Ultrastructure Expansion Microscopy (U-ExM) in Trypanosoma cruzi: localization of tubulin isoforms and isotypes

MARTINEZ PERALTA, GONZALO; ALONSO, VICTORIA LUCIA

Woods Hole

Congreso; XXXIII MOLECULAR PARASITOLOGY MEETING; 2022

Ultrastructure Expansion Microscopy (U-ExM) is a recently developed technique that enables the increase of the spatial resolution within a cell or a tissue for microscopic imaging by physically expanding the sample. Basically, biological specimens are included in swellable polymer hydrogels and then are physically expanded to resolve fine details. For the first time, we report a detailed protocol validating the use of U-ExM in Trypanosoma cruzi and quantifying the expansion factors of different subcellular compartments. Trypanosomatids are characterized by a precisely organized cytoskeletal constituted by stable microtubules (MT). Such MTs are present in the mitotic spindle, the basal body, the flagellar axoneme, and the subpellicular microtubules. The latter are connected to each other and to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. MTs are composed by α/β-tubulin heterodimers forming helical tubes and provide the basis for cytoskeletal architecture. They are regulated by interacting with a variety of MT-associated proteins (MAPs), by a differential expression of - and β-tubulin genes (tubulin isotypes), and by tubulin isoforms generated by the inclusion of different post-translational modifications (PTMs) in each isotype. In trypanosomatids, many PTMs were identified across the microtubular array. It is proposed that this could be used to control the differential recruitment of MAPs and motors during the cell cycle.We described the localization patterns of tubulin isoforms, such as -tubulin, -tubulin, validating this technique in all life cycle stages of T. cruzi. Also, we immunolocalized acetylated and tyrosinated -tubulin isotypes in epimastigotes to visualize subpellicular microtubules, the flagellar axoneme, the basal body, and the flagellar pocket. Furthermore, we validated the use of the cell-permeable dyes, such as Mitotracker, for U-ExM in T. cruzi. Finally, we obtained transgenic epimastigotes that express -tubulin with an HA-tag as a tool to study cytoskeleton dynamics in T. cruzi.