In Trypanosoma brucei, transition fibres (TF) form a nine-bladed pattern-like structure connecting the base of the flagellum to the flagellar pocket membrane. Despite the characterization of two TF proteins, CEP164C and TbRP2, little is known about the organization of these fibres. Here, we report the identification and characterization of the first kinetoplastid-specific TF protein named TFK1 (Tb927.6.1180). Bioinformatics and functional domain analysis identified three TFK1 distinct domains: an N-terminal domain of an unpredicted function, a coiled-coil domain involved in TFK1-TFK1 interaction and a C-terminal intrinsically disordered region potentially involved in protein interaction. Cellular immuno-localization showed that TFK1 is a newly identified basal body maturation marker. Further, using ultrastructure expansion and immuno-electron microscopies we localized CEP164C and TbRP2 at the TF and TFK1 on the distal appendage matrix of the TF. Importantly, RNAi knockdown of TFK1 in bloodstream form cells induced misplacement of basal bodies, a defect in the furrow or fold generation and eventually cell death. We hypothesize that TFK1 is a basal body positioning specific actor and a key regulator of cytokinesis in the bloodstream form Trypanosoma brucei.

Trypanosoma brucei belongs to a group of important zoonotic parasites. We investigated how these organisms develop their cytoskeleton (the internal skeleton that controls cell shape) and focused on an essential protein (BILBO1) first described in T. brucei .

Trypanosoma brucei belongs to a genus of protists that cause life-threatening and economically important diseases of human and animal populations in Sub-Saharan Africa. T. brucei cells are covered in surface glycoproteins some of which are used to escape the host immune system. Exo-/endocytotic trafficking of these and other molecules occurs via a single copy organelle called the flagellar pocket (FP). The FP is maintained and enclosed around the flagellum by the flagellar pocket collar (FPC). To date, the most important cytoskeletal component of the FPC is an essential, calcium-binding, polymer-forming protein called TbBILBO1. In searching for novel immune-tools to study this protein, we raised nanobodies against TbBILBO1. Nanobodies (Nb) that were selected according to their binding properties to TbBILBO1, were tested as immunofluorescence tools, and expressed as intrabodies (INb). One of them, Nb48, proved to be the most robust nanobody and intrabody. We further demonstrate that inducible, cytoplasmic expression of INb48 was lethal to these parasites, producing abnormal phenotypes resembling those of TbBILBO1 RNAi knockdown.; Our results validate the feasibility of generating functional single-domain antibody derived intrabodies to target trypanosome cytoskeleton proteins.