Septin crosstalk with microtubules and actin is regulated by a GSK3-dependent phosphoswitch.

Septins are cytoskeletal filaments that associate with the actin and microtubule cytoskeleton, but the mechanisms that govern septin crosstalk and function with these networks are largely unknown. Here, we show that glycogen synthase kinase 3 (GSK3) directly phosphorylates septin-9 (SEPT9), acting as a molecular switch that bidirectionally controls septin distribution between actin and microtubules. We show that GSK3 inhibition redistributes endogenous SEPT9 toward microtubules in multiple cell types. Phosphomimetic mutations at serines 82 and 85 reduce microtubule binding and enhance actin association in cells and in vitro, while phosphonull mutations promote microtubule binding and growth. In primary hippocampal neurons, GSK3β inactivation promotes SEPT9-microtubule association, and phosphomimetic mutations impair asymmetric neurite growth during neuronal polarization. These findings reveal a phosphorylation-dependent mechanism of septin partitioning between actin and microtubules, placing the cytoskeletal functions of septins under the control of GSK3 - a kinase linked to multiple signaling pathways of cell physiology and metabolism.