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.
- MeSH
- Biological Evolution MeSH
- Phylogeny MeSH
- Kinetoplastida * physiology genetics MeSH
- Publication type
- Journal Article MeSH
The major organelles of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) (~1.5 billion years ago). Their acquisitions were defining milestones during eukaryogenesis. Comparative cell biology and evolutionary analyses show multiple instances of homology in the protein machinery controlling distinct interorganelle trafficking routes. Resolving these homologous relationships allows us to explore processes underlying the emergence of additional, distinct cellular compartments, infer ancestral states predating LECA, and explore the process of eukaryogenesis itself. Here, we undertake a molecular evolutionary analysis (including providing a transcriptome of the jakobid flagellate Reclinomonas americana), exploring the origins of the machinery responsible for the biogenesis of lysosome-related organelles (LROs), the Biogenesis of LRO Complexes (BLOCs 1,2, and 3). This pathway has been studied only in animals and is not considered a feature of the basic eukaryotic cell plan. We show that this machinery is present across the eukaryotic tree of life and was likely in place prior to LECA, making it an underappreciated facet of eukaryotic cellular organisation. Moreover, we resolve multiple points of ancient homology between all three BLOCs and other post-endosomal retrograde trafficking machinery (BORC, CCZ1 and MON1 proteins, and an unexpected relationship with the "homotypic fusion and vacuole protein sorting" (HOPS) and "Class C core vacuole/endosomal tethering" (CORVET) complexes), offering a mechanistic and evolutionary unification of these trafficking pathways. Overall, this study provides a comprehensive account of the rise of the LROs biogenesis machinery from before the LECA to current eukaryotic diversity, integrating it into the larger mechanistic framework describing endomembrane evolution.
The utilization of nanoparticles for the intracellular delivery of theranostic agents faces one substantial limitation. Sequestration in intracellular vesicles prevents them from reaching the desired location in the cytoplasm or nucleus to deliver their cargo. We investigated whether three different cell-penetrating peptides (CPPs), namely, octa-arginine R8, polyhistidine KH27K and histidine-rich LAH4, could promote cytosolic and/or nuclear transfer of unique model nanoparticles-pseudovirions derived from murine polyomavirus. Two types of CPP-modified pseudovirions that carry the luciferase reporter gene were created: VirPorters-IN with CPPs genetically attached to the capsid interior and VirPorters-EX with CPPs noncovalently associated with the capsid exterior. We tested their transduction ability by luciferase assay and monitored their presence in subcellular fractions. Our results confirmed the overall effect of CPPs on the intracellular destination of the particles and suggested that KH27K has the potential to improve the cytosolic release of pseudovirions. None of the VirPorters caused endomembrane damage detectable by the Galectin-3 assay. Remarkably, a noncovalent modification was required to promote high transduction of the reporter gene and cytosolic delivery of pseudovirions mediated by LAH4. Together, CPPs in different arrangements have demonstrated their potential to improve pseudovirion invasion into cells, and these findings could be useful for the development of other nanoparticle-based delivery systems.
Endomembrane system compartments are significant elements in virtually all eukaryotic cells, supporting functions including protein synthesis, post-translational modifications and protein/lipid targeting. In terms of membrane area the endoplasmic reticulum (ER) is the largest intracellular organelle, but the origins of proteins defining the organelle and the nature of lineage-specific modifications remain poorly studied. To understand the evolution of factors mediating ER morphology and function we report a comparative genomics analysis of experimentally characterized ER-associated proteins involved in maintaining ER structure. We find that reticulons, REEPs, atlastins, Ufe1p, Use1p, Dsl1p, TBC1D20, Yip3p and VAPs are highly conserved, suggesting an origin at least as early as the last eukaryotic common ancestor (LECA), although many of these proteins possess additional non-ER functions in modern eukaryotes. Secondary losses are common in individual species and in certain lineages, for example lunapark is missing from the Stramenopiles and the Alveolata. Lineage-specific innovations include protrudin, Caspr1, Arl6IP1, p180, NogoR, kinectin and CLIMP-63, which are restricted to the Opisthokonta. Hence, much of the machinery required to build and maintain the ER predates the LECA, but alternative strategies for the maintenance and elaboration of ER shape and function are present in modern eukaryotes. Moreover, experimental investigations for ER maintenance factors in diverse eukaryotes are expected to uncover novel mechanisms.
- MeSH
- Endoplasmic Reticulum * metabolism MeSH
- Eukaryotic Cells * MeSH
- Protein Transport MeSH
- Publication type
- Journal Article MeSH
Giardia intestinalis is an enteric pathogen with an extremely modified membrane trafficking system, lacking canonical compartments such as the Golgi, endosomes, and intermediate vesicle carriers. By comparison the fornicate relatives of Giardia possess greater endomembrane system complexity. In eukaryotes, the ADP ribosylation factor (ARF) GTPase regulatory system proteins, which consist of the small GTPase ARF1, and its guanine exchange nucleotide factors (GEFs) and GTPase activating proteins (GAPs), coordinate temporal and directional trafficking of cargo vesicles by recognizing and interacting with heterotetrameric coat complexes at pre-Golgi and post-Golgi interfaces. To understand the evolution of this regulatory system across the fornicate lineage, we have performed comparative genomic and phylogenetic analyses of the ARF GTPases, and their regulatory GAPs and GEFs in fornicate genomes and transcriptomes. Prior to our analysis of the fornicates, we first establish that the ARF GAP sub-family ArfGAP with dual PH domains (ADAP) is sparsely distributed but present in at least four eukaryotic supergroups and thus was likely present in the Last Eukaryotic Common Ancestor (LECA). Next, our collective comparative genomic and phylogenetic investigations into the ARF regulatory proteins in fornicates identify a duplication of ARF1 GTPase yielding two paralogues of ARF1F proteins, ancestral to all fornicates and present in all examined isolates of Giardia. However, the ARF GEF and ARF GAP complement is reduced compared with the LECA. This investigation shows that the system was significantly streamlined prior to the fornicate ancestor but was not further reduced concurrent with a transition into a parasitic lifestyle.
- MeSH
- ADP-Ribosylation Factors * genetics metabolism MeSH
- Phylogeny MeSH
- Giardia lamblia * genetics metabolism MeSH
- GTPase-Activating Proteins genetics metabolism MeSH
- Guanine Nucleotide Exchange Factors genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The membrane-trafficking system is a defining facet of eukaryotic cells. The best-known organelles and major protein families of this system are largely conserved across the vast diversity of eukaryotes, implying both ancient organization and functional unity. Nonetheless, intriguing variation exists that speaks to the evolutionary forces that have shaped the endomembrane system in eukaryotes and highlights ways in which membrane trafficking in protists differs from that in our well-understood models of mammalian and yeast cells. Both parasites and free-living protists possess specialized trafficking organelles, some lineage specific, others more widely distributed - the evolution and function of these organelles begs exploration. Novel members of protein families are present across eukaryotes but have been lost in humans. These proteins may well hold clues to understanding differences in cellular function in organisms that are of pressing importance for planetary health.
The discovery that the protist Monocercomonoides exilis completely lacks mitochondria demonstrates that these organelles are not absolutely essential to eukaryotic cells. However, the degree to which the metabolism and cellular systems of this organism have adapted to the loss of mitochondria is unknown. Here, we report an extensive analysis of the M. exilis genome to address this question. Unexpectedly, we find that M. exilis genome structure and content is similar in complexity to other eukaryotes and less "reduced" than genomes of some other protists from the Metamonada group to which it belongs. Furthermore, the predicted cytoskeletal systems, the organization of endomembrane systems, and biosynthetic pathways also display canonical eukaryotic complexity. The only apparent preadaptation that permitted the loss of mitochondria was the acquisition of the SUF system for Fe-S cluster assembly and the loss of glycine cleavage system. Changes in other systems, including in amino acid metabolism and oxidative stress response, were coincident with the loss of mitochondria but are likely adaptations to the microaerophilic and endobiotic niche rather than the mitochondrial loss per se. Apart from the lack of mitochondria and peroxisomes, we show that M. exilis is a fully elaborated eukaryotic cell that is a promising model system in which eukaryotic cell biology can be investigated in the absence of mitochondria.
Formins are evolutionarily conserved eukaryotic proteins engaged in actin nucleation and other aspects of cytoskeletal organization. Angiosperms have two formin clades with multiple paralogs; typical plant Class I formins are integral membrane proteins that can anchor cytoskeletal structures to membranes. For the main Arabidopsis housekeeping Class I formin, FH1 (At3g25500), plasmalemma localization was documented in heterologous expression and overexpression studies. We previously showed that loss of FH1 function increases cotyledon epidermal pavement cell shape complexity via modification of actin and microtubule organization and dynamics. Here, we employ transgenic Arabidopsis expressing green fluorescent protein-tagged FH1 (FH1-GFP) from its native promoter to investigate in vivo behavior of this formin using advanced microscopy techniques. The fusion protein is functional, since its expression complements the fh1 loss-of-function mutant phenotype. Accidental overexpression of FH1-GFP results in a decrease in trichome branch number, while fh1 mutation has the opposite effect, indicating a general role of this formin in controlling cell shape complexity. Consistent with previous reports, FH1-GFP associates with membranes. However, the protein exhibits surprising actin- and secretory pathway-dependent dynamic localization and relocates between cellular endomembranes and the plasmalemma during cell division and differentiation in root tissues, with transient tonoplast localization at the transition/elongation zones border. FH1-GFP also accumulates in actin-rich regions of cortical cytoplasm and associates with plasmodesmata in both the cotyledon epidermis and root tissues. Together with previous reports from metazoan systems, this suggests that formins might have a shared (ancestral or convergent) role at cell-cell junctions.
BACKGROUND: Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts. RESULTS: We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids. CONCLUSIONS: Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the 'shopping bag' hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.
Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking, and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidylinositol-4-phosphate (PI4P), phosphatidic acidic (PA), and phosphatidylserine (PS) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of organelles not only defines the electrostatic territory but also distinguishes different functional compartments within this territory by specifying their varying surface charges.
- MeSH
- Arabidopsis growth & development metabolism MeSH
- Cell Membrane metabolism MeSH
- Phosphatidylinositol Phosphates metabolism MeSH
- Phosphatidylserines metabolism MeSH
- Plant Roots growth & development metabolism MeSH
- Phosphatidic Acids metabolism MeSH
- Organelles MeSH
- Arabidopsis Proteins metabolism MeSH
- Signal Transduction MeSH
- Static Electricity * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
