Morphology of the Trypanosome Bilobe, a Novel Cytoskeletal Structure

Abstract

The trypanosome bilobe is a cytoskeletal structure of unclear function. To date, four proteins have been shown to localize stably to it: TbMORN1, TbLRRP1, TbCentrin2, and TbCentrin4. In this study, a combination of immunofluorescence microscopy and electron microscopy was used to explore the morphology of the bilobe and its relationship to other nearby cytoskeletal structures in the African trypanosome procyclic trypomastigote. The use of detergent/salt-extracted flagellum preparations was found to be an effective way of discerning features of the cytoskeletal ultrastructure that are normally obscured. TbMORN1 and TbCentrin4 together define a hairpin structure comprising an arm of TbCentrin4 and a fishhook of TbMORN1. The two arms flank a specialized microtubule quartet and the flagellum attachment zone filament, with TbMORN1 running alongside the former and TbCentrin4 alongside the latter. The hooked part of TbMORN1 sits atop the flagellar pocket collar marked by TbBILBO1. The TbMORN1 bilobe occasionally exhibits tendrillar extensions that seem to be connected to the basal and probasal bodies. The TbMORN1 molecules present on these tendrils undergo higher rates of turnover than those for molecules on the main bilobe structure. These observations have been integrated with previous detailed descriptions of the cytoskeletal elements in trypanosome cells.

Structure of the TbBILBO1 Protein N-terminal Domain from Trypanosoma brucei Reveals an Essential Requirement for a Conserved Surface Patch

“…To analyze whether the detached flagella were proximally associated with a basal body, the cells were co-labeled with TbCentrin4. TbCentrin4 localizes to both the basal body and a cytoskeletal structure called the bilobe (21,22). In duplicating cells overexpressing Ty1-TbBILBO1-FL, TbCentrin4-labeled basal bodies were observed at the base of both old and new flagella (Fig.…”

Tubulin-binding cofactor C domain-containing protein TBCCD1 orchestrates cytoskeletal filament formation

“…Position and appearance of the elongate Ty YFP:TBCCD1 signal adjacent to basal bodies was immediately reminiscent of the trypanosome-specific filamentous 'bi-lobe' (Esson et al, 2012) implicated in providing a template for cell-cycle-regulated Golgi biogenesis and duplication in T. brucei (Ho et al, 2006). Fluorescence and immunoelectron microscopy of known bi-lobe proteins, including centrin isoforms and various trypanosomatidspecific proteins, reveal the bi-lobe to possess a complex sub-structure (Esson et al, 2012;Gheiratmand et al, 2013;Morriswood et al, 2013).…”

“…Fluorescence and immunoelectron microscopy of known bi-lobe proteins, including centrin isoforms and various trypanosomatidspecific proteins, reveal the bi-lobe to possess a complex sub-structure (Esson et al, 2012;Gheiratmand et al, 2013;Morriswood et al, 2013). Immunofluorescence with either the monoclonal pan-centrin antibody 20H5 (Fig.…”

Assembly and maintenance of the flagellum attachment zone filament in Trypanosoma brucei

“…Assembly of the new FAZ filament appears to be initiated from the bilobe region (Esson et al, 2012) by initially forming a short FAZ root around the bilobe, which likely represents the initial FAZ nucleation and is known to occur before, and independently of, the assembly of the new flagellum (Kohl et al, 2003(Kohl et al, , 1999. Through unknown mechanisms, this short FAZ root was maintained when FAZ filament proteins, such as FAZ2, CC2D or KMP11, were depleted (Figs 2, 3, 5 and 6) and when the flagellum protein FLAM3 was depleted (Rotureau et al, 2014).…”

“…Additionally, depletion of BIBLO-1, a flagellar pocket collar component, also completely disrupts the new FAZ filament and the short FAZ root (Bonhivers et al, 2008). Given the physical association between the fishhook of the bilobe structure and the flagellar pocket collar (Esson et al, 2012), it is unclear whether defective assembly of the flagellar pocket collar caused by BILBO-1 depletion disrupts bilobe duplication. Nevertheless, the involvement of bilobe and flagellar pocket collar in FAZ filament assembly might suggest that there is coordination between the assembly of the FAZ filament and the biogenesis of the bilobe and flagellar pocket collar.…”