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.

Spindle pole body duplication defective yeast cells are more prone to membrane damage.

Correct separation of chromosomes during mitosis is essential for preventing genetic instability and aneuploidy. Such separation is dependent on correct duplication of the nuclear-associated microtubular organizing center, i.e., spindle pole body (SPB), in fungi. MonoPolar Spindle 2 (MPS2) is an essential gene, encoding a membrane protein required for the insertion of SPB into the nuclear envelope. We recently reported that the SESA complex, which is composed of Smy2, Eap1, Scp160, and Asc1, suppresses the essential role of MPS2 (Sezen et al. 2009, Genes & Development 23:1559-1570), i.e., in SESA-active cells Mps2 becomes nonessential. We also proposed that the SESA network facilitates this insertion by altering the membrane lipid composition (Sezen 2015, FEMS Yeast Research 15:fov089). In addition, we are interested in the antifungal properties of essential oils and previously reported that membrane integrity of yeast cells is impaired upon exposure to turpentine, thyme, oregano, and orange peel essential oils (Konuk and Ergüden 2017, BioCell 41:13-18). Due to our continuing interest in the SESA system and the mechanisms by which essential oils affect yeast cells, we aimed to investigate the effects of essential oils on yeast cell membranes. Herein, we show that mps2∆ 2µm-SMY2 and mps2∆ pom34∆ cells, in which the SESA complex is active and SPB duplication is defective, are more prone to membrane damage upon treatment with essential oils.

Genetic analysis of Mps3 SUN domain mutants in Saccharomyces cerevisiae reveals an interaction with the SUN-like protein Slp1.

In virtually all eukaryotic cells, protein bridges formed by the conserved inner nuclear membrane SUN (for Sad1-UNC-84) domain-containing proteins and their outer nuclear membrane binding partners span the nuclear envelope (NE) to connect the nucleoplasm and cytoplasm. These linkages are important for chromosome movements within the nucleus during meiotic prophase and are essential for nuclear migration and centrosome attachment to the NE. In Saccharomyces cerevisiae, MPS3 encodes the sole SUN protein. Deletion of MPS3 or the conserved SUN domain is lethal in three different genetic backgrounds. Mutations in the SUN domain result in defects in duplication of the spindle pole body, the yeast centrosome-equivalent organelle. A genome-wide screen for mutants that exhibited synthetic fitness defects in combination with mps3 SUN domain mutants yielded a large number of hits in components of the spindle apparatus and the spindle checkpoint. Mutants in lipid metabolic processes and membrane organization also exacerbated the growth defects of mps3 SUN domain mutants, pointing to a role for Mps3 in nuclear membrane organization. Deletion of SLP1 or YER140W/EMP65 (for ER membrane protein of 65 kDa) aggravated growth of mps3 SUN domain mutants. Slp1 and Emp65 form an ER-membrane associated protein complex that is not required directly for spindle pole body duplication or spindle assembly. Rather, Slp1 is involved in Mps3 localization to the NE.