The food poison microbe Clostridium perfringens produces a toxin that is useful

Question

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?

Explanation / Answer

1) 50% of strands are bound with claudins only; while the rest 50% are bound with claudins as well as occludins. Occludins is another transmembrane protein of tight junctions. It functions together with Claudins; it helps in strengthening the tight junction.

So, it can be said that presence of occludins keeps the tight junctions of 50% cells intact. And these cells are observed as toxin resistant.

2) Addition of toxin to the basolateral surface disrupts the intercellular connections, while, this is not the case when the toxin is added to the top. This means that the extra proteins, other than Claudins (like Occludins) are present more on the apical surface than the basal surface.

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