Distinct mechanism of syntaxin3-dependent tight junction assembly in keratinocyte model cells

In the epidermis, differentiated keratinocytes form tight junctions (TJs) at a specific layer of the stratified epithelium, contributing to the skin barrier function. Previously, we demonstrated that the t-SNARE protein Syntaxin3 (Stx3) is necessary for TJ assembly in differentiated HaCaT keratinocytes. In simple epithelial cells, the apical targeting signal (ATS), located within helix a of Stx3, appears to be essential for TJ formation. Here, using Stx3-knockout HaCaT cells with inducible expression of various Stx3 mutants, we present a distinct mechanism of Stx3-dependent TJ assembly in epidermal keratinocytes that have lost apicobasal polarity. We found that in differentiated HaCaT cells, Stx3 lacking ATS still retains the ability to induce TJ formation, but replacing helices a and b with those of its paralogue epimorphin/Stx2 eliminated this ability. Intriguingly, a subpopulation of wild-type Stx3 appeared to be exposed to the extracellular space upon differentiation, and extracellular addition of a recombinant form of Stx3 supported TJ assembly. We also found that substituting only the six amino acids in helix b of Stx3 (SMEKHI) with the corresponding motif from Stx2, known as the functional core of extracellular Stx2, completely abolished this ability. These findings suggest that the differentiation-induced extrusion of Stx3 is crucial for TJ assembly in differentiated keratinocytes, with the possible core motif in helix b playing an essential role, providing new insights into the spatiotemporal dynamics of TJ formation in the stratified epidermis.