Forces and signal transduction by desmosomal cadherins

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Desmosomal cadherins (DCs) are transmembrane adhesion molecules located inside desmosomes and also found extradesmosomal in the membrane. Within desmosomes, DCs are coupled intracellularly to the plaque proteins plakophilin and plakoglobin. Desmoplakin connects the plaque to the intermediate filament cytoskeleton. This construct provides intercellular adhesive strength to tissues subjected to high degrees of mechanical stress such as the epidermis or the heart muscle which can be concluded from severe diseases arising from impaired DC function. However, the adhesive properties of DCs have not been characterized so far and the functions of extradesmosomal DCs in the membrane are largely unknown. Recent evidence demonstrates that DCs, in addition to their adhesive function, shape cell behavior via modulating intracellular signaling in an adhesion-dependent manner. Here, the extradesmosomal DCs may play a crucial role. To gain further insights into these functions, the proposal aims to characterize the adhesive and the signaling properties of DCs via biophysical and biochemical approaches in living keratinocytes. We will evaluate (i) the distribution, binding partners and binding characteristics of specific DC isoforms in living keratinocytes using atomic force microscopy (AFM) and optical trapping approaches and (ii) determine whether these parameters are dependent on the anchorage to the cytoplasmic plaque. Here we will apply murine keratinocytes deficient for plakoglobin, desmoplakin and keratin filaments. To investigate outside-in-signaling elicited by DCs in an adhesion-dependent manner, we will (iii) use FRET probes and biochemical assays to measure the activity of p38MAPK, RhoA, PKC and Src after interference with the binding of specific DC isoforms with antibodies or peptides. Because these molecules are also known to modulate keratinocyte cohesion, we finally will (iv) evaluate how the distribution and binding properties of DCs are altered by inside-out signaling via these signaling pathways. Taken together, these experiments will provide novel insights into the function of specific DCs and their roles as receptors and targets of intracellular signaling.