New dimensions in acidocalcisome research: the potential of cryo-EM to uncover novel aspects of protozoan parasite physiology

Abstract

Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by enabling high-resolution, near-native visualization of macromolecular structures and entire cells. Its application to etiologic agents of diseases is an expanding field, particularly for those caused by viruses or unicellular eukaryotes, such as protozoan parasites and fungi. This review focuses on acidocalcisomes-ion-rich, multifunctional organelles essential for cell physiology and survival in several pathogens. The structure and function of these organelles are examined through a range of electron microscopy techniques, using Trypanosoma cruzi as a model. The advantages and limitations of the methods employed to study acidocalcisome morphofunctional organization-such as chemical fixation, plunge and high-pressure freezing, cryo-electron microscopy of vitrified sections (CEMOVIS), freeze-drying, freeze substitution, tomography, and microanalysis using X rays and inelastic scattered electrons-are discussed, alongside their contributions to our current understanding of acidocalcisome structure and function. Recent advances in cryo-EM and its potential to address longstanding questions and fill existing gaps in our understanding of parasite ion mobilization mechanisms and physiology are also discussed.

Keywords: Trypanosoma cruzi; acidocalcisomes; biomolecular condensates; cryo electron microscopy; ion nanodomains.

Fig 1

Visualization of acidocalcisomes in T. cruzi using different methodologies. (A) TEM image of an unfixed whole amastigote observed in conventional bright-field mode (left) and in electron spectroscopic imaging using contrast tuning (right), where the inelastic imaging mode provides optimal visualization of the acidocalcisomes, which are arranged in alignment (arrows). Scale bars: 700 nm. (B) Virtual section from an electron tomogram showing acidocalcisomes with 3D elemental mapping models, revealing the presence of ion nanodomains within the organelles. Scale bars = 500, 300, and 100 nm, from top left to bottom. (Panel A was reproduced from reference with permission; panel B was reproduced from reference with permission.)

Fig 2

Visualization of acidocalcisomes in T. cruzi using cryo-EM and cryo-ET. (A and B) A single image and a virtual section from a whole cell tomogram, respectively. Both images show different patterns of acidocalcisome structure, the fully loaded matrix containing a well-preserved electrodense core (arrows) and the partially filled matrix with cores of different diameters (asterisks). In these images, the surrounding and intracellular interactions with the acidocalcisomes can be clearly observed. Scale bar = 500 and 200 nm, respectively. (C) TEM image of a ~200 nm lamella from an epimastigote form of T. cruzi. To minimize beam damage and acquire an overview for a search map, images of lamellae are initially acquired at low magnification, which reduces the signal-to-noise ratio (SNR). The nucleus (N), reservosomes (R), and acidocalcisomes are visible, with their matrix either fully (arrows) or partially filled (dotted square). (D) Cryo-ET virtual section of the acidocalcisomes from the dotted square region in panel A, showing both the membranes and electrodense cores with high resolution. Scale bar = 200 nm. (E) Cryo-ET of whole cell virtual section, displaying fine details of the acidocalcisome matrix texture (inset), potentially representing the in situ observation of aggregates of polyphosphate molecules. Scale bar = 100 nm. (F) 3D model based on the threshold of the acidocalcisome matrix shown in panel E. (G) The final 3D model reveals highly interconnected structures, which, when closely observed, resemble the conformation and size of polyphosphate chains in the presence of ions such as Mg²⁺. Conformation models obtained from molecular dynamics based on four polyphosphate chains with 50 phosphates and magnesium over a 100 ns simulation are shown. (Panel A was reproduced from reference with permission.)