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 distal end of the eukaryotic flagellum/cilium is important for axonemal growth and signaling and has distinct biomechanical properties. Specific flagellum tip structures exist, yet their composition, dynamics, and functions are largely unknown. We used biochemical approaches to identify seven constituents of the flagella connector at the tip of an assembling trypanosome flagellum and three constituents of the axonemal capping structure at the tips of both assembling and mature flagella. Both tip structures contain evolutionarily conserved as well as kinetoplastid-specific proteins, and component assembly into the structures occurs very early during flagellum extension. Localization and functional studies reveal that the flagella connector membrane junction is attached to the tips of extending microtubules of the assembling flagellum by a kinesin-15 family member. On the opposite side, a kinetoplastid-specific kinesin facilitates attachment of the junction to the microtubules in the mature flagellum. Functional studies also suggest roles of several other components and the definition of subdomains in the tip structures.

• Klíčová slova
• axonemal capping structure, flagella connector, flagellar distal end, structure immunoprecipitation, trypanosome,
• MeSH
• axonema chemie   metabolismus   MeSH
• flagella chemie   metabolismus   MeSH
• kineziny chemie   metabolismus   MeSH
• protozoální proteiny chemie   metabolismus   MeSH
• Trypanosoma brucei brucei chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kineziny MeSH
• 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

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