The septin cytoskeleton is a regulator of intestinal epithelial barrier integrity and mucosal inflammation.

Intestinal epithelial barrier integrity is essential for human health, and its disruption induces and exacerbates intestinal inflammatory disorders. While the epithelial cytoskeleton is critical for maintaining gut barrier-integrity, the role of septins - a family of GTP-binding, cytoskeletal proteins - is largely unknown. This highlights an important knowledge gap, as dysfunction of septins, and specifically septin 9 (SEPT9), is associated with intestinal pathologies. We determined that SEPT9 localizes to the apical junctions of intestinal epithelial cells (IECs), overlapping with both tight and adherens junctions. IEC-specific ablation of SEPT9 in mice resulted in leaky gut, due to mislocalization of junctional proteins, and increased susceptibility to experimental colitis. Consistently, SEPT9 expression was significantly reduced in intestinal mucosa of patients with inflammatory bowel disease (IBD). Using affinity-purification mass spectrometry, super-resolution imaging, and genetic KO, we determined that SEPT9 interacts with and is necessary to recruit nonmuscle myosin IIC (NMIIC) to the IEC perijunctional actomyosin belt. Loss of NMIIC also caused IEC barrier disruption. In summary, SEPT9 regulates intestinal barrier integrity by supporting the assembly of tight and adherens junctions through NMIIC recruitment to the actomyosin belt. The septin cytoskeleton safeguards the intestinal mucosa during acute inflammation, and its disruption in IBD suggests a loss of this protective function.