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
• chloroplasty ultrastruktura   MeSH
• cytoskelet * ultrastruktura   MeSH
• Euglena gracilis * ultrastruktura   MeSH
• flagella ultrastruktura   MeSH
• fluorescenční mikroskopie * metody   MeSH
• mitochondrie ultrastruktura   MeSH
• mitóza MeSH
• protilátky chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protilátky 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.

• Klíčová slova
• constructive neutral evolution, evolutionary divergence, evolutionary mechanisms, gene replacement, molecular evolution, protein complexes,
• MeSH
• Eukaryota * genetika   MeSH
• fylogeneze MeSH
• genomika MeSH
• molekulární evoluce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Euglenids represent a group of protists with diverse modes of feeding. To date, only a partial genomic sequence of Euglena gracilis and transcriptomes of several phototrophic and secondarily osmotrophic species are available, while primarily heterotrophic euglenids are seriously undersampled. In this work, we begin to fill this gap by presenting genomic and transcriptomic drafts of a primary osmotroph, Rhabdomonas costata. The current genomic assembly length of 100 Mbp is 14× smaller than that of E. gracilis. Despite being too fragmented for comprehensive gene prediction it provided fragments of the mitochondrial genome and comparison of the transcriptomic and genomic data revealed features of its introns, including several candidates for nonconventional types. A set of 39,456 putative R. costata proteins was predicted from the transcriptome. Annotation of the mitochondrial core metabolism provides the first data on the facultatively anaerobic mitochondrion of R. costata, which in most respects resembles the mitochondrion of E. gracilis with a certain level of streamlining. R. costata can synthetise thiamine by enzymes of heterogenous provenances and haem by a mitochondrial-cytoplasmic C4 pathway with enzymes orthologous to those found in E. gracilis. The low percentage of green algae-affiliated genes supports the ancestrally osmotrophic status of this species.

• MeSH
• biologická evoluce MeSH
• Chromatium genetika   metabolismus   MeSH
• Euglenida genetika   metabolismus   MeSH
• exony genetika   MeSH
• fylogeneze MeSH
• genom MeSH
• heterotrofní procesy MeSH
• introny genetika   MeSH
• mitochondrie genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• transkriptom genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered. RESULTS: We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome. CONCLUSIONS: Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

• Klíčová slova
• Evolution, Mitochondrial ribosome, Phylogeny, Single-cell amplified genome,
• MeSH
• Euglenida * genetika   MeSH
• Euglenozoa genetika   MeSH
• europium MeSH
• fylogeneze MeSH
• genom mitochondriální * genetika   MeSH
• genomika MeSH
• mitochondriální DNA MeSH
• RNA transferová MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• europium MeSH
• mitochondriální DNA MeSH
• RNA transferová MeSH

Mitochondrial protein import requires outer membrane receptors that evolved independently in different lineages. Here we used quantitative proteomics and in vitro binding assays to investigate the substrate preferences of ATOM46 and ATOM69, the two mitochondrial import receptors of Trypanosoma brucei The results show that ATOM46 prefers presequence-containing, hydrophilic proteins that lack transmembrane domains (TMDs), whereas ATOM69 prefers presequence-lacking, hydrophobic substrates that have TMDs. Thus, the ATOM46/yeast Tom20 and the ATOM69/yeast Tom70 pairs have similar substrate preferences. However, ATOM46 mainly uses electrostatic, and Tom20 hydrophobic, interactions for substrate binding. In vivo replacement of T. brucei ATOM46 by yeast Tom20 did not restore import. However, replacement of ATOM69 by the recently discovered Tom36 receptor of Trichomonas hydrogenosomes, while not allowing for growth, restored import of a large subset of trypanosomal proteins that lack TMDs. Thus, even though ATOM69 and Tom36 share the same domain structure and topology, they have different substrate preferences. The study establishes complementation experiments, combined with quantitative proteomics, as a highly versatile and sensitive method to compare in vivo preferences of protein import receptors. Moreover, it illustrates the role determinism and contingencies played in the evolution of mitochondrial protein import receptors.

• Klíčová slova
• Trichomonas, Trypanosoma, mitochondria, protein import, receptors,
• MeSH
• mitochondriální importní komplex MeSH
• mitochondriální proteiny genetika   MeSH
• mitochondrie genetika   metabolismus   MeSH
• molekulární evoluce * MeSH
• proteinové prekurzory genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• transport proteinů genetika   MeSH
• transportní proteiny mitochondriální membrány genetika   MeSH
• transportní proteiny genetika   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   patogenita   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální importní komplex MeSH
• mitochondriální proteiny MeSH
• proteinové prekurzory MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• TOM20 protein, S cerevisiae MeSH Prohlížeč
• TOM70 protein, S cerevisiae MeSH Prohlížeč
• transportní proteiny mitochondriální membrány MeSH
• transportní proteiny MeSH

The order Trypanosomatida has been well studied due to its pathogenicity and the unique biology of the mitochondrion. In Trypanosoma brucei, four DNA polymerases, namely PolIA, PolIB, PolIC, and PolID, related to bacterial DNA polymerase I (PolI), were shown to be localized in mitochondria experimentally. These mitochondrion-localized DNA polymerases are phylogenetically distinct from other family A DNA polymerases, such as bacterial PolI, DNA polymerase gamma (Polγ) in human and yeasts, "plant and protist organellar DNA polymerase (POP)" in diverse eukaryotes. However, the diversity of mitochondrion-localized DNA polymerases in Euglenozoa other than Trypanosomatida is poorly understood. In this study, we discovered putative mitochondrion-localized DNA polymerases in broad members of three major classes of Euglenozoa-Kinetoplastea, Diplonemea, and Euglenida-to explore the origin and evolution of trypanosomatid PolIA-D. We unveiled distinct inventories of mitochondrion-localized DNA polymerases in the three classes: (1) PolIA is ubiquitous across the three euglenozoan classes, (2) PolIB, C, and D are restricted in kinetoplastids, (3) new types of mitochondrion-localized DNA polymerases were identified in a prokinetoplastid and diplonemids, and (4) evolutionarily distinct types of POP were found in euglenids. We finally propose scenarios to explain the inventories of mitochondrion-localized DNA polymerases in Kinetoplastea, Diplonemea, and Euglenida.

• Klíčová slova
• DNA replication, Diplonemea, Euglenida, Kinetoplastea, Prokinetoplastina, Trypanosomatida, family A DNA polymerase, plant and protist organellar DNA polymerase,
• Publikační typ
• časopisecké články MeSH