Expansion microscopy of apicomplexan parasites.

Apicomplexan parasites comprise significant pathogens of humans, livestock and wildlife, but also represent a diverse group of eukaryotes with interesting and unique cell biology. The study of cell biology in apicomplexan parasites is complicated by their small size, and historically this has required the application of cutting-edge microscopy techniques to investigate fundamental processes like mitosis or cell division in these organisms. Recently, a technique called expansion microscopy has been developed, which rather than increasing instrument resolution like most imaging modalities, physically expands a biological sample. In only a few years since its development, a derivative of expansion microscopy known as ultrastructure-expansion microscopy (U-ExM) has been widely adopted and proven extremely useful for studying cell biology of Apicomplexa. Here, we review the insights into apicomplexan cell biology that have been enabled through the use of U-ExM, with a specific focus on Plasmodium, Toxoplasma and Cryptosporidium. Further, we summarize emerging expansion microscopy modifications and modalities and forecast how these may influence the field of parasite cell biology in future.

Ultrastructure expansion microscopy reveals the cellular architecture of budding and fission yeast.

The budding and fission yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have served as invaluable model organisms to study conserved fundamental cellular processes. Although super-resolution microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeasts has remained limited due to the specific know-how and instrumentation required, contrasted with the relative ease of endogenous tagging and live-cell fluorescence microscopy. To facilitate super-resolution microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling a 4-fold isotropic expansion. We demonstrate that U-ExM allows imaging of the microtubule cytoskeleton and its associated spindle pole body, notably unveiling the Sfi1p-Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by monitoring the homeostatic regulation of nuclear pore complex number through the cell cycle. Combined with NHS-ester pan-labelling, which provides a global cellular context, U-ExM reveals the subcellular organization of these two yeast models and provides a powerful new method to augment the already extensive yeast toolbox. This article has an associated First Person interview with Kerstin Hinterndorfer and Felix Mikus, two of the joint first authors of the paper.

Characterization of the novel mitochondrial genome segregation factor TAP110 in Trypanosoma brucei.

Proper mitochondrial genome inheritance is important for eukaryotic cell survival. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome, the kinetoplast (k)DNA. The kDNA is anchored to the basal body via the tripartite attachment complex (TAC) to ensure proper segregation. Several components of the TAC have been described; however, the connection of the TAC to the kDNA remains elusive. Here, we characterize the TAC-associated protein TAP110. We find that both depletion and overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks. Proteome analysis after TAP110 overexpression identified several kDNA-associated proteins that changed in abundance, including a TEX-like protein that dually localizes to the nucleus and the kDNA, potentially linking replication and segregation in the two compartments. The assembly of TAP110 into the TAC region seems to require the TAC but not the kDNA itself; however, once TAP110 has been assembled, it also interacts with the kDNA. Finally, we use ultrastructure expansion microscopy in trypanosomes for the first time, and reveal the precise position of TAP110 between TAC102 and the kDNA, showcasing the potential of this approach.This article has an associated First Person interview with the first author of the paper.

Improving the resolution of fluorescence nanoscopy using post-expansion labeling microscopy.

The visualization of the cell ultrastructure and molecular complexes has long been reserved for electron microscopy owing to its nanometric resolution. In recent years, this monopoly has been challenged by super-resolution (SR) fluorescence microscopy, which allows the visualization of cell structures with high spatial resolution, approaching virtually molecular dimensions. However, the resolution of current SR microscopy does not systematically reach the level of the ultrastructural information provided by electron microscopy. In this review, we are discussing the potential of revealing cell ultrastructure using the recent method of expansion microscopy (ExM). In particular, we are discussing the limitations that exist in SR and ExM methods that prevent the visualization of nanometric molecular assemblies and how post-labeling expansion could help alleviate them to reveal the molecular cartography of cells with unprecedented details.

Coupling Auxin-Inducible Degron System with Ultrastructure Expansion Microscopy to Accelerate the Discovery of Gene Function in Toxoplasma gondii.

Ultrastructure expansion microscopy (U-ExM) is an emerging technique allowing the localization of proteins and cellular structures, at a level of resolution only distinguishable previously via immunoelectron microscopy. U-ExM, as its name indicates, is based on the physical expansion of the sample in the three dimensions without altering its internal features. The proteins of interest are later immunostained for their detection. To accelerate the discovery of gene function in the protozoan parasite Toxoplasma gondii, U-ExM can be coupled to the auxin-inducible degron system (mAiD system). This pipeline led to the subcellular localization of the gene product at unprecedented resolution and simultaneously assessed the consequences of conditional gene disruption. In this chapter, we explain the specific U-ExM protocol used for T. gondii tachyzoite samples and provide non-trivial advice and tips to successfully perform the experiments.