Septin higher-order structure on yeast membranes in vitro

Septins are cytoskeletal proteins that form filaments and higher-order structures, and remodel membranes in a variety of processes. Structural and cell biological studies provided atomic- and micro-scale details, but the understanding of septin assembly at the mesoscale is limited. Here, we used high-speed atomic force microscopy (HS-AFM) to analyze yeast septin assembly on yeast supported lipid bilayers (SLBs). We found the coexistence of three lipid phases in yeast membranes, where septin polymerized selectively on the liquid-disordered phase. Septin filaments adhered to membranes with a conserved face; and paired filaments, previously reported in less native environments, were not observed. Additionally, septin filaments exhibited lateral and longitudinal alignment. We used HS-AFM force-sweep experiments to disrupt septin structures and observe organizational recovery through self-templating. Finally, septin filaments stacked, where higher layer filament alignment was templated by the layer below. Thus, septins encode their 3D-structural organization, likely tunable by the membrane and bulk environment.