Expansion Microscopy on <em>Saccharomyces cerevisiae</em>

Abstract

The unicellular eukaryote S. cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, due to its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross- linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi &amp;ndash; including yeast &amp;ndash; and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts are crucial steps to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae.

Ultrastructure Expansion Microscopy reveals the nanoscale cellular architecture of budding and fission yeast

“…However, such approaches include a full enzymatic treatment that digests the proteome, and more importantly, pre-expansion labeling which retains the linkage error (Zwettler et al, 2020). Post-labeling ExM protocol on as S. Cerevisiae has recently been reported (Korovesi et al, 2022); however, the resolution power of this method on different organelles, its compatibility with antibodies, and its feasibility in other yeast models remain to be assessed. Here, we develop a robust U-ExM protocol for budding and fission yeast, keeping the proteome intact and labelling post-expansion to reduce linkage errors (Hamel and Guichard, 2021).…”

Expansion Microscopy: Super-Resolution Imaging with Hydrogels

“…141 This approach was replicated almost identically by another group not much later. 142 A specific Chitinase seems unnecessary to obtain full 4-fold homogeneous expansion of yeast cells, likely owing to its low quantity in yeast cell walls.…”

“…Lessons from Cell Wall Homogenization: Adapting Existing Protocols. Of note, both the authors of μExM 37 and the groups that achieved expansion of Saccharomyces cerevisiae 39,142 were adapting protocols originally used for other methods relating to their model organisms. Bacteria are lysed with lysozyme and mutanolysin to obtain spheroblasts for transformations or other processes, or for DNA preparations� just as the protein digestion approach in classical expansion microscopy 1 was adapted from DNA-extraction protocols.…”

“…The treatment of Saccharomyces cerevisiae with lyticase/zymolyase is, equally, an established protocol for generating spheroblasts. Both μExM 37 and the groups that achieved expansion of Saccharomyces cerevisiae 39,142 stuck with these protocols so closely that they�perhaps somewhat counterintuitively when compared to other the protocols highlighted thus far� performed cell wall digestion even before hydrogel polymerization. However, this does not seem to compromise structural integrity of the sample, while facilitating access of monomers for a more homogeneous formation of the hydrogel/sample hybrid throughout the sample volume.…”

Experimental approaches to study evolutionary cell biology using yeasts

“…While traditionally considered suboptimal for the imaging of intracellular features, superresolution techniques were recently developed (Chen et al, 2021;Hinterndorfer et al, 2022;Korovesi et al, 2022), and used (Dey et al, 2020) in yeast to study cellular processes at higher resolution, and new tools have been provided for the imaging of several subcellular structures (Akhuli et al, 2022;Zhu et al, 2019). A large number of telomere-to-telomere assemblies recently revealed the landscape of structural variants of S. cerevisiae natural isolates (S. O'Donnell et al, 2022).…”