Maintenance of hook complex integrity and centrin arm assembly facilitates flagellum inheritance in Trypanosoma brucei

Abstract

Inheritance of the newly assembled flagellum in the human parasite, Trypanosoma brucei, depends on the faithful duplication and segregation of multiple flagellum-associated cytoskeletal structures, including the hook complex and its associated centrin arm. The biological functions of this unique hook complex-centrin arm assembly remain poorly understood. Here, we report a hook complex-associated protein named BOH2 that plays an essential role in promoting flagellum inheritance. BOH2 localizes to the hooked part of the hook complex by bridging the hook complex, the centrin arm, and the flagellum attachment zone filament. Depletion of BOH2 caused the loss of the shank part of the hook complex and its associated protein TbSmee1, disrupted the assembly of the centrin arm and the recruitment of centrin arm-associated protein CAAP1, inhibited the assembly of the flagellum attachment zone, and caused flagellum mis-positioning and detachment. These results demonstrate unusual roles of BOH2 in maintaining hook complex integrity and promoting centrin arm formation, and suggest that proper assembly of the hook complex-centrin arm structure facilitates flagellum inheritance.

Bhalin, an Essential Cytoskeleton-Associated Protein of Trypanosoma brucei Linking TbBILBO1 of the Flagellar Pocket Collar with the Hook Complex

“…The protocol was adapted from [20]. BSF trypanosomes were diluted to 1 × 10 5 cells/mL and grown overnight at 37 • C and induced with 1 µg/mL tetracycline.…”

“…The HC was initially described as a bilobe consisting of a hook structure and a parallel arm [17]. TbCentrin2, TbCentrin4 [18] and TbCAAP (Centrin Arm Associated Protein 1) [19,20] have been localised to the arm structure. Knockdown of TbCentrin2 or TbCentrin4 prevents Flagellum Attachment Zone (FAZ) assembly and affects flagellum attachment.…”

“…Uptake assays using fluorescent markers demonstrated that TbMORN1 functions to influence the entry of molecules into the flagellar pocket, suggesting a novel link between the cytoskeleton and the endomembrane system. Recently, two proteins BOH1 (Bait of Hook 1) and BOH2 were described as localising partly to the hook and extending distally between the hook and arm, and whose knockdown impaired flagellum inheritance and proper assembly of the hook complex [20,26].…”

Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei

“…The specific association of unique protein partners with individual eIF4F subunits may reflect alternative roles or possible mechanisms associated with specific mRNA targets. One such example is CAAP1, one of the topmost proteins associated with EIF4E3, previously implicated in flagellar biosynthesis (Pham et al, 2020). Intriguingly, both EIF4E3 and EIF4E4 co-precipitate specifically with AAA-ATPases (associated with various cellular activities).…”

A Spef1-interacting microtubule quartet protein in Trypanosoma brucei promotes flagellar inheritance by regulating basal body segregation

“…3D-SIM super-resolution microscopy 3D-SIM super-resolution microscopy was performed essentially as described in our previous publication (48). Briefly, T. brucei cells settled on coverslips were treated with 1% NP-40 in the PEME buffer (see previous sections), fixed in cold methanol (−20 C), and then incubated in PBS containing 1% BSA.…”