Abstract

Septins are GTP-binding cytoskeletal proteins that shape and compartmentalize the plasma membrane. Their complex interactome has made it difficult to understand the molecular factors that govern their assembly. Moreover, it is unclear whether human septin hexamers and octamers form distinct higher-order assemblies, especially at the plasma membrane. Here, we address this question by using label-free methods to probe binding and self-assembly of purified human septins on supported lipid bilayers. Quartz crystal microbalance with dissipation (QCM-D) monitoring revealed that septin-membrane binding is diffusion-limited and concentration-dependent. The viscoelastic properties differed between septin oligomers suggestive of structural differences. Imaging of membrane-bound septin networks by atomic force microscopy (AFM) revealed that septin hexamers formed aligned nematic filamentous networks, whereas septin octamers formed aligned curved structures including spirals. QCM-D and AFM measurements both showed that septins form double-layered filament networks. However, upon C-terminal truncation of the SEPT6 and SEPT7 subunits, hexamers no longer bound the membrane while octamers formed a single-layered network of filament spirals. Our findings reveal that human septin hexamers and octamers interact differently with membranes, providing a baseline to understand their functions in the cell.