Ashbya gossypii as a model system to study septin organization by single-molecule localization microscopy.

Heteromeric complexes of GTP-binding proteins from the septin family assemble into higher order structures that are essential for cell division in many organisms. The correct organization of the subunits into filaments, gauzes, and rings is the basis of septin function in this process. Electron microscopy and polarization fluorescence microscopy contributed greatly to the understanding of the dynamics and organization of such structures. However, both methods show technical limitations in resolution and specificity that do not allow the identification of individual septin complexes in assemblies in intact cells. Single-molecule localization-based fluorescence superresolution microscopy methods combine the resolution of cellular structures at the nanometer level with highest molecular specificity and excellent contrast. Here, we provide a protocol that enables the investigation of the organization of septin complexes in higher order structures in cells by combining advantageous features of the model organism Ashbya gossypii with single-molecule localization microscopy. Our assay is designed to investigate the general assembly mechanism of septin complexes in cells and is applicable to many cell types.