Souza-Melo, N. | Search bioRxiv

bioRxiv2026

Adaptation of Iterative Ultrastructure Expansion Microscopy (iU-ExM) enables high-resolution imaging of the cytoskeleton and subcellular architecture of Trypanosoma cruzi

Kinetoplastid parasites, including Trypanosoma cruzi, possess a highly organized subpellicular microtubule network essential for maintaining cell shape, organelle positioning, and dividing. Here we describe results obtained using immunofluorescence microscopy of cells processed using conventional ultrastructure expansion microscopy (U-ExM), that improved visualization of the cytoskeletal structures, with an ~4.5-fold expansion, as well as the recently described and iterative ultrastructure expansion microscopy (iU-ExM) with about 6.2-fold and 18-fold expansion of epimastigotes of T. cruzi. The optimized protocol involves the formation of an initial hydrogel crosslinked with N,N'(1,2-dihydroxyethylene) bisacrylamide (DHEBA), a temperature- and alkaline pH-sensitive cleavable crosslinker, followed by thermal denaturation at 85 degrees Celsius, intermediate immunostaining, neutral gel re-embedding, and the formation of a second hydrogel crosslinked with N,N' methylenebisacrylamide (BIS). Subsequent dissolution of the first hydrogel under alkaline conditions enables a second round of expansion. This approach achieved approximately 6.2-fold expansion during the first round and a final linear expansion of approximately 18-fold. Using conventional confocal microscopy, iU-ExM enabled high-resolution visualization of individual subpellicular microtubules and specialized internal microtubule arrangements, including those associated with the basal body, flagellar pocket, Golgi complex, and cytopharynx. Furthermore, the enhanced spatial resolution facilitated the examination of cytoskeletal reorganization at different stages of the cell cycle, revealing changes in microtubule organization associated with mitotic spindle formation and cytokinesis. Together, these findings establish iU-ExM as an accessible high-resolution imaging approach for investigating the complex subcellular architecture of T. cruzi, providing new opportunities to explore cytoskeletal organization and remodeling during parasite cell division using conventional confocal microscopy.

cell biology↗

bioRxiv2023

Trypanosoma cruzi VDU deubiquitinase mediates surface protein trafficking and infectivity

Ubiquitylation is a post-translational modification promoting protein degradation. Within the endomembrane system ubiquitylation marks proteins for lysosomal processing. Deubiquitinases (DUBs) cleave ubiquitin from modified proteins and, in the case of surface proteins, prevent lysosomal targeting and thus play a major role in controlling turnover. In Trypanosoma cruzi, the etiological agent of Chagas disease, acquisition of nutrients in parasites proliferating within the blood meal of insect vectors occurs via the cytostome, a unique structure connected to a long tubular cytopharynx. Contents are delivered to late endosomes and accumulate in reservosomes, equivalent to lysosomes. When starved, T. cruzi differentiates into mammalian infective trypomastigotes, which are cell cycle arrested. Here we asked what roles ubiquitylation plays in this unique endocytic process by interrogation of the T. cruzi ortholog of VDU (von Hippel-Lindau-interacting deubiquitylating enzyme)/USP33 (ubiquitin-specific protease). We found that TcVDU expression level inversely correlated with transferrin endocytosis, and that overexpression led to a longer retention of the endocytic cargo near the cytostome. TcVDU itself was found enriched in the anterior region of the parasite, in proximity to endocytic cargo. Most importantly, TcVDU overexpression reduced parasite invasion capacity and led to increased release of trans-sialidase. These alterations in the abundance of multiple surface proteins in TcVDU mutants indicate a key role of TcVDU in modulating the T. cruzi surface by affecting the endosomal traffic and consequently the host-parasite interface. Author summaryTrypanosoma cruzi is the cause of Chagas disease that affects large populations of Central and South America. Disease is spreading to other continents due to poor control of blood transfusion from donors with chronic and undiagnosed infection, migration and drug treatment with limited performance and undesired side effects. Proliferating forms of T. cruzi acquire nutrients through the cytostome, a cell surface opening connected to a long tubular cytopharynx. Internalized material is endocytosed in the cytopharynx. This is distinct from other members of the Trypanosomatids, which endocytose material exclusively through the flagellar pocket. By using CRISPR gene editing and overexpression we demonstrated that a conserved deubiquitinase (VDU) is a key control element for traffic from the cytopharynx to endosomal compartments in T. cruzi. Changed levels of VDU modify endosomal traffic and largely impact the parasite surface, causing changes in infectivity.

microbiology↗