Actin-cytoskeleton-dependent regulation of desmosomal adhesion in keratinocytes

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This is a renewal proposal for'Role of the cytoskeleton for pemphigus pathogenesis'. It aims to elucidate actin-dependent mechanisms regulating desmosomal adhesion in keratinocytes. Desmosomes are adhesive intercellular contacts and especially abundant in the epidermis. In desmosomes, the transmembrane cadherin-type adhesion molecules desmogleins (Dsg) and desmocollins mediate the coupling of adjacent cells. Data from the previous grant clearly demonstrated that the actin cytoskeleton and actin-binding proteins such as alpha-adducin are required for strong cell cohesion and modulate the amount of the important adhesion molecule Dsg3 in the desmosome. The mechanisms underlying this regulation are largely unknown. The proposal focusses on the role of the actin cytoskeleton for desmosome formation and is split in two parts. (I) We will investigate the mechanisms how the actin-binding protein alpha-adducin regulates the assembly of Dsg3 in the desmosome. We will apply keratinocytes isolated from alpha-adducin wildtype and knockout mice and evaluate desmosome assembly induced by Ca2+ elevation. Furthermore, we will study in detail whether alpha-adducin modulates the intracellular transport of Dsg3 or its lateral incorporation into the desmosome in the membrane. The transport to the membrane will be evaluated by life cell imaging of a light-inducible Dsg3 construct. Using atomic force microscopy and single molecule fluorescence, we will map the distribution and the mobility of Dsg3 molecules on the cell surface to test the hypothesis that actin and adducin-dependent lateral clustering is a prerequisite for desmosome formation, as it was shown for E-cadherin and adherens junctions. (II) Because the actin-binding protein cortactin associates with Dsg3, we will test in keratinocytes from cortactin wildtype and knockout mice whether this molecule and its phosphorylation is required for desmosome formation and the regulation of cell cohesion. Furthermore, we will study the observed complex of Dsg3, E-cadherin and the tyrosine kinase Src in more detail and test whether Src activity is required for desmosome formation. Moreover we will investigate the regulation how Src activity is controlled in this context (i.e. by tyrosine phosphatases) and which molecules are targeted by Src (i.e. cortactin). Using this approach we will extend our knowledge on the regulation of intercellular adhesion which is a prerequisite for a better understanding of severe diseases caused by impairment of desmosome function such as pemphigus.