Septin Periodicity And Bundling Arrangements In Mammalian Cells Revealed By Single-Molecule Localization Microscopy

Vissa, Adriano 1, 6 ; Giuliani, Maximiliano 1, 6 ; Froese, Carol D. 2 ; Kim, Moshe S. 2 ; Soroor, Forooz 2, 3 ; Kim, Peter K. 2, 3 ; Trimble, William S. 2, 3, 4 ; Yip, Christopher M. 1, 3, 5, 6

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Program in Cell Biology, The Hospital for Sick Children; 3. Department of Biochemistry, University of Toronto; 4. Department of Physiology, University of Toronto; 5. Department of Chemical Engineering and Applied Chemistry, University of Toronto; 6. Donnelly Centre for Cellular and Biomolecular Research, University of Toronto

Septins are a conserved family of GTPases that associate with numerous components of the cytoskeleton and the inner leaflet of the plasma membrane. These proteins are involved in many biological processes including cell division and membrane trafficking, and serving as a scaffolding component of the cytoskeleton used to recruit other proteins and form diffusion barriers to maintain the composition of membrane domains. In order to carry out their cellular functions, septins undergo interactions via their NC or G interfaces to form heteromeric rod-like structures that can polymerize into filaments and associate laterally into bundles. While electron microscopy studies of affinity-tagged and purified S. cerevisiae septin complexes have provided evidence for this periodic organization and in-registry lateral bundling in vitro, the in-vivo arrangement of stress fiber-associated septin bundles in mammalian cells remains poorly characterized. We report here on a Direct Stochastic Optical Reconstruction Microscopy (dSTORM) and Photoactivated Localization Microscopy (PALM) study of the 2D spatial distribution of septins in mammalian cells using Total Internal Relection Fluorescence (TIRF). From simulated and experimental results, we show the effects of labeling method, labeling efficiency, and fluorescent emitter photophysics on image reconstruction and interpretation. Our results suggest that septins organize by polymerization of hetero-octamers yielding an approximate 35 nm periodicity between subsequent units of SEPT2-SEPT2 or SEPT9-SEPT9, and that lateral bundling occurs with the homotypic septin proteins in registry. Furthermore, we observe a heterogeneous population of bundle sizes across and within cells, suggesting a local environment-specific mechanism is responsible for regulating filament bundling.