| Septins comprise a family of filament-forming GTPases that appear to be a novel component of the cytoskeleton. Septins form rod-shaped complexes that join end-on-end to polymerise into filaments, and assemble into higher-order structures such as bundled filaments and rings. These septin structures are thought to have two functions: one is to act as macromolecular scaffolds that recruit proteins to specific cellular locations and second, to serve as membrane diffusion barriers by confining distinct membrane domains. Both septin functions are required at the cleavage site of dividing cells, where septins recruit and confine membrane proteins that drive cytokinesis. Septin higher-order structures are found in diverse cellular contexts including neurons, migrating cells, cells with primary cilia, spermatozoa and host-pathogen interactions. The best insight into assembly of septin higher-order structures come from budding yeast. The four budding yeast septins are apolar octamers arranged as Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11, and two septin-septin interaction interfaces (NC and G) alternate within the octamer. These complexes join end-on-end to form filaments, and mutations in the terminal end subunit (Cdc11) prevent polymerisation and cytokinesis. It is not known how these octamers are assembled. In this thesis, I examine two aspects related to the structure of septins. In the first structural study, I show that septins form initial dimers at the G interface, which then permits NC interactions. This implies that the strongest G interface septin dimers might specify how octamers might be assembled. In the second structural study, I examine the positional arrangement of mammalian septins within a putative octamer. I show that Sept9 is a terminal septin subunit such that a putative mammalian septin octamer is arranged as Sept9-Sept7-Sept6-Sept2-Sept2-Sept6-Sept7-Sept9. Similar to yeast, I find that a mutation in the terminal end subunit (Sept9) prevents septin polymerisation and cytokinesis. Finally, I examine the role of septins in cells with primary cilia, an antennae-like cellular protrusion that act as efficient signaling organelles. I show that septins might regulate RhoA signaling at the base of cilia by binding and activating a RhoGEF, ArhGEF18. Collectively, my findings provide a step forward in understanding mammalian septin biology. |
