BONHIVERS, M. | bioRxiv Science

bioRxiv2025

Flagellar pocket collar biogenesis: Cytoskeletal organization and novel structures in a unicellular parasite

Understanding how cells build and organize their internal structures is a fundamental question in biology, with important implications for human health and disease. Trypanosomes are single-celled flagellated parasites that cause life-threatening diseases in human and animals. Their survival relies on a specialized compartment called the flagellar pocket (FP), which serves as a gateway for nutrient uptake, and immune evasion. The formation and function of the FP are supported by an intricate cytoskeletal structure known as the flagellar pocket collar (FPC). However, the mechanisms underlying its assembly remain poorly understood. In this study, we used cutting-edge ultrastructure expansion microscopy (U-ExM) to investigate FPC biogenesis in Trypanosoma brucei. We mapped the formation of the new microtubule quartet (nMtQ) alongside flagellum growth, providing new insights into its assembly. Additionally, we tracked the localization dynamics of key structural proteins - BILBO1, MORN1, and BILBO2 - during the biogenesis of the FPC and the hook complex (HC). Notably, we identified two previously undetected structures: the proFPC and the transient FPC-interconnecting fibre (FPC-IF), both of which appear to play crucial roles in linking and organizing cellular components during cell division. By uncovering these novel aspects of FPC biogenesis, our study significantly advances the understanding of cytoskeletal organization in trypanosomes and opens new avenues for exploring the functional significance of these structures.

microbiology↗

bioRxiv2021

Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar

The flagellar pocket (FP) is the only endo- and exocytic organelle in most trypanosomes and, as such, is essential throughout the life cycle of the parasite. The neck of the FP is maintained enclosed around the flagellum via the flagellar pocket collar (FPC). The FPC is a macromolecular cytoskeletal structure and is essential for the formation of the FP and cytokinesis. FPC biogenesis and structure are poorly understood, mainly due to the lack of information on FPC composition. To date, only two FPC proteins, BILBO1 and FPC4, have been characterized. BILBO1 forms a molecular skeleton upon which other FPC proteins can, theoretically, dock onto. We previously identified FPC4 as the first BILBO1 interacting partner and demonstrated that its C-terminal domain interacts with the BILBO1 N-terminal domain (NTD). Here, we report the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240) by yeast two-hybrid screen, bioinformatics, functional and structural studies. We show that BILBO2 colocalizes with BILBO1 and can modulate the shape of the BILBO1 filament by interacting with the BILBO1 EF-hand domains. Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD domain and that both domains interact with FPC4. We have determined a 1.9 [A] resolution crystal structure of the BILBO2 NTD in complex with the FPC4 BILBO1-binding domain. Together with mutational analyses, our studies reveal key residues for the function of the BILBO2 NTD and its interaction with FPC4 and evidenced a tripartite interaction between BILBO1, BILBO2, and FPC4. Our work sheds light on the first atomic structure of an FPC protein complex and represents a significant step in deciphering the FPC function in Trypanosoma brucei and other pathogenic kinetoplastids. Author summaryTrypanosomes belong to a group of zoonotic, protist, parasites that are found in Africa, Asia, South America, and Europe and are responsible for severe human and animal diseases. They all have a common structure called the flagellar pocket (FP). In most trypanosomes, all macromolecular exchanges between the trypanosome and the environment occur via the FP. The FP is thus essential for cell viability and evading the host immune response. We have been studying the flagellar pocket collar (FPC), an enigmatic macromolecular structure at the neck of the FP, and demonstrated its essentiality for the biogenesis of the FP. We demonstrated that BILBO1 is an essential protein of the FPC that interacts with other proteins including a microtubule-binding protein FPC4. Here we identify another bona fide FPC protein, BILBO2, so named because of close similarity with BILBO1 in protein organization and functional domains. We demonstrate that BILBO1 and BILBO2 share a common N-terminal domain involved in the interaction with FPC4, and illustrate a tripartite interaction between BILBO1, BILBO2, and FPC4. Our study also provides the first atomic view of two FPC components. These data represent an additional step in deciphering the FPC structure and function in T. brucei.

cell biology↗