Nuclear export of mRNAs requires loading the mRNP to the transporter Mex67/Mtr2 in the nucleoplasm, controlled access to the pore by the basket-localised TREX-2 complex and mRNA release at the cytoplasmic site by the DEAD-box RNA helicase Dbp5. Asymmetric localisation of nucleoporins (NUPs) and transport components as well as the ATP dependency of Dbp5 ensure unidirectionality of transport. Trypanosomes possess homologues of the mRNA transporter Mex67/Mtr2, but not of TREX-2 or Dbp5. Instead, nuclear export is likely fuelled by the GTP/GDP gradient created by the Ran GTPase. However, it remains unclear, how directionality is achieved since the current model of the trypanosomatid pore is mostly symmetric. We have revisited the architecture of the trypanosome nuclear pore complex using a novel combination of expansion microscopy, proximity labelling and streptavidin imaging. We could confidently assign the NUP76 complex, a known Mex67 interaction platform, to the cytoplasmic site of the pore and the NUP64/NUP98/NUP75 complex to the nuclear site. Having defined markers for both sites of the pore, we set out to map all 75 trypanosome proteins with known nuclear pore localisation to a subregion of the pore using mass spectrometry data from proximity labelling. This approach defined several further proteins with a specific localisation to the nuclear site of the pore, including proteins with predicted structural homology to TREX-2 components. We mapped the components of the Ran-based mRNA export system to the nuclear site (RanBPL), the cytoplasmic site (RanGAP, RanBP1) or both (Ran, MEX67). Lastly, we demonstrate, by deploying an auxin degron system, that NUP76 holds an essential role in mRNA export consistent with a possible functional orthology to NUP82/88. Altogether, the combination of proximity labelling with expansion microscopy revealed an asymmetric architecture of the trypanosome nuclear pore supporting inherent roles for directed transport. Our approach delivered novel nuclear pore associated components inclusive positional information, which can now be interrogated for functional roles to explore trypanosome-specific adaptions of the nuclear basket, export control, and mRNP remodelling.

• MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• jaderný pór * metabolismus   ultrastruktura   MeSH
• komplex proteinů jaderného póru metabolismus   MeSH
• messenger RNA * metabolismus   genetika   MeSH
• nukleocytoplazmatické transportní proteiny metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• protozoální proteiny metabolismus   genetika   MeSH
• transport RNA MeSH
• Trypanosoma brucei brucei * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• komplex proteinů jaderného póru MeSH
• messenger ribonucleoprotein MeSH Prohlížeč
• messenger RNA * MeSH
• nukleocytoplazmatické transportní proteiny MeSH
• proteiny vázající RNA MeSH
• protozoální proteiny MeSH
• ribonukleoproteiny MeSH

Conserved histone methyltransferases of the DOT1 family are involved in replication regulation, cell cycle progression, stage differentiation, and gene regulation in trypanosomatids. However, the specific functions of these enzymes depend on the host evasion strategies of the parasites. In this study, we investigated the role of DOT1B in Leishmania mexicana, focusing on life cycle progression and infectivity. In contrast to Trypanosoma brucei, in which DOT1B is essential for the differentiation of mammal-infective bloodstream forms to insect procyclic forms, L. mexicana DOT1B (LmxDOT1B) is not critical for the differentiation of promastigotes to amastigotes in vitro. Additionally, there are no significant differences in the ability to infect or differentiate in macrophages or sand fly vectors between the LmxDOT1B-depleted and control strains. These findings highlight the divergence of the function of DOT1B in these related parasites, suggesting genus-specific adaptations in the use of histone modifications for life cycle progression and host adaptation processes.

• Klíčová slova
• DOT1, Leishmania mexicana, differentiation, histone methyltransferase, sand fly, virulence,
• MeSH
• histonlysin-N-methyltransferasa * metabolismus   genetika   MeSH
• Leishmania mexicana * růst a vývoj   enzymologie   genetika   patogenita   MeSH
• makrofágy * parazitologie   MeSH
• myši MeSH
• protozoální proteiny * metabolismus   genetika   MeSH
• Psychodidae parazitologie   MeSH
• stadia vývoje MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• histonlysin-N-methyltransferasa * MeSH
• protozoální proteiny * MeSH

Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major, which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to the rapidness of image acquisition and three-dimensional reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic and multivesicular tubule-associated microtubules of L. major. We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. Three-dimensional reconstruction of an entire volume of these cells provided details on the morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.

• Klíčová slova
• Leishmania major, Trypanosoma brucei, expansion microscopy, microtubule-based cytoskeleton,
• MeSH
• elektronová mikroskopie metody   MeSH
• kinetochory metabolismus   ultrastruktura   MeSH
• Kinetoplastida metabolismus   ultrastruktura   MeSH
• Leishmania major metabolismus   ultrastruktura   MeSH
• mikrotubuly metabolismus   ultrastruktura   MeSH
• protozoální proteiny metabolismus   MeSH
• Trypanosoma brucei brucei metabolismus   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new-flagellum and the old-flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new-flagellum daughter in particular re-modelling rapidly and extensively in early G1. This re-modelling process involves an increase in cell body, flagellum and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old-flagellum daughter undergoes a different G1 re-modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that the two daughters of a proliferative division of T. brucei are non-equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non-equivalence.

• MeSH
• buněčné dělení MeSH
• cytokineze MeSH
• flagella genetika   metabolismus   MeSH
• proliferace buněk MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• stadia vývoje MeSH
• Trypanosoma brucei brucei cytologie   genetika   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite's life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.

• MeSH
• flagella genetika   metabolismus   MeSH
• Leishmania genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH
• protozoální proteiny MeSH

Leishmania kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for Leishmania pathogenicity. The Leishmania FP has a bulbous region posterior to the FP collar and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein, FAZ5, clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: Mutants were unable to develop late-stage infections in sand flies, and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.

• Klíčová slova
• Leishmania, flagellar pocket, morphogenesis, pathogenicity,
• MeSH
• buněčná membrána metabolismus   MeSH
• cilie genetika   fyziologie   ultrastruktura   MeSH
• delece genu MeSH
• endocytóza MeSH
• flagella genetika   fyziologie   ultrastruktura   MeSH
• interakce hostitele a parazita MeSH
• Leishmania genetika   patogenita   fyziologie   ultrastruktura   MeSH
• mezibuněčné spoje MeSH
• myši MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• virulence genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

Trypanosomes are masters of adaptation to different host environments during their complex life cycle. Large-scale proteomic approaches provide information on changes at the cellular level, and in a systematic way. However, detailed work on single components is necessary to understand the adaptation mechanisms on a molecular level. Here, we have performed a detailed characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation factor Tb927.11.2400, identified previously in a SILAC-based comparative proteome study. Tb927.11.2400 shares 38% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form (PCF) stage-specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin-like (TbFlabarinL), and demonstrate that it originates from a gene duplication event, which occurred in the African trypanosomes. TbFlabarinL is not essential for the growth of the parasites under cell culture conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We generated TbFlabarinL-specific antibodies, and showed that it localizes in the flagellum. Co-immunoprecipitation experiments together with a biochemical cell fractionation suggest a dual association of TbFlabarinL with the flagellar membrane and the components of the paraflagellar rod.

• MeSH
• duplikace genu MeSH
• flagella fyziologie   MeSH
• fylogeneze MeSH
• geneticky modifikované organismy MeSH
• myši inbrední C57BL MeSH
• proteinové domény MeSH
• protozoální proteiny chemie   genetika   imunologie   metabolismus   MeSH
• Trypanosoma brucei brucei genetika   patogenita   fyziologie   MeSH
• trypanozomóza africká parazitologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protozoální proteiny MeSH

The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics.

• Klíčová slova
• BBSome, MKS/B9 complex, cilium/flagellum, transition zone, trypanosome,
• MeSH
• Bardetův-Biedlův syndrom genetika   metabolismus   MeSH
• bazální tělíska metabolismus   ultrastruktura   MeSH
• cilie genetika   metabolismus   MeSH
• ciliopatie genetika   metabolismus   MeSH
• cytoskelet metabolismus   ultrastruktura   MeSH
• encefalokéla genetika   metabolismus   MeSH
• flagella genetika   metabolismus   ultrastruktura   MeSH
• fluorescenční mikroskopie MeSH
• kompartmentace buňky MeSH
• lidé MeSH
• mutace MeSH
• polycystická choroba ledvin genetika   metabolismus   MeSH
• poruchy ciliární motility genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• retinopathia pigmentosa MeSH
• RNA interference MeSH
• transmisní elektronová mikroskopie MeSH
• Trypanosoma genetika   metabolismus   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH
• protozoální proteiny MeSH

UNLABELLED: Trypanosomatid parasites are significant causes of human disease and are ubiquitous in insects. Despite the importance of Drosophila melanogaster as a model of infection and immunity and a long awareness that trypanosomatid infection is common in the genus, no trypanosomatid parasites naturally infecting Drosophila have been characterized. Here, we establish a new model of trypanosomatid infection in Drosophila--Jaenimonas drosophilae, gen. et sp. nov. As far as we are aware, this is the first Drosophila-parasitic trypanosomatid to be cultured and characterized. Through experimental infections, we find that Drosophila falleni, the natural host, is highly susceptible to infection, leading to a substantial decrease in host fecundity. J. drosophilae has a broad host range, readily infecting a number of Drosophila species, including D. melanogaster, with oral infection of D. melanogaster larvae resulting in the induction of numerous immune genes. When injected into adult hemolymph, J. drosophilae kills D. melanogaster, although interestingly, neither the Imd nor the Toll pathway is induced and Imd mutants do not show increased susceptibility to infection. In contrast, mutants deficient in drosocrystallin, a major component of the peritrophic matrix, are more severely infected during oral infection, suggesting that the peritrophic matrix plays an important role in mediating trypanosomatid infection in Drosophila. This work demonstrates that the J. drosophilae-Drosophila system can be a powerful model to uncover the effects of trypanosomatids in their insect hosts. IMPORTANCE: Trypanosomatid parasites are ubiquitous in insects and are significant causes of disease when vectored to humans by blood-feeding insects. In recent decades, Drosophila has emerged as the predominant insect model of infection and immunity and is also known to be infected by trypanosomatids at high rates in the wild. Despite this, there has been almost no work on their trypanosomatid parasites, in part because Drosophila-specific trypanosomatids have been resistant to culturing. Here, we present the first isolation and detailed characterization of a trypanosomatid from Drosophila, finding that it represents a new genus and species, Jaenimonas drosophilae. Using this parasite, we conducted a series of experiments that revealed many of the unknown aspects of trypanosomatid infection in Drosophila, including host range, transmission biology, dynamics of infection, and host immune response. Taken together, this work establishes J. drosophilae as a powerful new opportunity to study trypanosomatid infections in insects.

• MeSH
• biologické modely MeSH
• Drosophila imunologie   parazitologie   MeSH
• fylogeneze MeSH
• hostitelská specificita MeSH
• interakce hostitele a patogenu * MeSH
• molekulární sekvence - údaje MeSH
• protozoální DNA chemie   genetika   MeSH
• sekvenční analýza DNA MeSH
• shluková analýza MeSH
• Trypanosomatina klasifikace   růst a vývoj   imunologie   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální DNA MeSH

The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure - the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms.

• Klíčová slova
• Flagellum attachment zone, Morphogenesis, Trypanosomes,
• MeSH
• cilie genetika   metabolismus   MeSH
• cytokineze genetika   MeSH
• cytoskelet genetika   metabolismus   MeSH
• flagella genetika   metabolismus   MeSH
• mikrotubuly genetika   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• stadia vývoje genetika   MeSH
• Trypanosoma brucei brucei genetika   růst a vývoj   MeSH
• tvar buňky genetika   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH