The malaria parasite Plasmodium falciparum is an obligate intracellular organism that spends an important part of its lifecycle inside human erythrocytes. The endocytosis of host-cell cytosol and its delivery to a lysosome-like organelle called the food vacuole are critical for the parasite’s survival and proliferation. Recent work has started to identify some of the molecular players involved in this pathway, but much remains to be discovered. Evidence suggests that phosphatidylinositol-3-phosphate (PI3P) plays a central role in this process. In unicellular eukaryotes, such as yeast, PI3P is generated by a single PI3-kinase, whose activity is regulated by a pseudokinase called Vps15. P. falciparum also possesses a PI3K that generates PI3P and bioinformatics analysis has revealed the presence of an uncharacterized putative orthologue of Vps15. We here present our characterization of PfVps15. We first show that it is constitutively expressed throughout the asexual erythrocytic cycle and that it interacts with PfPI3K, but unlike in yeast and mammalian cells, it is potentially not part of a heterotetrameric complex. The removal of PfVps15 from its site of action by knock sideways led to rapid parasite death. Phenotypic analyses revealed a decrease in PI3P levels, the abrogation of the delivery of host-cell cytosol containing vesicles to the food vacuole, and defects in apicoplast biogenesis and mitochondrial fission. Collectively, our data has identified a protein critical for the synthesis of PI3P and provides molecular evidence for the importance of this lipid in the vesicular trafficking pathway of host-cell cytosol, and apicoplast and mitochondrion dynamics.

Learned societies play a vital role in fostering interactions that are important in scholarly discourse and the advancement of biological sciences. However, they now face threats from declining funding and membership, shifting disciplinary boundaries, changing approaches towards digital communication, and academic marketization. We outline the historical development of these societies and propose ways to sustain them. Key considerations include improving meetings, adapting publishing models, ensuring financial stability, expanding membership, strengthening outreach, and managing increasingly broad remits. Our main aim is to examine how regional learned societies can maintain their roles to support scientific progress and enrich broader society.

The Canadian province of Alberta contains substantial oilsands reservoirs, consisting of bitumen, clay and sand. Extracting oil involves separating bitumen from inorganic particles using hot water and chemical diluents, resulting in liquid tailings waste with ecotoxicologically significant compounds. Ongoing efforts aim to reclaim tailings-affected areas, with protist colonisation serving as one assessment method of reclamation progress. Oilsands-associated protist communities have mainly been evaluated using amplicon sequencing of the 18S rRNA V4 region; however, this barcode may overlook important protist groups. This study examined how community assessment methods between the V4 and V9 regions differ in representing protist diversity across four oilsands-associated environments. The V9 barcode identified more operational taxonomical units (OTUs) for Discoba, Metamonada and Amoebozoa compared with the V4. A comparative shotgun metagenomics approach revealed few eukaryotic contigs but did recover a complete Paramicrosporidia mitochondrial genome, only the second publicly available from microsporidians. Both V4 and V9 markers were informative for assessing community diversity in oilsands-associated environments and are most effective when combined for a comprehensive taxonomic estimate, particularly in anoxic environments.

Diplonemids are among the most abundant and species-rich protists in the oceans. Marine heterotrophic flagellates, including diplonemids, have been suggested to play important roles in global biogeochemical cycles. Diplonemids are also the sister taxon of kinetoplastids, home to trypanosomatid parasites of global health importance, and thus are informative about the evolution of kinetoplastid biology. However, the genomic and cellular complement that underpins diplonemids' highly successful lifestyle is underexplored. At the same time, our framework describing cellular processes may not be as broadly applicable as presumed, as it is largely derived from animal and fungal model organisms, a small subset of extant eukaryotic diversity. In addition to uniquely evolved machinery in animals and fungi, there exist components with sporadic (i.e., "patchy") distributions across other eukaryotes. A most intriguing subset are components ("jötnarlogs") stochastically present in a wide range of eukaryotes but lost in animal and/or fungal models. Such components are considered exotic curiosities but may be relevant to inferences about the complexity of the last eukaryotic common ancestor (LECA) and frameworks of modern cell biology. Here, we use comparative genomics and phylogenetics to comprehensively assess the membrane-trafficking system of diplonemids. They possess several proteins thought of as kinetoplastid specific, as well as an extensive set of patchy proteins, including jötnarlogs. Diplonemids apparently function with endomembrane machinery distinct from existing cell biological models but comparable with other free-living heterotrophic protists, highlighting the importance of including such exotic components when considering different models of ancient eukaryotic genomic complexity and the cell biology of non-opisthokont organisms.

Phosphoinositides (PIPs), are key regulators of membrane identity and vesicular trafficking. By dynamically shaping the lipid composition of intracellular membranes, PIPs help ensure the specificity of cargo delivery. In apicomplexan parasites such as Plasmodium falciparum, the biogenesis of the specialized secretory organelles involved in erythrocyte invasion (named rhoptries, micronemes, and dense granules), remains poorly understood, particularly regarding how proteins are sorted and specifically targeted to their respective destinations. Our hypothesis is that PIPs might play a role in this process. We here present our characterization of the P. falciparum protein Pf3D7_0704400, a putative PIP-binding protein containing a PX domain. We named this protein PfPX2, following the previously characterized PX domain-containing protein PfPX1. In silico structural analysis revealed that the PfPX2 PX domain contains both canonical and non-canonical PIP-binding motifs and a positively charged binding pocket. Lipid binding assays showed that the PfPX2 PX domain can bind all species of PIPs with a preference for PI3P, PI5P and PI(3,5)P2. Immunofluorescence assays demonstrated that PfPX2 localized to the Golgi apparatus and the micronemes in developing schizonts. Moreover, proximity labelling enabled the identification of protein such as PfSortilin, the clathrin heavy chain and PfDyn1 as potential interactors of PfPX2. Globally, these data suggest that PfPX2 is a PIP-binding protein potentially implicated in vesicular trafficking between the Golgi apparatus and the micronemes. Our bioinformatics analyses identified PX2 orthologues across apicomplexans and indeed other alveolates, raising the possibility that this protein plays a role in a broad range of medically, agriculturally, and environmentally relevant organisms.

The emergence of eukaryotes from their prokaryotic ancestors (eukaryogenesis) marked a fundamental shift in cellular organisation, with the appearance of intracellular compartments including the nucleus, the Golgi apparatus and endosomes. These organelles are part of the endomembrane system of eukaryotic cells, which mediates many processes, including secretion of proteins to the exterior of the cell, uptake of material by endocytosis, and compartmentalized degradation of cellular components. The period of eukaryogenesis after the merger of prokaryotic lineages but preceding the last eukaryotic common ancestor, is inferred to have involved a progressive increase in cellular complexity through expansion of organelle-specific protein machineries. However, the steps and stages of organelle emergence during this period are poorly understood as no extant organisms exist from this period, precluding the use of comparative genomics to determine the properties of ancestral proteins present. Membrane trafficking pathways linking organelles are regulated by Arf family GTPases, including Arf1 and Arf6, both present in the last eukaryotic common ancestor. Here we use ancestral sequence reconstruction and molecular cell biological characterization to explore the properties of the ancestor of the Arf1 and Arf6 GTPases. Arf1 has a major function at the Golgi apparatus in regulation of the secretory pathway, whereas Arf6 regulates endocytic pathways at the plasma membrane and endosomes. Our results indicate that the ancestral Arf1/6 protein localizes to both the Golgi and the plasma membrane. We find that localization to the plasma membrane is due to a C-terminal polybasic motif that unexpectedly is also found in a number of modern Arf1 proteins from a wide diversity of eukaryotes. Our data suggest that the ancestral Arf protein acted at both internal compartments and the cell periphery, a feature preserved in a number of modern Arf1 proteins.

Apicomplexan parasites are obligate intracellular pathogens possessing unique organelles but lacking several components of the membrane trafficking machinery conserved in other eukaryotes. While some of these components have been lost during evolution, others remain undetectable by standard bioinformatics approaches. Using a conditional splitCas9 system in Toxoplasma gondii, we previously identified TGGT1_301410, a hypothetical gene conserved among apicomplexans, as a potential trafficking factor. Here, we show that TGGT1_301410 is a distant ortholog of T. gondii tepsin (TgTEP), localized to the trans-Golgi and functioning as an accessory protein of the adaptor protein complex 4 (AP4). We demonstrate that AP4-TgTEP is essential for the actin-dependent transport of vesicles to the plant-like vacuole (PLVAC) and Golgi organization. Notably, our findings reveal that, unlike in metazoans, the AP4 complex in T. gondii utilizes clathrin as a coat protein, a mechanism more closely aligned with that of plants. These results underscore a conserved yet functionally adapted vesicular transport system in Apicomplexa.

Arf and Rab family small GTPases and their regulators, GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), play a central role in membrane trafficking. In this study, we focused on a recently reported GAP for Arf (and potentially Rab) proteins, the CSW complex, a part of a small family of longin domain-containing proteins that form complexes with GAP activity. This family also includes folliculin and GATOR1, which are GAPs for the Rag/Gtr GTPases. All three complexes are associated with lysosomes and play a role in nutrient signaling, the latter two being directly involved in the mTOR pathway. The role of CSW is not clear, but in addition to having GAP activity on Arf proteins in vitro, its mutation causes severe neurodegenerative diseases. Here we update the reported pan-eukaryotic presence of folliculin and GATOR1, and demonstrate that CSW is also found throughout eukaryotes, though with sporadic distribution. We identify highly conserved motifs in all CSW subunits, some shared with the catalytic subunits of folliculin and GATOR1, that provide new potential avenues for experimental exploration. Remarkably, one such conserved sequence, the "GP" motif, is also found in structurally related longin proteins present in the archaeal ancestor of eukaryotes.

The origin of meiotic sex was a key milestone in the evolution of the eukaryotic cell. The paralogous DNA recombinases Rad51 and meiosis-specific DMC1 are nearly universal among eukaryotes and have been used previously to trace the timing and origins of the meiotic machinery. Here we perform comparative genomics and phylogenetic analyses of Rad51 and DMC1 drawn from diverse eukaryotes with RadA recombinase sequences from a broad sampling of archaeal taxa, focusing on the recently sequenced diversity of Asgard archaeal taxa. We show that even with increased and new sampling, the eukaryotic Rad51 and DMC1 proteins still resolve separately from any archaeal RadA sequences. These findings suggest that the duplication of RadA into general and meiosis-specific paralogues occurred after the divergence of the eukaryotic progenitor and did not evolve at an earlier stage. These findings raise the important question of how the evolution of meiotic sex was linked to genome size expansion and the acquisition of the mitochondrial endosymbiont in early eukaryotes.

Classical cell biology paradigms are largely established on animal, fungal, and plant models which constitute a small fraction of eukaryotic diversity. Some important cellular machinery has been historically overlooked due to their absence from animals and fungi, e.g., the membrane-trafficking complex TSET involved in plant cell division and endocytosis. Here, we document TSET complexes in distantly related photosynthetic eukaryotic groups (green algae, red algae, haptophytes, cryptophytes, and stramenopiles including diatoms). 3D modeling predicts that at least some stramenopile-encoded subunits share conserved structural features with plant orthologues, and gene expression analysis from the diatom Phaeodactylum tricornutum shows that they are co-expressed with endomembrane trafficking proteins. Finally, diatom TSET genes are detectable in meta-transcriptomic data from Tara Oceans, suggesting functional roles in the wild. These results support the importance of integrating non-model organisms into our understanding of eukaryotic cell biology, as they may reveal underappreciated protein complexes essential for cellular and ecosystem functions.

Surface mining and extraction of oil sands in Canada produces fluid tailings that contain several compounds of concern for the environment. One option for mine reclamation is the construction of Pit Lakes (PLs) to contain and remediate these tailings. Ultimately, PLs should support food webs typical of boreal lakes. From 2015 to 2021, we applied 16S/18S rRNA gene amplicon sequencing and metagenomics to monitor prokaryotic and eukaryotic microbes in the only full-scale PL of the oil sands industry (Base Mine Lake or BML), and compared it to two control environments: a freshwater reservoir unaffected by tailings, and active tailings ponds receiving regular industrial input. Microbial communities in BML were always intermediate to the two control environments based on alpha and beta diversity analyses. BML communities were highly variable with year, season, and water depth, and contained fewer core species than the freshwater reservoir. Several hydrocarbon degraders and sulfur cycling bacteria were identified as indicator species of tailings ponds, while several phototrophs were indicative of freshwater. However, all of these species were abundant in BML, suggesting that the PL supports food webs characteristic of each control environment. Over the 6-year study, the relative abundances of some common freshwater phytoplankton (Cryptomonas, Mychonastes, Trebouxiophyceae, Cyanobium) and heterotrophic bacteria (Sporichthyaceae, Ca. Fonsibacter, Ilumatobacteraceae, Microbacteriaceae, Ca. Planktophila) increased in BML. The results suggest that microbial communities and processes in BML represent an intermediate state between a tailings pond and a natural freshwater system, and did not stabilize within 10 years of its creation.

The intestinal parasite Giardia intestinalis , the causative agent of the globally distributed diarrheal disease Giardiasis, is one of the few genetically tractable members of the phylum Fornicata, which includes both parasitic and free-living species. The diversity of membrane traffic machinery in this lineage is of special interest in relation to the evolution of parasitism and the emergence of specialized organelles as possible adaptations to parasitism. Here, we performed a functional characterization of the ARF family of regulatory GTPases and their regulators in Giardia traffic, including three ARF paralogues, one ARF GAP and one ARF GEF. Using a combination of bioinformatic tools, protein network discovery and validation, and confocal light microscopy, we show that the Giardia ARF complement studied here is robustly associated with peripheral endocytic compartments (PECs), essential feeding organelles that are unique to Giardia parasites. Intersection of the interactomes of this ARF complement with previously published data for PECs-proteins (including membrane adaptors, predicted retromer subunits and SNAREs), revealed a complex crosstalk between the ARF complement and several membrane traffic processes. ### Competing Interest Statement The authors have declared no competing interest. CIHR Doctoral Scholarship, 175863 Alberta Innovates Graduate Student Scholarship NSERC Discovery Grants, RES0043758, RES0046091 Swiss National Science Foundation, https://ror.org/00yjd3n13, PR00P3_179813, PR00P3_179813/2, PR00P3_179813/3

The evolution of eukaryotes, eukaryogenesis, marked a fundamental shift in cellular organisation for life on Earth, with the advent of the nucleus, mitochondria, and other compartmentalization. The organelles and machinery of the endomembrane system, being responsible for material synthesis, intracellular targeting, uptake and secretion, are key eukaryotic hallmark to be explained. The period of eukaryogenesis after the merger of prokaryotic lineages but preceding the Last Eukaryotic Common Ancestor, is inferred to have involved a rise in cellular complexity through expansion of organelle-specific protein machinery, but the steps and stages of organelle emergence is essentially a blackbox. This is particularly problematic since ancestral protein function is not necessarily inferable from modern components. Here we apply Ancient Protein Resurrection (Ancestral Sequence Reconstruction and molecular cell biological characterization) to explore the ancient evolution of the Arf GTPases. Arf1 is canonically understood to act in eukaryotic cells at the Golgi apparatus, while Arf6 acts at endosomes and the plasma membrane. We find that the inferred ancestral Arf1/6 protein acts at both Golgi apparatus and plasma membrane in both mammalian and yeast cells. Unexpectedly, we find that the mechanism of localization to the plasma membrane is due to a C-terminal motif found in Arf1 proteins from the wide diversity of modern eukaryotes, though not the best characterized models of yeast, plants, and humans. Our data suggests that the ancestral Arf protein acted at multiple locations in eukaryotic cells prior to the diversification of modern lineages and Arf1 proteins may do so to a greater extent than appreciated in organisms of medical and ecological importance today. ### Competing Interest Statement The authors have declared no competing interest. Natural Sciences and Engineering Research Council, https://ror.org/01h531d29, RES0043758, RES0046091 Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-20-CE13-0007

The evolution of eukaryotes is a fundamental event in the history of life. The closest prokaryotic lineage to eukaryotes, the Asgardarchaeota, encode proteins previously found only in eukaryotes, providing insight into their archaeal ancestor. Eukaryotic cells are characterized by endomembrane organelles, and the Arf family GTPases regulate organelle dynamics by recruiting effector proteins to membranes upon activation. The Arf family is ubiquitous among eukaryotes, but its origins remain elusive. Here we report a group of prokaryotic GTPases, the ArfRs, which are widely present in Asgardarchaeota. Phylogenetic analyses reveal that eukaryotic Arf family proteins arose from the ArfR group. Expression of representative Asgardarchaeota ArfR proteins in yeast and X-ray crystallographic studies show that ArfR GTPases possess the mechanism of membrane binding and structural features unique to Arf family proteins. Our results indicate that Arf family GTPases originated in the archaeal ancestor of eukaryotes, consistent with aspects of the endomembrane system evolving early in eukaryogenesis. Eukaryotic Arf family proteins involved in endomembrane organelle dynamics arose from the Asgard archaeal ArfR GTPases, indicating this capability likely originated in the archaeal ancestor of eukaryotes.

The organelle paralogy hypothesis (OPH) aims to explain the evolution of non-endosymbiotically derived organelles. It predicts that lineage-specific pathways or organelles should result when identity-encoding membrane-trafficking components duplicate and co-evolve. Here, we investigate the presence of such lineage-specific membrane-trafficking machinery paralogs in Apicomplexa, a globally important parasitic lineage. We are able to identify 18 paralogs of known membrane-trafficking machinery, in several cases co-incident with the presence of new endomembrane organelles in apicomplexans or their parent lineage, the Alveolata. Moreover, focused analysis of the apicomplexan Arf-like small GTPases (i.e., ArlX3) revealed a specific post-Golgi trafficking pathway. This pathway appears involved in delivery of proteins to micronemes and rhoptries, with knockdown demonstrating reduced invasion capacity. Overall, our data have identified an unforeseen post-Golgi trafficking pathway in apicomplexans and are consistent with the OPH mechanism acting to produce endomembrane pathways or organelles at various evolutionary stages across the alveolate lineage.

Early Endosomes sort transmembrane cargo whether for lysosomal degradation or retrieval to the plasma membrane or the Golgi complex. Endosomal retrieval in eukaryotes is governed by the anciently homologous Retromer or Retriever complexes. Each comprises a core tri-protein subcomplex, membrane-deformation proteins, and interacting partner complexes, together retrieving a variety of known cargo proteins. Trichomonas vaginalis; a sexually transmitted human parasite uses the endomembrane system for pathogenesis. It has massively and selectively expanded its endomembrane protein complement, the evolutionary path of which has been largely unexplored. Our molecular evolutionary study of Retromer, Retriever and associated machinery in parabasalids and its free-living sister lineage of Anaeramoeba, demonstrates specific expansion of the Retromer machinery, contrasting with the Retriever components. We also observe partial loss of Commander complex and Sorting Nexins in Parabasalia but complete retention in Anaeramoeba. Notably, we identify putative parabasalid Sorting Nexin analogues. Finally, we report the first Retriever protein localization in a non- metazoan group along with Retromer protein localization in T. vaginalis. Therefore, we provide a unique genome expansion study of endomembrane trafficking system in parasitic and free- living protists alike.

Understanding the origin of eukaryotic cells is one of the most difficult problems in all of biology. A key challenge relevant to the question of eukaryogenesis is reconstructing the gene repertoire of the last eukaryotic common ancestor (LECA). As data sets grow, sketching an accurate genomics-informed picture of early eukaryotic cellular complexity requires provision of analytical resources and a commitment to data sharing. Here, we summarise progress towards understanding the biology of LECA and outline a community approach to inferring its wider gene repertoire. Once assembled, a robust LECA gene set will be a useful tool for evaluating alternative hypotheses about the origin of eukaryotes and understanding the evolution of traits in all descendant lineages, with relevance in diverse fields such as cell biology, microbial ecology, biotechnology, agriculture, and medicine. In this Consensus View, we put forth the status quo and an agreed path forward to reconstruct LECA’s gene content.

Summary The emergence of eukaryotes from their prokaryotic ancestors is one of the most fundamental evolutionary events in the history of life. Little is robustly known about how eukaryogenesis occurred, but a major breakthrough came with the identification of the Asgardarchaeota, the closest prokaryotic lineage to eukaryotes yet discovered. Endomembrane organelles, and the capacity to transport material between them, are major hallmarks of eukaryotic cells. The Arf family GTPases are crucial regulators of organelle dynamics in eukaryotes, functioning in vesicle budding, membrane tethering and membrane-cytoskeleton interactions. Although an expanded GTPase complement has been reported in the Asgardarchaeota, the specific origins of the Arf family remain elusive. Here we report a new group of prokaryotic GTPases, the ArfRs. Widely present in Asgardarchaeota and almost exclusive to them, it is the clade from which all eukaryotic Arf family proteins are derived. Heterologous expression of representative Asgardarchaeota ArfR proteins in the model eukaryote Saccharomyces cerevisiae and X-ray crystallographic studies demonstrate that ArfR GTPases possess the mechanism of membrane binding and structural features unique to Arf family proteins. Our results show that Arf family GTPases are present in Asgardarchaeota, and strongly suggest that they originated in the archaeal contributor to eukaryogenesis, providing support for nascent endomembrane system capacity evolving early in eukaryogenesis.

Background Base Mine Lake (BML) is the first full-scale end pit lake for the oil sands mining industry in Canada. BML sequesters oil sands tailings under a freshwater cap and is intended to develop into a functional ecosystem that can be integrated into the local watershed. The first stage of successful reclamation requires the development of a phytoplankton community supporting a typical boreal lake food web. To assess the diversity and dynamics of the phytoplankton community in BML at this reclamation stage and to set a baseline for future monitoring, we examined the phytoplankton community in BML from 2016 through 2021 using molecular methods (targeting the 23S, 18S, and 16S rRNA genes) and microscopic methods. Nearby water bodies were used as controls for a freshwater environment and an active tailings pond. Results The phytoplankton community was made up of diverse bacteria and eukaryotes typical of a boreal lake. Microscopy and molecular data both identified a phytoplankton community comparable at the phylum level to that of natural boreal lakes, dominated by Chlorophyta , Cryptophyta , and Cyanophyta , with some Bacillariophyta , Ochrophyta , and Euglenophyta . Although many of the same genera were prominent in both BML and the control freshwater reservoir, there were differences at the species or ASV level. Total diversity in BML was also consistently lower than the control freshwater site, but consistently higher than the control tailings pond. The phytoplankton community composition in BML changed over the 5-year study period. Some taxa present in 2016–2019 (e.g., Choricystis ) were no longer detected in 2021, while some dinophytes and haptophytes became detectable in small quantities starting in 2019–2021. Different quantification methods (qPCR analysis of 23S rRNA genes, and microscopic estimates of populations and total biomass) did not show a consistent directional trend in total phytoplankton over the 5-year study, nor was there any consistent increase in phytoplankton species diversity. The 5-year period was likely an insufficient time frame for detecting community trends, as phytoplankton communities are highly variable at the genus and species level. Conclusions BML supports a phytoplankton community composition somewhat unique from control sites (active tailings and freshwater lake) and is still changing over time. However, the most abundant genera are typical of natural boreal lakes and have the potential to support a complex aquatic food web, with many of its identified major phytoplankton constituents known to be primary producers in boreal lake environments.

AbstractSymbiotic relationships between eukaryotes and prokaryotes played pivotal roles in the evolution of life and drove the emergence of specialized symbiotic structures in animals, plants and fungi. The host-evolved symbiotic structures of microbial eukaryotes – the vast majority of such hosts in nature – remain largely unstudied. Here we describe highly structured symbiosomes within three free-living anaerobic protists (Anaeramoeba spp.). We dissect this symbiosis using complete genome sequencing and transcriptomics of host and symbiont cells coupled with fluorescence in situ hybridization, and 3D reconstruction using focused-ion-beam scanning electron microscopy. The emergence of the symbiosome is underpinned by expansion of gene families encoding regulators of membrane trafficking and phagosomal maturation and extensive bacteria-to-eukaryote lateral transfer. The symbionts reside deep within a symbiosomal membrane network that enables metabolic syntrophy by precisely positioning sulfate-reducing bacteria alongside host hydrogenosomes. Importantly, the symbionts maintain connections to the Anaeramoeba plasma membrane, blurring traditional boundaries between ecto- and endosymbiosis.