The epidermis is an effective barrier between humans and the environment, providing protection from environmental insult and dehydration as well as mechanical resistance. This fundamental feature of skin is derived from the presence of mechanically robust cell-cell and cell-matrix adhesive junctions called desmosomes. A variety of human disorders, both inherited and acquired, are caused by defects in the adhesive function or signaling activity of desmosomes. These specialized membrane domains are comprised of many proteins carrying out different functions. Adhesion between cells is mediated by interaction of the extracellular domain of the desmosomal cadherins, desmocollins and desmogleins. The desmosome can exist in different adhesive states dependent on the interactions of these proteins, including disrupted and hyper- adhesive. Pemphigus vulgaris is an autoimmune blistering disease that disrupts the desmosome. The organization and dynamic properties of these proteins within the desmosome in live cells remains elusive. A significant challenge in studying the organizational behavior of these proteins in vivo is lack of suitable techniques. Here we propose to apply a powerful emerging imaging technique, fluorescence polarization microscopy. This will allow us to identify order and organization of desmosomal proteins in cells, as well as dynamic changes in this organization caused by pemphigus vulgaris autoantibodies and hyperadhesion. Polarization microscopy therefore allows us to overcome the difficulties inherent in measuring the orientations and organizational behavior of single proteins within macromolecular complexes in living cells. This ordering of desmosomal cadherins is hypothesized to be critical to function and adhesive state in both healthy and diseased epidermis. The experiments described in this proposal allow, for the first time, identification of protein order in normal, disrupted, and hyperadhesive desmosomes providing novel insight into the structure and function of these critical complexes.
A variety of inherited and acquired human disorders are caused by defects in the adhesive function or signaling activity of desmosomes. The goal of this proposal is to provide novel insight into the structure and function of desmosomes by applying novel methods to measure protein order. These studies will have important implications for future therapeutic strategies to modulate the structure of desmosomes in epithelial disorders.
