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 1CL Delta FY and 1CL Delta PL 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 NH2-terminus (EGFP1CL Delta FY and EGFP1CL Delta PL) or the myc-epitope at the COOH-terminus (1CL Delta FYmyc and 1CL Delta PLmyc). The expression of EGFP1CL Delta FY and EGFP1CL Delta PL in TJ-free HEK293 cells formed TJ strands resembling those formed by wild-type claudin-1. The expression of 1CL Delta PLmyc 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, 1CL Delta FYmyc formed aggregates of short continuous strands which were frequently associated with vesicle-like structures. Coculture experiments with MDCK II cells showed that 1CL Delta PLmyc was localized at heterotypic cell-cell junctions but 1CL Delta FYmyc was not. These results suggest that changes in the TJ morphology due to the expression of either 1CL Delta FYmyc or 1CL Delta PLmyc may be caused by some factors specific to epithelial MDCK II cells including endogenous claudins.