Two types of tight junctions (TJ) can be distinguished: between two neighboring cells, the bicellular TJ (bTJ) is forming cell-cell contacts, while at the meeting point of three cells, a tubular structure is formed by the tricellular TJ (tTJ). Tricellulin is preferentially localized in tTJs, but also in the bTJ. The longest isoform of tricellulin, TRIC-a, is supposed to maintain the stability of the TJ, but the exact function was not clearly solved yet.

Methods: 

Tricellulin (TRIC-a) was cloned and overexpressed into the low resistance kidney tubule cell line MDCK II. Expression of TRIC-a and possible changes in other TJ proteins were checked by Western blot and immunofluorescent stainings, which were observed by confocal microscopy. Effects of tricellular and bicellular localization of TRIC-a were analyzed in detail, employing two-path impedance spectroscopy, biionic and dilution potential measurements, and flux studies with paracellular markers of different sizes. Macromolecule passage was directly observed by fluorescence life cell imaging microscopy. TJ ultrastructure was analyzed by freeze fracture electron microscopy.

Results: 

The localization of TRIC-a turned out to be dependent on the expression rate. Moderate overexpression led to localization mainly in tTJ and caused a substantial reduction of permeabilities for macromolecules between 4 and 10 kDa. At that location, TRIC-a did not alter tight junction resistance and permeabilities for mid-sized solutes. Strong overexpression led to TRIC-a localization also in bTJs. At this, also paracellular resistance was increased and permeability for mono- and divalent ions was decreased without charge preference. Fluorescence life cell imaging revealed the passage of 3 kDa but not 70 kDa dextran across tTJs and bTJs. Analysis of the TJ ultrastructure showed that overexpression of TRIC-a in bTJs leads to more linear bTJ strands with less strand breaks while in tTJ the ultrastructure was unchanged.

Conclusions: 

Tricellulin proves to be a barrier-forming TJ protein. If localized in bTJ, it consolidates the bTJ strand meshwork by closing of strand breaks, and reduces the paracellular permeability for ions. The main effect at its predominant location, the tTJ, is to decrease the passage of macromolecules. These findings, in turn, indicate that the tTJ central tube forms a pathway for macromolecules.