Septin architecture dictates size-dependent diffusion barriers on biomimetic membranes

Abstract Septins are cytoskeletal filaments bound to the inner leaflet of the plasma membrane, essential for cell division. They are involved in the formation of diffusion barriers for membrane-bound components. Whether septins directly function as barriers or as part of a broader regulatory cascade of events remains unclear. We addressed this using in vitro reconstituted assay of biomimetic synthetic membranes. We probed whether the diffusion of biomimetic fluorescent objects with tunable steric hindrance – mimicking membrane bound proteins – is constrained by septins. Our results indicate that (i) lipids and transmembrane proteins lacking cytosolic domains diffuse freely in the presence of septins (ii) membrane-bound objects with large cytosolic domains experience size-dependent diffusion constraints; and (iii) the ability of septins to act as diffusion barriers is finely tuned by their intrinsic filament organization. These findings demonstrate that septins can directly impose size-selective diffusion barriers and that their filament organization critically tunes this function.