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Fig. 10. Formation of `reporter' disulphide bonds during cadherin-mediated cell adhesion. K562 cells expressing wild-type or mutant N-cadherin were allowed to adhere, under reducing conditions, to a panel of monomeric N-cadherin-Fc molecules bearing the same set of mutations. Oxidising conditions were then restored and the formation of disulphide bonds was assessed by SDS-PAGE and immunoblotting as described. (a) The blot was developed with antibody to cellular cadherin cytoplasmic domain. The upper panels show gels run under non-reducing conditions. Disulphide-bonded trans-dimers form only when cadherin molecules bearing complementary cysteine point mutations were apposed. In contrast, the D1C and D27C mutations allowed formation of disulphide-bonded cis-dimers on the cell surface. With wild-type cells (Wt, right panel), no disulphide-bonded species are seen. The track labelled `uncoated' in this series reflects a small degree of background adhesion to wells lacking cadherin and shows the position of the cis-dimer. The lower panel shows the same preparations run under reducing conditions. (b) In a similar experiment, the blot was developed with anti-Fc. Again the trans-dimer can be seen when mutations D1C and R25C were apposed. As in (a), cis-dimers were formed by D1C and D27C mutants but not by R25C. Cis-dimers formed by monomeric N-cadherin-Fc are seen to run slightly below the trans-dimer in contrast to the situation in (a) where the cellular cis-dimer runs above the trans-species.
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