Separation of cell-cell adhesion complexes by differential centrifugation.

The number of proteins found associated with cell-cell adhesion substructures is growing rapidly. Based on potential protein-protein interactions, complex protein networks at cell-cell contacts can be modeled. Traditional studies to examine protein-protein interactions include co-immunoprecipitation or pull-down experiments of tagged proteins. These studies provide valuable information that proteins can associate directly or indirectly through other proteins in a complex. However, they do not clarify if a given protein is part of other protein complexes or inform about the specificity of those interactions in the context of adhesion substructures. Thus, it is not clear if models compiled from these types of studies reflect the combination of protein interactions in the adhesion complex in vivo for a specific cell type. Therefore, we present here a method to separate cell-cell contact membrane substructures with their associated protein complexes based on their buoyant behavior in iodixanol density gradients. Analysis of 16 proteins of the apical junctional complex (AJC) in epithelial Madin-Darby canine kidney cells revealed a more simple organization of the AJC adhesion complex than that predicted from the combination of all possible protein-protein interactions defined from co-immunoprecipitation and pull-down experiments.