African trypanosomes are medically important parasites that cause sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible polymerase chain reaction-based protein tagging in trypanosomes, which enabled the subcellular mapping of 89% of the trypanosome proteome. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an extended set of >100 plasmids that incorporate universal primer annealing sequences, enabling protein tagging with a range of fluorescent, biochemical and epitope tags, using five different selection markers. We evaluated the suitability of various fluorescent proteins for live and fixed cell imaging, fluorescent movies, and we demonstrate the use of tagging plasmids encoding tandem epitope tags to support expansion microscopy approaches. We show that this series of plasmids is functional in other trypanosomatid parasites, significantly increasing its value. Finally, we developed a new plasmid for tagging glycosylphosphatidylinositol-anchored proteins. We anticipate that this will be an important toolset for investigating trypanosomatid protein localization and function.

• Klíčová slova
• expansion microscopy, protein tagging, toolkit, trypanosomatid, trypanosome,
• MeSH
• epitopy metabolismus   MeSH
• lidé MeSH
• plazmidy * metabolismus   genetika   MeSH
• protozoální proteiny * metabolismus   genetika   MeSH
• transport proteinů MeSH
• Trypanosoma brucei brucei metabolismus   genetika   MeSH
• Trypanosoma genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• epitopy MeSH
• protozoální proteiny * MeSH

The exon junction complex (EJC) is a key player in metazoan mRNA quality control and is placed upstream of the exon-exon junction after splicing. Its inner core is composed of Magoh, Y14, eIF4AIII and BTZ and the outer core of proteins involved in mRNA splicing (CWC22), export (Yra1), translation (PYM) and nonsense mediated decay (NMD, UPF1/2/3). Trypanosoma brucei encodes only two genes with introns, but all mRNAs are processed by trans-splicing. The presence of three core EJC proteins and a potential BTZ homologue (Rbp25) in trypanosomes has been suggested to adapt of the EJC function to mark trans-spliced mRNAs. We analysed trypanosome EJC components and noticed major differences between eIF4AIII and Magoh/Y14: (i) whilst eIF4AIII is essential, knocking out both Magoh and Y14 elicits only a mild growth phenotype (ii) eIF4AIII localization is mostly nucleolar, while Magoh and Y14 are nucleolar and nucleoplasmic but excluded from the cytoplasm (iii) eIF4AIII associates with nucleolar proteins and the splicing factor CWC22, but not with Y14 or Magoh, while Magoh and Y14 associate with each other, but not with eIF4AIII, CWC22 or nucleolar proteins. Our data argue against the presence of a functional EJC in trypanosomes, but indicate that eIF4AIII adopted non-EJC related, essential functions, while Magoh and Y14 became redundant. Trypanosomes also possess homologues to the NMD proteins UPF1 and UPF2. Depletion of UPF1 causes only a minor reduction in growth and phylogenetic analyses show several independent losses of UPF1 and UPF2, as well as complete loss of UPF3 in the Kinetoplastida group, indicating that UPF1-dependent NMD is not essential. Regardless, we demonstrate that UPF1 depletion restores the mRNA levels of a PTC reporter. Altogether, we show that the almost intron-less trypanosomes are in the process of losing the canonical EJC/NMD pathways: Y14 and Magoh have become redundant and the still-functional UPF1-dependent NMD pathway is not essential.

• MeSH
• eukaryotický iniciační faktor 4A metabolismus   genetika   MeSH
• exony genetika   MeSH
• messenger RNA genetika   metabolismus   MeSH
• nonsense mediated mRNA decay * MeSH
• protozoální proteiny * metabolismus   genetika   MeSH
• sestřih RNA MeSH
• Trypanosoma brucei brucei * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• eukaryotický iniciační faktor 4A MeSH
• messenger RNA MeSH
• protozoální proteiny * MeSH

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
• jaderný pór * metabolismus   MeSH
• komplex proteinů jaderného póru * metabolismus   genetika   MeSH
• messenger RNA * metabolismus   genetika   MeSH
• nukleocytoplazmatické transportní proteiny metabolismus   genetika   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 RNA * MeSH
• nukleocytoplazmatické transportní proteiny MeSH
• protozoální proteiny * MeSH

Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an 'all in one' solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.

• Klíčová slova
• BioID, Trypanosoma brucei, TurboID, cell biology, human, nuclear pore, phase separation, streptavidin imaging,
• MeSH
• biotinylace MeSH
• fluorescenční mikroskopie metody   MeSH
• lidé MeSH
• protilátky metabolismus   MeSH
• protozoální proteiny imunologie   metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   genetika   imunologie   MeSH
• streptavidin * chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protilátky MeSH
• protozoální proteiny MeSH
• rekombinantní fúzní proteiny MeSH
• streptavidin * MeSH

Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread. One of the key life cycle stages is the haptomonad form, which attaches to insect tissues through its flagellum. This adhesion, conserved across kinetoplastid parasites, is implicated in having an important function within their life cycles and hence in disease transmission. Here, we discover the kinetoplastid-insect adhesion proteins (KIAPs), which localise in the attached Leishmania flagellum. Deletion of these KIAPs impairs cell adhesion in vitro and prevents Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. Additionally, loss of parasite adhesion in the sand fly results in reduced physiological changes to the fly, with no observable damage of the stomodeal valve and reduced midgut swelling. These results provide important insights into a comprehensive understanding of the Leishmania life cycle, which will be critical for developing transmission-blocking strategies.

• MeSH
• buněčná adheze MeSH
• flagella * metabolismus   MeSH
• hmyz - vektory parazitologie   MeSH
• hmyzí proteiny metabolismus   genetika   MeSH
• interakce hostitele a parazita MeSH
• Leishmania * fyziologie   genetika   metabolismus   MeSH
• leishmanióza parazitologie   přenos   MeSH
• protozoální proteiny metabolismus   genetika   MeSH
• Psychodidae * parazitologie   MeSH
• stadia vývoje MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hmyzí proteiny MeSH
• protozoální proteiny MeSH

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.

• Klíčová slova
• Trypanosoma, Cilia, Ciliogenesis, Distal appendages, Flagella, Transition fibres,
• MeSH
• bazální tělíska metabolismus   MeSH
• časové faktory MeSH
• cilie genetika   metabolismus   MeSH
• flagella genetika   metabolismus   MeSH
• konzervovaná sekvence MeSH
• protozoální proteiny * genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• transport proteinů MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny * 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
• endoribonukleasy * metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• RNA čepičky * genetika   metabolismus   MeSH
• stabilita RNA MeSH
• Trypanosoma * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endoribonukleasy * MeSH
• messenger RNA MeSH
• mRNA decapping enzymes MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA čepičky * MeSH

Unc-51-like kinase (ULK) family serine-threonine protein kinase homologues have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicate a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localization throughout the cell body and flagellum, with enrichment near the flagellar base and tip. The stable expression of LmxULK4 was dependent on the presence of LmxFused. Fused/STK36 was previously shown to localize to mammalian motile cilia, and we demonstrate here that ULK4 also localizes to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/ STK36 in a pathway required for stable assembly of motile cilia.

• MeSH
• cilie metabolismus   MeSH
• flagella * metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• protein-serin-threoninkinasy * metabolismus   MeSH
• savci metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein-serin-threoninkinasy * MeSH
• Stk36 protein, mouse MeSH Prohlížeč

Control of protein levels is vital to cellular homeostasis, for maintaining a steady state, to coordinate changes during differentiation and other roles. In African trypanosomes surface proteins contribute to immune evasion, drug sensitivity and environmental sensing. The trypanosome surface is dominated by the GPI-anchored variant surface glycoprotein, but additional GPI-anchored and trans-membrane domain proteins are present with known roles as nutrient receptors and signal transducers. The evolutionarily conserved deubiquitinase orthologs of Usp7 and Vdu1 in trypanosomes modulate abundance of many surface proteins, including the invariant surface glycoproteins, which have roles in immune evasion and drug sensitivity. Here we identify multiple trypanosome Skp1 paralogs and specifically a divergent paralog SkpZ. Affinity isolation and LCMSMS indicates that SkpZ forms a heterotrimeric complex with TbUsp7 and TbTpr86, a tetratricopeptide-repeat protein. Silencing SkpZ decreases TbUsp7 and TbTpr86 abundance, confirming a direct association. Further, SkpZ knockdown decreases the abundance of multiple trans-membrane domain (TMD) proteins but increases GPI-anchored surface protein levels. Hence, a heterotrimeric complex of TbTpr86, TbUsp7 and SkpZ (TUSK) regulates expression levels of a significant cohort of trypanosome surface proteins mediating coordination between TMD and GPI-anchored protein expression levels.

• Klíčová slova
• cullin complex, deubiquitinase, drug sensitivity, surface proteins, trafficking, trypanosomes, ubiquitylation,
• Publikační typ
• časopisecké články MeSH

Leishmaniases are neglected diseases caused by protozoans of the genus Leishmania that threaten millions of people worldwide. Cutaneous leishmaniasis (CL) caused by L. major is a typical zoonosis transmitted by phlebotomine sand flies and maintained in rodent reservoirs. The female sand fly was assumed to become infected by feeding on the skin lesion of the host, and the relative contribution of asymptomatic individuals to disease transmission was unknown. In this study, we infected 32 Meriones shawi, North African reservoirs, with a natural dose of L. major obtained from the gut of infected sand flies. Skin manifestations appeared in 90% of the animals, and xenodiagnosis with the proven vector Phlebotomus papatasi showed transmissibility in 67% of the rodents, and 45% were repeatedly infectious to sand flies. Notably, the analysis of 113 xenodiagnostic trials with 2189 sand flies showed no significant difference in the transmissibility of animals in the asymptomatic and symptomatic periods; asymptomatic animals were infectious several weeks before the appearance of skin lesions and several months after their healing. These results clearly confirm that skin lesions are not a prerequisite for vector infection in CL and that asymptomatic animals are an essential source of L. major infection. These data are important for modeling the epidemiology of CL caused by L. major.

• Klíčová slova
• Leishmania, Meriones, Phlebotomus, asymptomatic infection, reservoir host, xenodiagnosis,
• Publikační typ
• časopisecké články MeSH