The Role of the Actin‐binding Protein α‐Adducin for Desmosome Turnover and Keratinocyte Adhesive Function

Abstract

Desmosomes are cell‐cell junctions which provide strong intercellular adhesion and couple the intermediate filament cytoskeleton to sites of cell‐cell contact. We have previously shown that the actin‐binding protein α‐adducin is required for desmosome‐mediated intercellular adhesion. We here tested in more detail the mechanisms underlying α‐adducin‐dependent regulation of desmosomal turnover and adhesion. Experiments were conducted in human keratinocytes with siRNA‐mediated knock‐down of α‐adducin, in knock‐out cell lines obtained with the CRISPR/Cas9 system and in murine knock‐out cells. Dispase‐based dissociation assays confirmed reduced intercellular binding strength in cells lacking α‐adducin. A more immature desmosome state in α‐adducin knock‐outs was indicated by reduced Ca2+‐insensitivity. α‐adducin knockout led to elevated levels of desmoglein (Dsg) 2 and Dsg3 in the membrane. In atomic force microscopy (AFM) single molecule experiments, an increased interaction probability of Dsg3 was detectable in α‐adducin knock‐out cells. This was accompanied by increased amounts of membrane tethers which is in line with a less rigid membrane structure and a role of α‐adducin in organizing the cortical actin cytoskeleton. Ca2+‐depletion/repletion experiments revealed enhanced fragmentation of Dsg3 staining in the membrane in α‐adducin knock‐out cells, indicating dysfunctional desmosome turnover. Indeed, life cell imaging experiments showed less directed intracellular transport of Dsg3‐containing vesicles to the cell membrane whereas fluorescence recovery after photobleaching (FRAP) studies and single molecule imaging demonstrated increased mobility of extradesmosomal Dsg3 in the cell membrane. Interestingly, despite their decreased intercellular cohesion, cells lacking α‐adducin showed slower cell migration in in vitro scratch and ex vivo skin punch biopsy assays. Together, our data indicate that α‐adducin contributes to intercellular adhesion by regulating Dsg3‐dependent adhesive functions, Dsg3 trafficking and desmosome stability.Support or Funding InformationGerman Research Council SP1300/1‐3This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.