Deciphering the molecular composition of tricellular junctions

Tight Junctions (TJs) are intercellular adhesion complexes that contain an elaborate network of both transmembrane and cytosolic proteins, regulating epithelium integrity. In this thesis, I studied the interactome of a transmembrane protein tricellulin in Madin Darby Canine Kidney (MDCK) epithelial cells. It was the first protein found at specialized tricellular junctions (TCJ), intersection at the apical vertices of three or more epithelial cells, implicated to regulate the mechanical stress and help to maintain the epithelial monolayer mechano-homeostasis. We examined tricellulin localization at the TCJs and established a list of interaction partners of this protein via proximity dependent biotin ligation (BioID) approach. BioID-analysis identified both known and novel TCJ components: angulin, MAGI3, afadin and occludin. Through immunofluorescence localization studies, these candidates were identified at apical junction complexes, co-localizing with the well-defined TJ marker protein occludin. Subsequently, CRISPR-Cas9 knockout (KO) cell lines of these interactors were studied for their potential effects on the actin cytoskeleton and TJ morphology. In cells lacking either angulin, MAGI3 or tricellulin, the actin cytoskeleton morphology was altered, and TJ morphology appeared tortuous. Moreover, absence of afadin drastically affected the actin cytoskeleton and TJ morphology, resulting in physical openings within the TJs. The effect of afadin depletion was further studied through examining the localization pattern of TJ transmembrane proteins. The results provided evidence that afadin is essential for maintaining tissue integrity and barrier function at the epithelial TJ. Previous knockdown studies have suggested angulin’s importance for recruiting tricellulin to the TCJ. In support to these earlier studies, we utilized our KO cultures to confirm that accumulation of tricellulin at TCJ was compromised upon absence of angulin. However, depletion of tricellulin did not drastically affect the localization of angulin, reflecting an upstream role of angulin for tricellulin localization. Future investigations using these KO cell lines will further elucidate the molecular mechanisms underlying the formation, mechanosensation and maintenance of the TCJs