be in contact with this competitive inhibitor. Overall, our results are a comprehensive portrait of this DHFRs resistance to methotrexate.