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, GSK3{beta} 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. HighlightsO_LIGSK3{beta} phosphorylates SEPT9, and its activity gates septin-cytoskeleton association C_LIO_LIS82/S85 phosphorylation reduce microtubule binding and increase actin localization C_LIO_LIUnphosphorylated SEPT9 binds preferentially to microtubules, promoting their growth C_LIO_LIGSK3{beta} inactivation drives SEPT9 to microtubules to establish neuronal polarity C_LI