Cellular Architecture and Osmoregulation in Trypanosoma cruzi: Bridging Structure and Function with Cryo-ET

Trypanosoma cruzi, the causative agent of Chagas disease, relies on sophisticated osmoregulatory mechanisms to survive diverse environmental conditions across its life cycle. Central to this system are the contractile vacuole complex (CVC) and acidocalcisomes (Acc). The contractile vacuole complex (CVC) is responsible for the uptake and efflux of intracellular water and compost by a central vacuole and a network of tubules and vesicles called spongiome [1]. Acc are organelles rich in polyphosphates (polyP), calcium, and other cations organized in clusters as nanodomains playing in the ion homeostasis [2]. Using high-pressure freezing and freezing substitution techniques associated with volume electron microscopy, we previously described the adhesion plaque (AP), an electrodense domain with a filament network connecting the CVC and the flagellar pocket membranes [3]. Continuing analysis using a mutant knockout for a mechanical sensitive channel (TcMscS KO) showed evidence of the AP serving as the site of water efflux through membrane fusion pores. However, specific mechanisms such as the dynamics of the adhesion plaque functioning and molecular identity, the contractile vacuole biogenesis and the ion dynamics into and throughout the acidocalcisome matrix are still not yet fully understood. Aiming to unveil the molecular and structural architecture of the osmoregulatory system of Trypanosoma cruzi closer to its native state, we analyzed epimastigote cells using high-performance cryo-microscopy (cryo-EM) and cryo-electron tomography (cryo-ET). Cells were applied onto Quantifoil 1.2/1.3R grids and cryofixed by plunge freezing. Whole cells were directly observed in a 300 kV Titan Krios G4 or transferred to a Cryo-FIB SEM Aquilos 2 microscope to lamellae production. Cryo-ET were acquired with a maximum dose of 120 electron e-/Å2 s on the 300 kV Titan Krios G4 equipped with a Selectris energy filter and a Falcon4i Direct Electron Detector camera. We observed the CVC in both lamellar and round shapes, corresponding to different stages of its pulsation cycle. Cryo-EM images also revealed evidence of fusion pores, corroborating our previous data obtained from freeze-substituted samples. Cryo-ET of lamellae further resolved membrane proteins of the CVC at higher resolution, resembling the structure of the vacuolar proton ATPase and retromers proteins [4,5] (Figure 1). The potential presence of retromers in the CVC, along with other endo-lysosomal proteins as AP180, clathrin, and Rab 11, supports the participation of the CVC in protein trafficking as well as its biogenesis from the Golgi apparatus [6,7]. The acidocalcisomes (Accs) exhibited distinct structural organization patterns: i) fully loaded with an electron-dense core, ii) displaying varying electron densities, potentially representing ionic phase transition and iii) partially filled. These patterns may correspond to different elemental clustering patterns recently described (Figure 2) [2]. Additionally, the electrodense core of the Accs showed a unique texture characterized by fiber-like structures measuring between 15Å and 75Å in diameter (Figure 2A). This size range aligns with polyphosphate aggregates interacting with ions, as suggested by molecular dynamic simulations. Furthermore, Accs were observed connected to the mitochondria via electron-dense elements (Figure 3). These elements could represent the TcIP3R, VDAC, and MCU channels, which mediate calcium transfer between the organelles [8]. Mitochondria also displayed random dense granules in their matrix, similar to granules found in mammalian mitochondria, which have been shown to contain phosphorus and calcium [9]. These findings provide new insights into the dynamic interplay between Accs and mitochondria, as well as the structure and function of the Acc and CVC in T. cruzi. Although, this work highlights the potential of cryo-EM and cryo-ET in advancing our understanding of the structural and molecular biology of T. cruzi, further and advanced analyses will be done to unravel the intricate details of these interactions and to deepen our comprehension of the underlying mechanisms at a molecular level [10]. Protein membranes on the contractile vacuole complex of T. cruzi. (A) Cryo lamella of epimastigote cell, where the flagellum (F), kinetoplast (K), and nucleus (N) can be identified. In the center of the image, near the kinetoplast, the CVC (square) is observed. (B) Cryo-ET virtual section displaying high-resolution CVC protein membranes. Proteins with structures resembling V-H+ ATPase (blue arrows) and retromers (arrowheads) are identifiable. Cryo-ET virtual sections show various acidocalcisome ultrastructures in T. cruzi. (A) Acidocalcisome with a fully loaded matrix, with fiber-like structures in the core (inset). (B) Acidocalcisome with varying electron density in the matrix. (C) Partially filled acidocalcisomes. Cryo-ETs of whole cells revealing acidocalcisome-mitochondria interactions in T. cruzi. (A) Membrane contact site between the acidocalcisome (Ac) and mitochondria (M). The electron-dense elements linking both membranes are indicated by the yellow arrow. (B) High magnification of the electron-dense granules within the mitochondrial matrix. (C) Another cell showing acidocalcisome-mitochondria contact. In the inset, the yellow arrow points to a structure traversing both membranes. (D) Three-dimensional model of the cell in (C). The electron-dense granules (dark red) are dispersed inside the mitochondrial matrix, mainly close to the membrane (pink) and cristae. In the top, insets show different angles of the acidocalcisome (orange) connected by the pointed structure (black) to the mitochondrial membrane.