Immunolocalization and 3D modeling of three unique proteins belonging to the costa of Tritrichomonas foetus

Nowadays, even in light of all the massive advances in cell biology, we still find some cellular structures that are not entirely understood. Among those, we highlight the costa, a structure from the mastigont system existent only in some members of the orders Trichomonadida and Tritrichomonadida, including the pathogens of venereal diseases in humans and cattle, Trichomonas vaginalis (T. vaginalis) and Tritrichomonas foetus (T. foetus), respectively. The costa is a prominent striated fiber and, although part of the cytoskeleton, differs from its classical components, and its molecular composition is still not fully characterized. Using proteomics of T. foetus's costa fraction, we previously identified hypothetic proteins, and among these, the protein ARM19800.1 positively localized in the costa and named costain-1. In this study, two other protein candidates were analyzed. To achieve the specific localization of 11810 and 32137 proteins in T. foetus's cells, it was used expansion microscopy and immunocytochemistry. The immunofluorescence revealed the presence of both proteins throughout the whole costa but with different intensities. Immunocytochemistry using negative staining, LR-White, and Epon embedding revealed further analyses of the protein's localization. All techniques confirmed the distinct and distributed localization of both proteins: costain-2 (11810) and costain-3 (32137). Also, AlfaFold3 was used to generate 3D models of the three identified proteins, showing a major prevalence of α-helical spans. Nonetheless, the identification and further characterization of these unique proteins can help understand their functional role in the assembled costa and, therefore, better understand the organization and function of this structure in these organisms.