Figure Q19–2 Effects of Clostridium toxin on the barrier function of MDCK cells (problem 19–7). (A) Addition of toxin from the basolateral side of the epithelial sheet. (B) Addition of toxin from the apical side of the epithelial sheet. For a given voltage, a higher resistance (ohms cm2) gives less paracellular current.
the absence of claudin-4, the cells remain healthy and the epithelial sheet appears intact. The mean number of strands in the tight junctions that link the cells also decreases over 24 hours from about four to about two, and they are less highly branched. A functional assay for the integrity of the tight junctions shows that transepithelial resistance decreases dramatically in the presence of the toxin, but the resistance can be restored by washing out the toxin (Figure Q19–2A). Curiously, the toxin produces these effects only when it is added to the basolateral side of the sheet; it has no effect when added to the apical surface (Figure Q19–2B).
A. How can it be that two tight-junction strands remain, even though all of the claudin-4 has disappeared?
b. Why do you suppose the toxin works when it is added to the basolateral side of the epithelial sheet, but not when added to the apical side?
19–8 It is not an easy matter to assign particular functions to specific components of the basal lamina, since the overall structure is a complicated composite material with both mechanical and signaling properties. Nidogen, for example, cross-links two central components of the basal lamina by binding to the laminin γ-1 chain and to type IV collagen. Given such a key role, it was surprising that mice with a homozygous knockout of the gene for nidogen-1 were entirely healthy, with no abnormal phenotype. Similarly, mice homozygous for a knockout of the gene for nidogen-2 also appeared completely normal. By contrast, mice that were homozygous for a defined mutation in the gene for laminin γ-1, which eliminated just the binding site for nidogen, died at birth with severe defects in lung and kidney formation. The mutant portion of the laminin γ-1 chain is thought to have no other function than to bind nidogen, and does not affect laminin structure or its ability to assemble into the basal lamina. How would you explain these genetic observations, which are summarized in Table Q19–1? What would you predict would be the phenotype of a mouse that was homozygous for knockouts of both nidogen genes?
19–9 Discuss the following statement: “The basal lamina of muscle fibers serves as a molecular bulletin board, in which adjoining cells can post messages that direct the differentiation and function of the underlying cells.” 19–10 The affinity of integrins for matrix components can be modulated by changes to their cytoplasmic domains: a process known as inside-out signaling. You have identified a key region in the cytoplasmic domains of αIIbβ3 integrin that seems to be required for inside-out signaling (Figure Q19–3). Substitution of alanine for either D723 in the β chain or R995 in the α chain leads to a high level of spontaneous activation, under conditions where the wild-type chains are inactive. Your advisor suggests that you convert the aspartate in the β chain to an arginine (D723R) and the arginine in the α chain to an aspartate (R995D). You compare all three α chains (R995, R995A, and R995D) against all three β chains (D723, D723A, and D723R). You find that all pairs have a high level of spontaneous activation, except D723 vs R995 (the wild type) and D723R vs R995D, which have low levels. Based on these results, how do you think the αIIbβ3 integrin is held in its inactive state?