Septin Hierarchical Assembly Revealed by High-Speed Atomic Force Microscopy(HS-AFM)

Abstract

Septins are GTP-binding proteins that form palyndromic linear hetero-oligomeric complexes, which in turn are able to assemble into filaments and higher-order structures, such as filaments pairs and bundles. Septins are essential for processes that involve cell division, cell polarity maintenance and membrane remodeling. Recent studies have provided structural information about the different levels of septin organization; however, the assembly mechanism how septin filaments polymerize and further assemble into higher hierarchical structures, and crucial determinants of septin assembly remain unclear. Here we used high-speed atomic force microscopy (HS-AFM) to explore these questions. We image septins from their monomolecular state to layers of several filaments thickness. We determine that the assembly of septin filaments and filament pairs are diffusion-controlled, while multilayer septin assembly is a more complex process, partly diffusion-controlled and partly dependent on the structural optimization to form pairs and bundles. The KCl concentration in the environment influences septin filament assembly, oriented packing, pairing and multilayer formation. Septin filament pairing is pH-dependent around physiological pH. We further propose that filament-pairing is mediated by the face opposite to the C-terminal coiled-coil domains.