An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris.

Evidence has accumulated that changes in intracellular signaling downstream of desmoglein 3 (Dsg3) may have a significant role in epithelial blistering in the autoimmune disease pemphigus vulgaris (PV). Currently, most studies on PV involve passive transfer of pathogenic antibodies into neonatal mice that have not finalized epidermal morphogenesis, and do not permit analysis of mature hair follicles (HFs) and stem cell niches. To investigate Dsg3 antibody-induced signaling in the adult epidermis at defined stages of the HF cycle, we developed a model with passive transfer of AK23 (a mouse monoclonal pathogenic anti-Dsg3 antibody) into adult 8-week-old C57Bl/6J mice. Validated using histopathological and molecular methods, we found that this model faithfully recapitulates major features described in PV patients and PV models. Two hours after AK23 transfer, we observed widening of intercellular spaces between desmosomes and EGFR activation, followed by increased Myc expression and epidermal hyperproliferation, desmosomal Dsg3 depletion, and predominant blistering in HFs and oral mucosa. These data confirm that the adult passive transfer mouse model is ideally suited for detailed studies of Dsg3 antibody-mediated signaling in adult skin, providing the basis for investigations on novel keratinocyte-specific therapeutic strategies.

Desmosomes: adhesive strength and signalling in health and disease.

Desmosomes are intercellular junctions whose primary function is strong intercellular adhesion, known as hyperadhesion. In the present review, we discuss how their structure appears to support this function as well as how they are assembled and down-regulated. Desmosomal components also have signalling functions that are important in tissue development and remodelling. Their adhesive and signalling functions are both compromised in genetic and autoimmune diseases that affect the heart, skin and mucous membranes. We conclude that much work is required on structure-function relationships within desmosomes in vivo and on how they participate in signalling processes to enhance our knowledge of tissue homoeostasis and human disease.

Tissue section AFM: In situ ultrastructural imaging of native biomolecules.

Conventional approaches for ultrastructural high-resolution imaging of biological specimens induce profound changes in bio-molecular structures. By combining tissue cryo-sectioning with non-destructive atomic force microscopy (AFM) imaging we have developed a methodology that may be applied by the non-specialist to both preserve and visualize bio-molecular structures (in particular extracellular matrix assemblies) in situ. This tissue section AFM technique is capable of: i) resolving nm-microm scale features of intra- and extracellular structures in tissue cryo-sections; ii) imaging the same tissue region before and after experimental interventions; iii) combining ultrastructural imaging with complimentary microscopical and micromechanical methods. Here, we employ this technique to: i) visualize the macro-molecular structures of unstained and unfixed fibrillar collagens (in skin, cartilage and intervertebral disc), elastic fibres (in aorta and lung), desmosomes (in nasal epithelium) and mitochondria (in heart); ii) quantify the ultrastructural effects of sequential collagenase digestion on a single elastic fibre; iii) correlate optical (auto fluorescent) with ultrastructural (AFM) images of aortic elastic lamellae.

Visualization of desmosomes in the electron microscope.

Desmosomes are intercellular junctions found in epithelia and some other tissues. Their primary function is strong cell-cell adhesion. They also link the intermediate filament (IF) cytoskeletons between cells and have roles in cell signaling, tissue morphogenesis, and wound repair. Because of their size (0.2-0.5 microm), details of their ultrastructure can only be resolved at the electron microscope (EM) level. Desmosomes have been visualized using a variety of ultrastructural techniques including lanthanum infiltration, freeze-fracture, electron tomography, cryo-electron microscopy and immunogold labeling. This chapter describes protocols for conventional transmission electron microscopy and for immunogold labeling of ultrathin cryosections. We also discuss the statistical analysis of immunogold particle distribution for low resolution molecular mapping.

Assays for the calcium sensitivity of desmosomes.

Epithelial cells in vivo exist as confluent cell sheets, but this confluence is disrupted if the sheets are wounded, if the cells are undergoing morphogenesis, or if they are taking part in invasion and metastasis. Desmosomes are one of the principal types of adhesive junctions in epithelia and are responsible for maintaining tissue integrity. It is likely that modulation of desmosomal adhesion is required to facilitate cell motility in response to alterations in the tissue architecture. Desmosomal adhesion changes from a calcium-dependent state to a calcium-independent state when cells become confluent. Our laboratory has shown that the alpha isoform of protein kinase C is involved in signaling the response of desmosomes to calcium concentration and wounding, in cultured epithelial cells and in mouse epidermis (in vivo).