Abstract
Septins are GTP-binding proteins that associate with the microtubule (MT) and actin cytoskeleton. Septins affect MT organization and posttranslational modifications, but their role in MT dynamics is less understood. Here, we reconstituted MT dynamics in the presence of the MT-binding septin (SEPT9) using an in vitro cell-free assay, which images the polymerization of tubulin from guanosine-5'-[(α,β)-methyleno]triphosphate (GMPCPP)-stabilized MT seeds. We found that submicromolar concentrations of SEPT9 suppress MT catastrophe and enhance the growth of MT plus ends to great lengths, while low micromolar concentrations of SEPT9 stabilize MTs by inhibiting dynamic instability. We show that SEPT9 associates preferentially with the lattice of GMPCPP-stabilized MT seeds and surprisingly recruits soluble tubulin to the MT lattice. Notably, the effects of SEPT9 on MT dynamics are dependent on its G-G dimerization interface, which is formed by the pockets of the GTP-binding domains. A mutation (H530D) that disrupts G-G dimerization abrogates the effects of SEPT9 on MT dynamics and diminishes its ability to recruit tubulin to the MT lattice. Taken together, these results suggest that SEPT9 promotes the formation and maintenance of long stable MTs through a mechanism that may involve recruitment of unpolymerized tubulin to the MT lattice.