The structure of desmosomes and desmosomal cadherins

We are investigating the molecular mechanism that binds cells in tissues so strongly together and makes tissues so tough.
Strong adhesion is especially important in tissues that are constantly subjected to physical stress such as cardiac muscle and the outer layer of skin, the epidermis. Failure of adhesion can result in heart failure or extremely unpleasant, potentially lethal skin disease.
To cope with everyday wear and tear normal cells must vary their adhesion. In order to repair a wound, epidermal cell must weaken their adhesion so that they can rearrange themselves and spread over the wound surface.
We believe that strong adhesion is mediated by minute structures called desmosomes that are like little Velcro patches on the cells. Our hypothesis is that desmosomes have a special property that distinguishes them from other cell adhesions and Velcro. Once they have formed initial weak adhesion between cells they can lock themselves into a strongly adhesive state in which they remain unless the tissue is wounded when they revert to the weaker state.
We will use electron microscopy and X-ray crystallography to investigate how desmosomes are able to do this.
Our results may suggest novel disease treatments and ways to accelerate wound healing.

The cells of tissues subject to mechanical stress, e.g. epidermis and cardiac muscle, are bound together by adhesive intercellular junctions known as desmosomes. Defects in desmosomes cause the potentially lethal human diseases epidermolysis bullosa, pemphigus and arrhythmogenic right ventricular cardiomyopathy. We recently proposed the concept of hyper-adhesion to explain how desmosomes mediate strong adhesion. Tissue desmosomes are hyper-adhesive apparently because they have an organised, quasi-crystalline arrangement of the extracellular (EC) domains of their adhesion molecules, the desmosomal cadherins (DCs). Hyper-adhesive desmosomes resist disruption by chelating agents. Hyper-adhesiveness is lost in wounded cell sheets, accompanied by loss of organisation and acquisition of sensitivity to chelating agents. Loss of hyper-adhesiveness appears to be mediated by protein kinase C alpha. Our recent work shows that the DCs desmocollin and desmoglein exhibit homophilic, isoform-specific adhesive binding that may be fundamental to desmosome structural organisation. Our hypothesis is that the organised arrangement of the DC EC domains relates to an organised molecular arrangement within the plaque and that modulation of the latter by PKCa causes concomitant disorganisation of the former and loss of hyper-adhesiveness. To provide evidence for this hypothesis we will investigate the structure of the plaque of isolated bovine nasal epidermal desmosomes by immunogold-labelling and electron microscopy (EM), and by atomic force microscopy (AFM), and determine the crystal structure of the desmocollin and desmoglein EC domains. Isolated desmosomes will be adsorbed to EM grids, labelled with antibodies to desmosomal plaque components and gold particles distributions analysed by multivariate analysis to map the 2D arrangement of plaque molecules. Desmosomal plaque architecture will be analysed by AFM including dissection of the plaque by application of increasing force via the tip stylus. AFM examination of freeze fracture replicas of desmosomes will be used to analyse structure within the membrane and intercellular space. Isolated desmosomes will be treated with PKCa and examined by EM and AFM to characterise configurational changes in the plaque, and will be used to study phosphorylation of plaque components and calcium binding. The structure of the EC domains of desmocollin and desmoglein will be determined by X-ray crystallography to reveal features involved in homophilic binding and hyper-adhesion. The effect of calcium concentration on the configuration of DC EC domains will be determined by small angle X-ray solution scattering and AFM.