Most cited article - PubMed ID 30732613
Transcriptome, proteome and draft genome of Euglena gracilis
Diplonemids are highly diverse and abundant marine plankton with significant ecological importance. However, little is known about their biology, even in the model diplonemid Paradiplonema papillatum whose genome sequence is available. Examining the subcellular localization of proteins using fluorescence microscopy is a powerful approach to infer their putative function. Here, we report a plasmid-based method that enables YFP-tagging of a gene at the endogenous locus. By examining the localization of proteins whose homologs are involved in chromosome organization or segregation in other eukaryotes, we discovered several notable features in mitotically dividing P. papillatum cells. Cohesin is enriched on condensed interphase chromatin. During mitosis, chromosomes organize into two rings (termed mitotic rings herein) that surround the elongating nucleolus and align on a bipolar spindle. Homologs of chromosomal passenger complex components (INCENP, two Aurora kinases and KIN-A), a CLK1 kinase, meiotic chromosome axis protein SYCP2L1, spindle checkpoint protein Mad1 and microtubule regulator XMAP215 localize in between the two mitotic rings. In contrast, a Mad2 homolog localizes near basal bodies as in trypanosomes. By representing the first molecular characterization of mitotic mechanisms in P. papillatum and raising many questions, this study forms the foundation for dissecting mitotic mechanisms in diplonemids.
- Keywords
- Euglenozoa, chromosome, diplonemid, kinetochore, kinetoplastid,
- MeSH
- Spindle Apparatus metabolism MeSH
- Chromosomal Proteins, Non-Histone metabolism MeSH
- Chromosomes metabolism MeSH
- Dinoflagellida * genetics metabolism cytology MeSH
- Mitosis * MeSH
- Cell Cycle Proteins metabolism MeSH
- Chromosome Segregation MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Chromosomal Proteins, Non-Histone MeSH
- Cell Cycle Proteins MeSH
This article explores the use of expansion microscopy, a technique that enhances resolution in fluorescence microscopy, on the autotrophic protist Euglena gracilis A modified protocol was developed to preserve the cell structures during fixation. Using antibodies against key cytoskeletal and organelle markers, α-tubulin, β-ATPase, and Rubisco activase, the microtubular structures, mitochondria, and chloroplasts were visualised. The organisation of the cytoskeleton corresponded to the findings from electron microscopy while allowing for the visualisation of the flagellar pocket in its entirety and revealing previously unnoticed details. This study offered insights into the shape and development of mitochondria and chloroplasts under varying conditions, such as culture ages and light cycles. This work demonstrated that expansion microscopy is a robust tool for visualising cellular structures in E. gracilis, an organism whose internal structures cannot be stained using standard immunofluorescence because of its complex pellicle. This technique also serves as a complement to electron microscopy, facilitating tomographic reconstructions in a routine fashion.
- MeSH
- Chloroplasts ultrastructure MeSH
- Cytoskeleton * ultrastructure MeSH
- Euglena gracilis * ultrastructure MeSH
- Flagella ultrastructure MeSH
- Microscopy, Fluorescence * methods MeSH
- Mitochondria ultrastructure MeSH
- Mitosis MeSH
- Antibodies chemistry MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Antibodies MeSH
UNLABELLED: Transmission of genetic material from one generation to the next is a fundamental feature of all living cells. In eukaryotes, a macromolecular complex called the kinetochore plays crucial roles during chromosome segregation by linking chromosomes to spindle microtubules. Little is known about this process in evolutionarily diverse protists. Within the supergroup Discoba, Euglenozoa forms a speciose group of unicellular flagellates-kinetoplastids, euglenids, and diplonemids. Kinetoplastids have an unconventional kinetochore system, while euglenids have subunits that are conserved among most eukaryotes. For diplonemids, a group of extremely diverse and abundant marine flagellates, it remains unclear what kind of kinetochores are present. Here, we employed deep homology detection protocols using profile-versus-profile Hidden Markov Model searches and AlphaFold-based structural comparisons to detect homologies that might have been previously missed. Interestingly, we still could not detect orthologs for most of the kinetoplastid or canonical kinetochore subunits with few exceptions including a putative centromere-specific histone H3 variant (cenH3/CENP-A), the spindle checkpoint protein Mad2, the chromosomal passenger complex members Aurora and INCENP, and broadly conserved proteins like CLK kinase and the meiotic synaptonemal complex proteins SYCP2/3 that also function at kinetoplastid kinetochores. We examined the localization of five candidate kinetochore-associated proteins in the model diplonemid, Paradiplonema papillatum. PpCENP-A shows discrete dots in the nucleus, implying that it is likely a kinetochore component. PpMad2, PpCLKKKT10/19, PpSYCP2L1KKT17/18, and PpINCENP reside in the nucleus, but no clear kinetochore localization was observed. Altogether, these results point to the possibility that diplonemids evolved a hitherto unknown type of kinetochore system. IMPORTANCE: A macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplonemids except for a putative centromere-specific histone H3 variant. Our results suggest that diplonemids evolved kinetochores that do not resemble previously known ones.
- Keywords
- Diplonemea, Kinetoplastea, Paradiplonema, cell division, cenH3/CENP-A, kinetochore,
- MeSH
- Euglenozoa * genetics metabolism MeSH
- Phylogeny MeSH
- Kinetochores * metabolism MeSH
- Protozoan Proteins metabolism genetics MeSH
- Chromosome Segregation MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Protozoan Proteins MeSH
BACKGROUND: In trypanosomatids, a group of unicellular eukaryotes that includes numerous important human parasites, cis-splicing has been previously reported for only two genes: a poly(A) polymerase and an RNA helicase. Conversely, trans-splicing, which involves the attachment of a spliced leader sequence, is observed for nearly every protein-coding transcript. So far, our understanding of splicing in this protistan group has stemmed from the analysis of only a few medically relevant species. In this study, we used an extensive dataset encompassing all described trypanosomatid genera to investigate the distribution of intron-containing genes and the evolution of splice sites. RESULTS: We identified a new conserved intron-containing gene encoding an RNA-binding protein that is universally present in Kinetoplastea. We show that Perkinsela sp., a kinetoplastid endosymbiont of Amoebozoa, represents the first eukaryote completely devoid of cis-splicing, yet still preserving trans-splicing. We also provided evidence for reverse transcriptase-mediated intron loss in Kinetoplastea, extensive conservation of 5' splice sites, and the presence of non-coding RNAs within a subset of retained trypanosomatid introns. CONCLUSIONS: All three intron-containing genes identified in Kinetoplastea encode RNA-interacting proteins, with a potential to fine-tune the expression of multiple genes, thus challenging the perception of cis-splicing in these protists as a mere evolutionary relic. We suggest that there is a selective pressure to retain cis-splicing in trypanosomatids and that this is likely associated with overall control of mRNA processing. Our study provides new insights into the evolution of introns and, consequently, the regulation of gene expression in eukaryotes.
- Keywords
- Introns, Kinetoplastea, Poly(A) polymerase, RNA helicase, RNA-binding protein, Splicing, Trypanosomatidae,
- MeSH
- Phylogeny MeSH
- Introns * genetics MeSH
- Kinetoplastida genetics MeSH
- Evolution, Molecular MeSH
- Genes, Protozoan genetics MeSH
- Protozoan Proteins genetics MeSH
- Trans-Splicing * genetics MeSH
- Trypanosomatina genetics MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Protozoan Proteins MeSH
The nuclear envelope (NE) separates translation and transcription and is the location of multiple functions, including chromatin organization and nucleocytoplasmic transport. The molecular basis for many of these functions have diverged between eukaryotic lineages. Trypanosoma brucei, a member of the early branching eukaryotic lineage Discoba, highlights many of these, including a distinct lamina and kinetochore composition. Here, we describe a cohort of proteins interacting with both the lamina and NPC, which we term lamina-associated proteins (LAPs). LAPs represent a diverse group of proteins, including two candidate NPC-anchoring pore membrane proteins (POMs) with architecture conserved with S. cerevisiae and H. sapiens, and additional peripheral components of the NPC. While many of the LAPs are Kinetoplastid specific, we also identified broadly conserved proteins, indicating an amalgam of divergence and conservation within the trypanosome NE proteome, highlighting the diversity of nuclear biology across the eukaryotes, increasing our understanding of eukaryotic and NPC evolution.
- Keywords
- AlphaFold, Nucleus, comparative genomics, molecular evolution, nuclear lamina, nuclear pore complex,
- MeSH
- Nuclear Envelope * metabolism MeSH
- Nuclear Pore metabolism MeSH
- Nuclear Pore Complex Proteins metabolism MeSH
- Humans MeSH
- Saccharomyces cerevisiae metabolism MeSH
- Trypanosoma * metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Nuclear Pore Complex Proteins MeSH
Dinoflagellates are a diverse group of ecologically significant micro-eukaryotes that can serve as a model system for plastid symbiogenesis due to their susceptibility to plastid loss and replacement via serial endosymbiosis. Kareniaceae harbor fucoxanthin-pigmented plastids instead of the ancestral peridinin-pigmented ones and support them with a diverse range of nucleus-encoded plastid-targeted proteins originating from the haptophyte endosymbiont, dinoflagellate host, and/or lateral gene transfers (LGT). Here, we present predicted plastid proteomes from seven distantly related kareniaceans in three genera (Karenia, Karlodinium, and Takayama) and analyze their evolutionary patterns using automated tree building and sorting. We project a relatively limited ( ~ 10%) haptophyte signal pointing towards a shared origin in the family Chrysochromulinaceae. Our data establish significant variations in the functional distributions of these signals, emphasizing the importance of micro-evolutionary processes in shaping the chimeric proteomes. Analysis of plastid genome sequences recontextualizes these results by a striking finding the extant kareniacean plastids are in fact not all of the same origin, as two of the studied species (Karlodinium armiger, Takayama helix) possess plastids from different haptophyte orders than the rest.
- Keywords
- Automated Tree Sorting, Myzozoa, Post-Endosymbiotic Organelle Evolution, Protists, Shopping Bag Model,
- MeSH
- Dinoflagellida * genetics metabolism MeSH
- Phylogeny MeSH
- Plastids genetics MeSH
- Proteome genetics metabolism MeSH
- Symbiosis genetics MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Proteome MeSH
Genetic variation is the major mechanism behind adaptation and evolutionary change. As most proteins operate through interactions with other proteins, changes in protein complex composition and subunit sequence provide potentially new functions. Comparative genomics can reveal expansions, losses and sequence divergence within protein-coding genes, but in silico analysis cannot detect subunit substitutions or replacements of entire protein complexes. Insights into these fundamental evolutionary processes require broad and extensive comparative analyses, from both in silico and experimental evidence. Here, we combine data from both approaches and consider the gamut of possible protein complex compositional changes that arise during evolution, citing examples of complete conservation to partial and total replacement by functional analogues. We focus in part on complexes in trypanosomes as they represent one of the better studied non-animal/non-fungal lineages, but extend insights across the eukaryotes by extensive comparative genomic analysis. We argue that gene loss plays an important role in diversification of protein complexes and hence enhancement of eukaryotic diversity.
- Keywords
- constructive neutral evolution, evolutionary divergence, evolutionary mechanisms, gene replacement, molecular evolution, protein complexes,
- MeSH
- Eukaryota * genetics MeSH
- Phylogeny MeSH
- Genomics MeSH
- Evolution, Molecular * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Archamoebae comprises free-living or endobiotic amoebiform protists that inhabit anaerobic or microaerophilic environments and possess mitochondrion-related organelles (MROs) adapted to function anaerobically. We compared in silico reconstructed MRO proteomes of eight species (six genera) and found that the common ancestor of Archamoebae possessed very few typical components of the protein translocation machinery, electron transport chain and tricarboxylic acid cycle. On the other hand, it contained a sulphate activation pathway and bacterial iron-sulphur (Fe-S) assembly system of MIS-type. The metabolic capacity of the MROs, however, varies markedly within this clade. The glycine cleavage system is widely conserved among Archamoebae, except in Entamoeba, probably owing to its role in catabolic function or one-carbon metabolism. MRO-based pyruvate metabolism was dispensed within subgroups Entamoebidae and Rhizomastixidae, whereas sulphate activation could have been lost in isolated cases of Rhizomastix libera, Mastigamoeba abducta and Endolimax sp. The MIS (Fe-S) assembly system was duplicated in the common ancestor of Mastigamoebidae and Pelomyxidae, and one of the copies took over Fe-S assembly in their MRO. In Entamoebidae and Rhizomastixidae, we hypothesize that Fe-S cluster assembly in both compartments may be facilitated by dual localization of the single system. We could not find evidence for changes in metabolic functions of the MRO in response to changes in habitat; it appears that such environmental drivers do not strongly affect MRO reduction in this group of eukaryotes.
- Keywords
- anaerobiosis, comparative genomics, mitochondrion-related organelles, reductive evolution,
- MeSH
- Anaerobiosis MeSH
- Eukaryota * MeSH
- Mitochondria * genetics MeSH
- Sulfates MeSH
- Iron MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Sulfates MeSH
- Iron MeSH
Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.
- MeSH
- Endoribonucleases * metabolism MeSH
- RNA, Messenger genetics metabolism MeSH
- RNA-Binding Proteins genetics metabolism MeSH
- RNA Caps * genetics metabolism MeSH
- RNA Stability MeSH
- Trypanosoma * genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Endoribonucleases * MeSH
- RNA, Messenger MeSH
- mRNA decapping enzymes MeSH Browser
- RNA-Binding Proteins MeSH
- RNA Caps * MeSH
The β-propeller protein Sec13 plays roles in at least three distinct processes by virtue of being a component of the COPII endoplasmic reticulum export vesicle coat, the nuclear pore complex (NPC) and the Seh1-associated (SEA)/GATOR nutrient-sensing complex. This suggests that regulatory mechanisms coordinating these cellular activities may operate via Sec13. The NPC, COPII and SEA/GATOR are all ancient features of eukaryotic cells, and in the vast majority of eukaryotes, a single Sec13 gene is present. Here we report that the Euglenozoa, a lineage encompassing the diplonemid, kinetoplastid and euglenid protists, possess two Sec13 paralogues. Furthermore, based on protein interactions and localization studies we show that in diplonemids Sec13 functions are divided between the Sec13a and Sec13b paralogues. Specifically, Sec13a interacts with COPII and the NPC, while Sec13b interacts with Sec16 and components of the SEA/GATOR complex. We infer that euglenozoan Sec13a is responsible for NPC functions and canonical anterograde transport activities while Sec13b acts within nutrient and autophagy-related pathways, indicating a fundamentally distinct organization of coatomer complexes in euglenozoan flagellates.
