The role of the flagellum attachment zone in Leishmania mexicana flagellar pocket architecture
Leishmania spp are flagellate protozoan parasites, with a digenetic life cycle, alternating between a mammalian host and insect vector. Within both the host and vector Leishmania adopts different morphologies and cell types, which are adapted to that ecological niche. One of the key architectural features in the determination of cell morphology is the flagellar pocket, which is the sole site of endo/exocytosis. It was shown that a large cytoskeletal structure, the flagellum attachment zone (FAZ), which connects the flagellum to the cell body, was an important contributor to the maintenance of the flagellar pocket architecture and its function. However, a poor understanding of FAZ molecular organisation and the specific roles of its constituent proteins remains. Here, 28 FAZ proteins were discovered by the endogenous tagging of the orthologs of T. brucei FAZ proteins identified through TrypTag. These proteins were categorised into five classes based on their localisation patterns. The classes sit within the three structural domains, the flagellum, intracellular and cell body of the FAZ structure. A deletion screen of these proteins revealed functional groups responsible for flagellum attachment and cell morphogenesis, with the specific function of these proteins connected to their exact location within the FAZ. Analyses of the flagellum domain proteins FAZ27 and FAZ34 revealed their importance for flagellum attachment. Moreover, the cell body domain CC2D was shown to be required for flagellum attachment and anterior cell tip morphogenesis and was dependent on FAZ2 for assembly. Importantly, the analysis of these proteins defined the assembly hierarchy of the three FAZ structural domains, with the cell body domain required for the assembly of the intracellular and flagellum domains and FAZ2 of the cell body domain being a key foundational protein. Together these results have provided crucial insights into the molecular organisation and function of the FAZ in Leishmania.
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