Authors

M Zelená²; E Casas⁴; C Lambert³; N Landrein³; D Dacheux⁵; E Bertiaux³; KI Abesamis¹; G Dong¹; V Varga²; DR Robinson³; M Bonhivers³;  ¹ Max F. Perutz Labs, Vienna Biocenter, Austria;  ² Institute of Molecular Genetics of the Czech Academy of Sciences, Czechia;  ³ Laboratoire de Microbiologie Fondamentale et Pathogénicité UMR-CNRS 5234, France;  ⁴ Univ. Bordeaux, INSERM, Biologie des maladies cardiovasculaires, U1034., France;  ⁵ Laboratoire de Microbiologie Fondamentale et Pathogénicité UMR-CNRS 5234, ENSTBB-Bordeaux INP., France

Discussion

Cytoskeletal architecture governs how unicellular pathogens such as Trypanosoma brucei organise their essential surface structures for survival. The flagellar pocket (FP), vital for nutrient uptake and immune evasion, relies on the flagellar pocket collar (FPC) (a specialised cytoskeletal scaffold) whose biogenesis mechanisms have been elusive due to imaging limitations. Here, we harness ultrastructure expansion microscopy (U-ExM) to dissect FPC assembly at nanoscale resolution within intact parasite cells, highlighting expansion methods as a game-changer for visualising complex cytoskeletal dynamics in parasites. U-ExM reveals the stepwise formation of the proximal new microtubule quartet (nMtQ) coordinated with flagellum extension, alongside dynamic localisations of core cytoskeletal proteins BILBO1, MORN1, and BILBO2 during joint FPC and hook complex (HC) biogenesis. Strikingly, this uncovers two novel cytoskeletal elements: a precursor FPC (proFPC) and a transient FPC-interconnecting fibre (FPC-IF). Our findings illuminate cytoskeletal biogenesis in trypanosomes, demonstrating how expansion microscopy unveils hidden scaffolds and paves the way for targeted functional studies of these structures.