The food poison microbe Clostridium perfringens produces a toxin that is useful for studies of epithelial sheet formation. A portion of the toxin binds to the extracellular domain of certain claudin isoforms and prevents their homotypic interactions. This toxin binds to claudin-4, which happens to be expressed in the tight junctions formed by the clonal dog kidney cell line called MDCK. The presence of tight junctions in MDCK cells can be assayed using microscopy and by measurement of the paracellular electrical resistance between the apical and basolateral sides of MDCK cell sheets (more resistance=tighter seal).
(1) Addition of the toxin to the media of MDCK cells reduces the average number of strands "stitching together" the membranes in tight junctions by about 50%. However, the remaining 50% of strands appear perfectly normal. If claudin-4 is involved in tight junction formation in these cells, why would 50% of strands be completely resistant to the toxin?
(2) Careful addition of toxin only to the basolateral surface of an MDCK cell sheet causes a partial drop in paracellular resistance (indicating a weaker, but still functional seal), while addition of toxin to only the apical surface causes no decrease in resistance. What does this tell you about the organization of different isoforms of tight junction molecules in a tight junction?