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

Abstract 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. Competing Interest Statement The authors have declared no competing interest. Abbreviations - BB - basal body - FP - flagellar pocket - FPC - flagellar pocket collar - FPC-IF - flagellar pocket collar interconnecting fibre - HC - hook complex - MtQ - microtubule quartet - nBB - new basal body - nFg - new flagellum - nFp - new flagellar pocket - nHC - new hook complex - nMtQ - new microtubule quartet - oBB - old basal body - oFg - old flagellum - oHC - old hook complex - oMtQ - old microtubule quartet - proBB - pro-basal body - proFPC - pro-flagellar pocket collar - U-ExM - ultra-structure expansion microscopy