Why and how trypanosomes build a Flagellar Pocket Collar – Structu-Ring

Trypanosoma brucei (T. brucei) is a flagellated protist responsible for tropical diseases. This organism is also an important cellular tool to study the generic organization and function of organelles, structures and cellular processes common to other pathogenic trypanosomatids such as T. cruzi responsible for Chagas disease, and Leishmania spp. (the latter being responsible for leishmaniasis). This is especially relevant because the G1 cell cycle stage has single copy organelles including the flagellum, Golgi, lysosome, and an endo- exocytosis organelle called the flagellar pocket (FP).
The cytoskeleton plays an essential role in all eukaryotes and in T. brucei during both the cell division cycle and the life cycle the biogenesis of the microtubule-based cytoskeleton, the flagellum, and the flagellar pocket are all critical events. The FP is an invagination of the plasma membrane containing the flagellum transition zone and is the sole site for endo- and exocytosis processes; as such the FP is essential for the survival of these parasites. At the site where the flagellum exits the cell, the FP tightly encircles the flagellum at the FP neck. A selective dynamic barrier is maintained at this site by the Flagellar Pocket Collar (FPC), an essential cytoskeleton-associated structure. The mechanisms behind FPC biogenesis and its functions remain elusive, mostly due to poor knowledge of the FPC's macromolecular composition and architecture. Importantly, BILBO1 is present in all pathogenic kinetoplastids. This raises the question why and how does the cell build a new daughter FPC and FP?
We previously identified the first FPC protein component in T. brucei - BILBO1. Prior collaborative studies between the Bonhivers (Bordeaux) and Dong (Vienna) laboratories have revealed that BILBO1 is a structural protein essential for the biogenesis of the FPC and other nearby cytoskeletal structures. We demonstrated that BILBO1 is a multi-domain protein that forms antiparallel dimers and oligomers both in vitro and filaments in a heterogeneous U-2 OS cellular context. However, the organisation of the filament(s) within the FPC is still unknown. This raises a second question of what is the molecular organization of the FPC?
BILBO1 has four main functional domains. The N-terminal domain (NTD) has a ubiquitin-like fold and is involved in interactions with partner proteins. The NTD is followed by two calcium-binding EF-hand motifs, a long coiled-coil domain, and a leucine zipper, the latter two being required for protein dimerisation and oligomerisation. Further, we have recently identified several BILBO1 interaction partners. One of these, FPC4, is involved in the interplay between the FPC and the microtubules in T. brucei.
Based on our robust preliminary data, this collaborative proposal aims at defining the 1) Proteomic mapping of the FPC; 2) The architecture of the FPC; 3) The real-time biogenesis, live imaging, quantification and the dynamics of the FPC. This will be done by further identifying FPC proteins and characterising their structure and their functional role in the architecture and the biogenesis of the FPC employing cutting-edge real-time, live-cell imaging, structural and super-resolution light and cryo-electronic microscopy and tomography means complemented with functional means. The data obtained from these structural and functional studies will explain how a new FPC is formed, why it is essential, what polymers/proteins are needed, how they interact, and in part, explain remodelling of the FPC in the context of flagellum and FP biogenesis.