Evolution of the ribbon-like organization of the Golgi apparatus in animal cells.

The "ribbon," a structural arrangement in which Golgi stacks connect to each other, is considered to be restricted to vertebrate cells. Although ribbon disruption is linked to various human pathologies, its functional role in cellular processes remains unclear. In this study, we investigate the evolutionary origin of the Golgi ribbon. We observe a ribbon-like architecture in the cells of several metazoan taxa suggesting its early emergence in animal evolution predating the appearance of vertebrates. Supported by AlphaFold2 modeling, we propose that the evolution of Golgi reassembly and stacking protein (GRASP) binding by golgin tethers may have driven the joining of Golgi stacks resulting in the ribbon-like configuration. Additionally, we find that Golgi ribbon assembly is a shared developmental feature of deuterostomes, implying a role in embryogenesis. Overall, our study points to the functional significance of the Golgi ribbon beyond vertebrates and underscores the need for further investigations to unravel its elusive biological roles.

The Origin of Metazoan Multicellularity: A Potential Microbial Black Swan Event.

The emergence of animals from their unicellular ancestors is a major evolutionary event. Thanks to the study of diverse close unicellular relatives of animals, we now have a better grasp of what the unicellular ancestor of animals was like. However, it is unclear how that unicellular ancestor of animals became the first animals. To explain this transition, two popular theories, the choanoblastaea and the synzoospore, have been proposed. We will revise and expose the flaws in these two theories while showing that, due to the limits of our current knowledge, the origin of animals is a biological black swan event. As such, the origin of animals defies retrospective explanations. Therefore, we should be extra careful not to fall for confirmation biases based on few data and, instead, embrace this uncertainty and be open to alternative scenarios. With the aim to broaden the potential explanations on how animals emerged, we here propose two novel and alternative scenarios. In any case, to find the answer to how animals evolved, additional data will be required, as will the hunt for microscopic creatures that are closely related to animals but have not yet been sampled and studied.

Poly(A)-binding protein is an ataxin-2 chaperone that regulates biomolecular condensates.

Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs), which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar of a few scaffold proteins that make up these phases, but how the partitioning of hundreds of SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, an SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14 aa sequence that acts as a condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal an unexpected molecular function for ancient poly(A)-binding proteins as regulators of biomolecular condensate proteins. These findings may inspire approaches to therapeutically target aberrant phases in disease.

Chemical factors induce aggregative multicellularity in a close unicellular relative of animals.

Regulated cellular aggregation is an essential process for development and healing in many animal tissues. In some animals and a few distantly related unicellular species, cellular aggregation is regulated by diffusible chemical cues. However, it is unclear whether regulated cellular aggregation was part of the life cycles of the first multicellular animals and/or their unicellular ancestors. To fill this gap, we investigated the triggers of cellular aggregation in one of animals' closest unicellular living relatives-the filasterean Capsaspora owczarzaki. We discovered that Capsaspora aggregation is induced by chemical cues, as observed in some of the earliest branching animals and other unicellular species. Specifically, we found that calcium ions and lipids present in lipoproteins function together to induce aggregation of viable Capsaspora cells. We also found that this multicellular stage is reversible as depletion of the cues triggers disaggregation, which can be overcome upon reinduction. Our finding demonstrates that chemically regulated aggregation is important across diverse members of the holozoan clade. Therefore, this phenotype was plausibly integral to the life cycles of the unicellular ancestors of animals.

Evolution of promoter-proximal pausing enabled a new layer of transcription control.

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here we analyzed transcription in species across the tree of life. We found that unicellular eukaryotes display a slow acceleration of Pol II near transcription start sites. This proto-paused-like state transitioned to a longer, focused pause in derived metazoans which coincided with the evolution of new subunits in the NELF and 7SK complexes. Depletion of NELF reverts the mammalian focal pause to a proto-pause-like state and compromises transcriptional activation for a set of heat shock genes. Collectively, this work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

Taxonomic composition, community structure and molecular novelty of microeukaryotes in a temperate oligomesotrophic lake as revealed by metabarcoding.

Microbial eukaryotes are diverse and ecologically important organisms, yet sampling constraints have hindered the understanding of their distribution and diversity in freshwater ecosystems. Metabarcoding has provided a powerful complement to traditional limnological studies, revealing an unprecedented diversity of protists in freshwater environments. Here, we aim to expand our knowledge of the ecology and diversity of protists in lacustrine ecosystems by targeting the V4 hypervariable region of the 18S rRNA gene in water column, sediment and biofilm samples collected from Sanabria Lake (Spain) and surrounding freshwater ecosystems. Sanabria is a temperate lake, which are relatively understudied by metabarcoding in comparison to alpine and polar lakes. The phylogenetic diversity of microbial eukaryotes detected in Sanabria spans all currently recognized eukaryotic supergroups, with Stramenopiles being the most abundant and diverse supergroup in all sampling sites. Parasitic microeukaryotes account for 21% of the total protist ASVs identified in our study and were dominated by Chytridiomycota, both in terms of richness and abundance, in all sampling sites. Sediments, biofilms and water column samples harbour distinct microbial communities. Phylogenetic placement of poorly assigned and abundant ASVs indicates molecular novelty inside Rhodophyta, Bigyra, early-branching Nucletmycea and Apusomonadida. In addition, we report the first freshwater incidence of the previously exclusively marine genera Abeoforma and Sphaeroforma. Our results contribute to a deeper understanding of microeukaryotic communities in freshwater ecosystems, and provide the first molecular reference for future biomonitoring surveys in Sanabria Lake.

On the origin and evolution of RNA editing in metazoans.

Extensive adenosine-to-inosine (A-to-I) editing of nuclear-transcribed mRNAs is the hallmark of metazoan transcriptional regulation. Here, by profiling the RNA editomes of 22 species that cover major groups of Holozoa, we provide substantial evidence supporting A-to-I mRNA editing as a regulatory innovation originating in the last common ancestor of extant metazoans. This ancient biochemistry process is preserved in most extant metazoan phyla and primarily targets endogenous double-stranded RNA (dsRNA) formed by evolutionarily young repeats. We also find intermolecular pairing of sense-antisense transcripts as an important mechanism for forming dsRNA substrates for A-to-I editing in some but not all lineages. Likewise, recoding editing is rarely shared across lineages but preferentially targets genes involved in neural and cytoskeleton systems in bilaterians. We conclude that metazoan A-to-I editing might first emerge as a safeguard mechanism against repeat-derived dsRNA and was later co-opted into diverse biological processes due to its mutagenic nature.

Evolutionary analysis of p38 stress-activated kinases in unicellular relatives of animals suggests an ancestral function in osmotic stress.

p38 kinases are key elements of the cellular stress response in animals. They mediate the cell response to a multitude of stress stimuli, from osmotic shock to inflammation and oncogenes. However, it is unknown how such diversity of function in stress evolved in this kinase subfamily. Here, we show that the p38 kinase was already present in a common ancestor of animals and fungi. Later, in animals, it diversified into three JNK kinases and four p38 kinases. Moreover, we identified a fifth p38 paralog in fishes and amphibians. Our analysis shows that each p38 paralog has specific amino acid substitutions around the hinge point, a region between the N-terminal and C-terminal protein domains. We showed that this region can be used to distinguish between individual paralogs and predict their specificity. Finally, we showed that the response to hyperosmotic stress in Capsaspora owczarzaki, a close unicellular relative of animals, follows a phosphorylation-dephosphorylation pattern typical of p38 kinases. At the same time, Capsaspora's cells upregulate the expression of GPD1 protein resembling an osmotic stress response in yeasts. Overall, our results show that the ancestral p38 stress pathway originated in the root of opisthokonts, most likely as a cell's reaction to salinity change in the environment. In animals, the pathway became more complex and incorporated more stimuli and downstream targets due to the p38 sequence evolution in the docking and substrate binding sites around the hinge region. This study improves our understanding of p38 evolution and opens new perspectives for p38 research.

Divergent genomic trajectories predate the origin of animals and fungi.

Animals and fungi have radically distinct morphologies, yet both evolved within the same eukaryotic supergroup: Opisthokonta1,2. Here we reconstructed the trajectory of genetic changes that accompanied the origin of Metazoa and Fungi since the divergence of Opisthokonta with a dataset that includes four novel genomes from crucial positions in the Opisthokonta phylogeny. We show that animals arose only after the accumulation of genes functionally important for their multicellularity, a tendency that began in the pre-metazoan ancestors and later accelerated in the metazoan root. By contrast, the pre-fungal ancestors experienced net losses of most functional categories, including those gained in the path to Metazoa. On a broad-scale functional level, fungal genomes contain a higher proportion of metabolic genes and diverged less from the last common ancestor of Opisthokonta than did the gene repertoires of Metazoa. Metazoa and Fungi also show differences regarding gene gain mechanisms. Gene fusions are more prevalent in Metazoa, whereas a larger fraction of gene gains were detected as horizontal gene transfers in Fungi and protists, in agreement with the long-standing idea that transfers would be less relevant in Metazoa due to germline isolation3-5. Together, our results indicate that animals and fungi evolved under two contrasting trajectories of genetic change that predated the origin of both groups. The gradual establishment of two clearly differentiated genomic contexts thus set the stage for the emergence of Metazoa and Fungi.

A phylogenetic and proteomic reconstruction of eukaryotic chromatin evolution.

Histones and associated chromatin proteins have essential functions in eukaryotic genome organization and regulation. Despite this fundamental role in eukaryotic cell biology, we lack a phylogenetically comprehensive understanding of chromatin evolution. Here, we combine comparative proteomics and genomics analysis of chromatin in eukaryotes and archaea. Proteomics uncovers the existence of histone post-translational modifications in archaea. However, archaeal histone modifications are scarce, in contrast with the highly conserved and abundant marks we identify across eukaryotes. Phylogenetic analysis reveals that chromatin-associated catalytic functions (for example, methyltransferases) have pre-eukaryotic origins, whereas histone mark readers and chaperones are eukaryotic innovations. We show that further chromatin evolution is characterized by expansion of readers, including capture by transposable elements and viruses. Overall, our study infers detailed evolutionary history of eukaryotic chromatin: from its archaeal roots, through the emergence of nucleosome-based regulation in the eukaryotic ancestor, to the diversification of chromatin regulators and their hijacking by genomic parasites.

Regulation of sedimentation rate shapes the evolution of multicellularity in a close unicellular relative of animals.

Significant increases in sedimentation rate accompany the evolution of multicellularity. These increases should lead to rapid changes in ecological distribution, thereby affecting the costs and benefits of multicellularity and its likelihood to evolve. However, how genetic and cellular traits control this process, their likelihood of emergence over evolutionary timescales, and the variation in these traits as multicellularity evolves are still poorly understood. Here, using isolates of the ichthyosporean genus Sphaeroforma-close unicellular relatives of animals with brief transient multicellular life stages-we demonstrate that sedimentation rate is a highly variable and evolvable trait affected by at least 2 distinct physical mechanisms. First, we find extensive (>300×) variation in sedimentation rates for different Sphaeroforma species, mainly driven by size and density during the unicellular-to-multicellular life cycle transition. Second, using experimental evolution with sedimentation rate as a focal trait, we readily obtained, for the first time, fast settling and multicellular Sphaeroforma arctica isolates. Quantitative microscopy showed that increased sedimentation rates most often arose by incomplete cellular separation after cell division, leading to clonal "clumping" multicellular variants with increased size and density. Strikingly, density increases also arose by an acceleration of the nuclear doubling time relative to cell size. Similar size- and density-affecting phenotypes were observed in 4 additional species from the Sphaeroforma genus, suggesting that variation in these traits might be widespread in the marine habitat. By resequencing evolved isolates to high genomic coverage, we identified mutations in regulators of cytokinesis, plasma membrane remodeling, and chromatin condensation that may contribute to both clump formation and the increase in the nuclear number-to-volume ratio. Taken together, this study illustrates how extensive cellular control of density and size drive sedimentation rate variation, likely shaping the onset and further evolution of multicellularity.

Rel/NF-κB Transcription Factors Emerged at the Onset of Opisthokonts.

The Rel/NF-κB transcription factor family has myriad roles in immunity, development, and differentiation in animals, and was considered a key innovation for animal multicellularity. Rel homology domain-containing proteins were previously hypothesized to have originated in a last common ancestor of animals and some of their closest unicellular relatives. However, key taxa were missing from previous analyses, necessitating a systematic investigation into the distribution and evolution of these proteins. Here, we address this knowledge gap by surveying taxonomically broad data from eukaryotes, with a special emphasis on lineages closely related to animals. We report an earlier origin for Rel/NF-κB proteins than previously described, in the last common ancestor of animals and fungi, and show that even in the sister group to fungi, these proteins contain elements that in animals are necessary for the subcellular regulation of Rel/NF-κB.

Txikispora philomaios n. sp., n. g., a micro-eukaryotic pathogen of amphipods, reveals parasitism and hidden diversity in Class Filasterea.

This study provides a morphological, ultrastructural, and phylogenetic characterization of a novel micro-eukaryotic parasite (2.3-2.6 µm) infecting amphipod genera Echinogammarus and Orchestia. Longitudinal studies across two years revealed that infection prevalence peaked in late April and May, reaching 64% in Echinogammarus sp. and 15% in Orchestia sp., but was seldom detected during the rest of the year. The parasite infected predominantly hemolymph, connective tissue, tegument, and gonad, although hepatopancreas and nervous tissue were affected in heavier infections, eliciting melanization and granuloma formation. Cell division occurred inside walled parasitic cysts, often within host hemocytes, resulting in hemolymph congestion. Small subunit (18S) rRNA gene phylogenies including related environmental sequences placed the novel parasite as a highly divergent lineage within Class Filasterea, which together with Choanoflagellatea represent the closest protistan relatives of Metazoa. We describe the new parasite as Txikispora philomaios n. sp. n. g., the first confirmed parasitic filasterean lineage, which otherwise comprises four free-living flagellates and a rarely observed endosymbiont of snails. Lineage-specific PCR probing of other hosts and surrounding environments only detected T. philomaios in the platyhelminth Procerodes sp. We expand the known diversity of Filasterea by targeted searches of metagenomic datasets, resulting in 13 previously unknown lineages from environmental samples.

Evolution of a histone variant involved in compartmental regulation of NAD metabolism.

NAD metabolism is essential for all forms of life. Compartmental regulation of NAD+ consumption, especially between the nucleus and the mitochondria, is required for energy homeostasis. However, how compartmental regulation evolved remains unclear. In the present study, we investigated the evolution of the macrodomain-containing histone variant macroH2A1.1, an integral chromatin component that limits nuclear NAD+ consumption by inhibiting poly(ADP-ribose) polymerase 1 in vertebrate cells. We found that macroH2A originated in premetazoan protists. The crystal structure of the macroH2A macrodomain from the protist Capsaspora owczarzaki allowed us to identify highly conserved principles of ligand binding and pinpoint key residue substitutions, selected for during the evolution of the vertebrate stem lineage. Metabolic characterization of the Capsaspora lifecycle suggested that the metabolic function of macroH2A was associated with nonproliferative stages. Taken together, we provide insight into the evolution of a chromatin element involved in compartmental NAD regulation, relevant for understanding its metabolism and potential therapeutic applications.

Stable transfection in protist Corallochytrium limacisporum identifies novel cellular features among unicellular animals relatives.

The evolutionary path from protists to multicellular animals remains a mystery. Recent work on the genomes of several unicellular relatives of animals has shaped our understanding of the genetic changes that may have occurred in this transition.1-3 However, the specific cellular modifications that took place to accommodate these changes remain unclear. To address this, we need to compare metazoan cells with those of their extant relatives, which are choanoflagellates, filastereans, ichthyosporeans, and corallochytreans/pluriformeans. Interestingly, these lineages display a range of developmental patterns potentially homologous to animal ones. Genetic tools have already been established in three of those lineages.4-7 However, there are no genetic tools available for Corallochytrea. We here report the development of stable transfection in the corallochytrean Corallochytrium limacisporum. Using these tools, we discern previously unknown biological features of C. limacisporum. In particular, we identify two different paths for cell division-binary fission and coenocytic growth-that reveal a non-linear life cycle. Additionally, we found that C. limacisporum is binucleate for most of its life cycle, and that, contrary to what happens in most eukaryotes, nuclear division is decoupled from cellular division. Moreover, its actin cytoskeleton shares characteristics with both fungal and animal cells. The establishment of these tools in C. limacisporum fills an important gap in the unicellular relatives of animals, opening up new avenues of research to elucidate the specific cellular changes that occurred in the evolution of animals.

The origin of animals: an ancestral reconstruction of the unicellular-to-multicellular transition.

How animals evolved from a single-celled ancestor, transitioning from a unicellular lifestyle to a coordinated multicellular entity, remains a fascinating question. Key events in this transition involved the emergence of processes related to cell adhesion, cell-cell communication and gene regulation. To understand how these capacities evolved, we need to reconstruct the features of both the last common multicellular ancestor of animals and the last unicellular ancestor of animals. In this review, we summarize recent advances in the characterization of these ancestors, inferred by comparative genomic analyses between the earliest branching animals and those radiating later, and between animals and their closest unicellular relatives. We also provide an updated hypothesis regarding the transition to animal multicellularity, which was likely gradual and involved the use of gene regulatory mechanisms in the emergence of early developmental and morphogenetic plans. Finally, we discuss some new avenues of research that will complement these studies in the coming years.

Towards understanding the origin of animal development.

Almost all animals undergo embryonic development, going from a single-celled zygote to a complex multicellular adult. We know that the patterning and morphogenetic processes involved in development are deeply conserved within the animal kingdom. However, the origins of these developmental processes are just beginning to be unveiled. Here, we focus on how the protist lineages sister to animals are reshaping our view of animal development. Most intriguingly, many of these protistan lineages display transient multicellular structures, which are governed by similar morphogenetic and gene regulatory processes as animal development. We discuss here two potential alternative scenarios to explain the origin of animal embryonic development: either it originated concomitantly at the onset of animals or it evolved from morphogenetic processes already present in their unicellular ancestors. We propose that an integrative study of several unicellular taxa closely related to animals will allow a more refined picture of how the last common ancestor of animals underwent embryonic development.

Integrin-Mediated Adhesion in the Unicellular Holozoan Capsaspora owczarzaki.

In animals, cell-matrix adhesions are essential for cell migration, tissue organization, and differentiation, which have central roles in embryonic development [1-6]. Integrins are the major cell surface adhesion receptors mediating cell-matrix adhesion in animals. They are heterodimeric transmembrane proteins that bind extracellular matrix (ECM) molecules on one side and connect to the actin cytoskeleton on the other [7]. Given the importance of integrin-mediated cell-matrix adhesion in development of multicellular animals, it is of interest to discover when and how this machinery arose during evolution. Comparative genomic analyses have shown that core components of the integrin adhesome pre-date the emergence of animals [8-11]; however, whether it mediates cell adhesion in non-metazoan taxa remains unknown. Here, we investigate cell-substrate adhesion in Capsaspora owczarzaki, the closest unicellular relative of animals with the most complete integrin adhesome [11, 12]. Previous work described that the life cycle of C. owczarzaki (hereafter, Capsaspora) includes three distinct life stages: adherent; cystic; and aggregative [13]. Using an adhesion assay, we show that, during the adherent life stage, C. owczarzaki adheres to surfaces using actin-dependent filopodia. We show that integrin ß2 and its associated protein vinculin localize as distinct patches in the filopodia. We also demonstrate that substrate adhesion and integrin localization are enhanced by mammalian fibronectin. Finally, using a specific antibody for integrin ß2, we inhibited cell adhesion to a fibronectin-coated surface. Our results suggest that adhesion to the substrate in C. owczarzaki is mediated by integrins. We thus propose that integrin-mediated adhesion pre-dates the emergence of animals.

Antifungal and antiprotozoal green amino acid-based rhamnolipids: Mode of action, antibiofilm efficiency and selective activity against resistant Candida spp. strains and Acanthamoeba castellanii.

Nowadays, infections caused by fungi and protists constitute a serious problem for public health services. The limited number of treatment options coupled with the increasing number of resistant microorganisms makes necessary the development of new non-toxic antifungal and antiprotozoal agents. Cationic amino acid-based rhamnolipids have been recently prepared by our group and exhibited good antibacterial activity. In this work, the antifungal, antibiofilm and antiprotozoal activity of these new rhamnolipids was investigated against a collection of fluconazole-resistant strains of different Candida species and Acanthamoeba castellanii, respectively. The arginine-RLs exhibited good antifungal activity against all fluconazole-resistant Candida spp. strains tested at MICs ranging from 6.5 to 20.7 mg/L. Their mechanism of action involves alterations in the permeability of the cell membranes that provoke death by apoptosis. The Arginine based-RLs also disperse Candida biofilms at low concentrations, similar to the MICs. All RLs tested (anionic and cationic) showed antiprotozoal activity, the arginine derivatives had the best activity killing the Acanthamoeba castellanii at concentrations of 4 mg/L. Interestingly, these surfactants have a wide range of action against yeast and A. castellanii in which they do not show toxicity against keratinocytes and fibroblasts. These results indicate that these new rhamnolipids have a sufficiently wide safety margin to be considered good candidates for several pharmaceutical applications such as combating fungal resistance and microbial biofilms and the formulation of antiprotozoal drugs.