The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

Naydenov, N. G.; Hu, G.; Zafar, A.; Robak, D.; Makhmudova, K.; Lechuga, S.; Sagwan, N.; Bandyopadhay, S.; Musich, R.; Jeffrey, E.; Sun, L.; Marino-Melendez, A.; Rieder, F.; Sheynkman, G.; Ivanov, A. I.; Ebrahim, S.

doi:10.1101/2024.12.20.629767

bioRxiv · 10.1101/2024.12.20.629767

The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

Naydenov, N. G.·Hu, G.·Zafar, A.·Robak, D.·Makhmudova, K.·Lechuga, S.·Sagwan, N.·Bandyopadhay, S.·Musich, R.·Jeffrey, E.·Sun, L.·Marino-Melendez, A.·Rieder, F.·Sheynkman, G.·Ivanov, A. I.·Ebrahim, S.

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Abstract

Background and AimsIntestinal epithelial barrier-integrity is essential for human health, and its disruption induces and exacerbates intestinal inflammatory disorders. While the cytoskeleton is critical for maintaining gut barrier-integrity, the role of the septins- the newest family of cytoskeletal proteins- is unknown. To address this knowledge gap, we evaluate the role of SEPT9- a critical component of the septin-cytoskeleton- in intestinal epithelial cell (IEC) barrier permeability and inflammation. MethodsWe developed SEPT9-NeonGreen knockin mice, inducible intestinal epithelial cell (IEC)-specific SEPT9 knockout (KO) mice, and SEPT9-KO human IEC lines. SEPT9 localization was analyzed using super-resolution microscopy. Barrier-integrity was assessed via transepithelial electrical resistance, FITC-dextran flux, and visualization of tight junction (TJ) and adherens junction (AJ) proteins. Dextran sodium sulfate-induced experimental colitis was evaluated in control and KO mice through measuring cytokine expression, immune cell infiltration, and IEC death. SEPT9 expression was examined in intestinal tissue of IBD patients. ResultsSEPT9 overlapped with TJs and AJs at IEC apical junctions. SEPT9 loss resulted in a leaky epithelial barrier due to mislocalization of junctional proteins. SEPT9 interacted with non-muscle myosin IIC (NMIIC) at the IEC apical-junctional actomyosin belt, and its ablation displaced NMIIC from IEC junctions. Loss of NMIIC also caused barrier disruption. SEPT9 KO mice exhibited increased susceptibility to experimental-colitis. SEPT9 expression was significantly reduced in intestinal mucosa of IBD patients. ConclusionSEPT9 regulates intestinal barrier integrity, supporting TJ and AJ assembly through NMIIC recruitment to the actomyosin belt. SEPT9 safeguards the intestinal mucosa during acute inflammation, and its reduced expression in IBD suggests a loss of this protective function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/629767v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@5141dcorg.highwire.dtl.DTLVardef@bae524org.highwire.dtl.DTLVardef@19c8eaorg.highwire.dtl.DTLVardef@d5b218_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Naydenov, N. G., Hu, G., Zafar, A., Robak, D., Makhmudova, K., Lechuga, S., Sagwan, N., Bandyopadhay, S., Musich, R., Jeffrey, E., Sun, L., Marino-Melendez, A., Rieder, F., Sheynkman, G., Ivanov, A. I., Ebrahim, S.. 2024-12-22. The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation. https://doi.org/10.1101/2024.12.20.629767

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