Freeze-fracture electron microscopic study of tight junction strands in HEK293 cells and MDCK II cells expressing claudin-1 mutants in the second extracellular loop.

Claudins constitute tight junction (TJ) strands. In order to examine the function of the second extracellular loop (ECL2), we constructed 1CLDeltaFY and 1CLDeltaPL in which highly conserved amino acids, FY or PL, in the ECL2 of mouse claudin-1 were deleted. They were then tagged with either EGFP at the NH(2)-terminus (EGFP1CLDeltaFY and EGFP1CLDeltaPL) or the myc-epitope at the COOH-terminus (1CLDeltaFYmyc and 1CLDeltaPLmyc). The expression of EGFP1CLDeltaFY and EGFP1CLDeltaPL in TJ-free HEK293 cells formed TJ strands resembling those formed by wild-type claudin-1. The expression of 1CLDeltaPLmyc in TJ-bearing MDCK II cells induced aberrant TJ strands in the lateral plasma membranes whose intramembranous particles were almost equally distributed in the P- and E-face. In contrast, 1CLDeltaFYmyc formed aggregates of short continuous strands which were frequently associated with vesicle-like structures. Coculture experiments with MDCK II cells showed that 1CLDeltaPLmyc was localized at heterotypic cell-cell junctions but 1CLDeltaFYmyc was not. These results suggest that changes in the TJ morphology due to the expression of either 1CLDeltaFYmyc or 1CLDeltaPLmyc may be caused by some factors specific to epithelial MDCK II cells including endogenous claudins.