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.

The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast1. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT2. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid3 and glucose4 signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.

The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have served as invaluable model organisms to study various fundamental and highly conserved cellular processes. While super-resolution (SR) microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeast models has remained limited due to the specific know-how and specialized instrumentation required, contrasted with the relative ease of endogenous tagging and live cell fluorescence microscopy in these systems. To facilitate SR microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling 4-fold isotropic expansion in both systems. We demonstrate here that U-ExM allows the nanoscale imaging of the microtubule cytoskeleton and its associated spindle pole body (SPB), notably unveiling a conserved Sfi1p/Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by quantifying the homeostatic regulation of nuclear pore complex (NPC) number through the cell cycle. Combined with pan-labelling (NHS ester), which provides a global cellular context, U-ExM unveils the subcellular organization of the eukaryote yeast models S. cerevisiae and S. pombe. This easy-to-implement imaging with conventional microscopes provides nanoscale resolution and adds a powerful new method to the already extensive yeast toolbox.