The cytoskeleton is a master organizer of the cellular cortex and membrane trafficking and therefore plays a crucial role in apico–basal polarity. Septins form a family of GTPases that assemble into non-polar filaments, which bind to membranes and recruit cytoskeletal elements such as microtubules and actin using their polybasic (PB) domains, to perform their broad biological functions. Nevertheless, the role of septins and the significance of their membrane-binding ability in apico–basal polarity remains under-investigated. Here, using 3D cultures, we demonstrated that septin 9 localizes to the basolateral membrane (BM). Its depletion induces an inverted polarity phenotype, decreasing β-catenin at BM and increasing transforming growth factor (TGFβ) and Epithelial–Mesenchymal Transition (EMT) markers. Similar effects were observed after deleting its two PB domains. The mutant became cytoplasmic and apical. The cysts with an inverted polarity phenotype displayed an invasive phenotype, with src and cortactin accumulating at the peripheral membrane. The inhibition of TGFβ-receptor and RhoA rescued the polarized phenotype, although the cysts from overexpressed septin 9 overgrew and presented a filled lumen. Both phenotypes corresponded to tumor features. This suggests that septin 9 expression, along with its assembly through the two PB domains, is essential for establishing and maintaining apico–basal polarity against tumor development.

The cytoskeleton is a master organizer of cell cortex, organelles positioning and membrane trafficking. Therefore, playing a decisive role in establishment of apico-basal polarity. Septins are critical cytoskeleton components which function in apico-basal polarity remain weakly investigated. Here, using 3D culture system, we demonstrate that septin9 localizes at basolateral membrane (BM). Its depletion induces an inverted polarity phenotype, decreasing -catenin at BM and increasing the expression of the transforming growth factor (TGF) and the epithelial-to-mesenchymal transition (EMT) markers. Similar effects were observed upon deleting its two polybasic domains, and the mutant become cytoplasmic and apical. The cysts with an inverted polarity phenotype display an invasive phenotype, with src and cortactin accumulating at the peripheric membrane. Inhibition of TGF-receptor and RhoA rescues the polarized phenotype. Although the cysts from overexpressed-septin 9, overgrowth and present a filled lumen. Both phenotypes correspond to tumor features. This suggests that septin 9 expression and assembly through the two PB domains are essential for establishing and maintaining apico-basal polarity against tumor development through a mechanism involving TGF-dependent EMT and RhoA activity.