Investigating the Mechanical Loading of Desmosomes

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Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells and are essential for the mechanical integrity of mammalian tissues. Mutations in desmosomal proteins cause severe human pathologies including epithelial blistering and heart muscle dysfunction, thus current models assume a central role of desmosomes in transmitting mechanical force between cells. However, direct evidence for the load-bearing nature of these macromolecular structures is still lacking.In the previous funding period, we therefore focused our efforts on the development and analysis of Förster resonance energy transfer (FRET)-based tension sensors to measure the piconewton-scale forces experienced by desmoplakin, an obligate desmosomal protein that links the junctional desmosomal plaque to the intermediate filament cytoskeleton. Our experiments showed that our desmoplakin tension sensors are functional in cells, and quantitative live cell FRET analyses revealed that desmoplakin does not experience significant tension under homeostatic conditions. However, our experiments demonstrate that desmoplakin becomes mechanically loaded in response to external mechanical stresses. The stress-induced loading of desmoplakin is force-specific, transient and sensitive to the magnitude and orientation of applied tissue deformation. Our data indicate that desmosomes fulfill a distinct mechanical function than previously analyzed cell adhesion complexes and seem to act as molecular stress absorbers in epithelial tissues. Here, we propose to use our newly developed technologies to elucidate the molecular mechanisms governing force transduction through desmoplakin, and by extension, the desmosome. We will test how previously described post-translational desmoplakin modifications affect desmosome loading, and to which extend different keratin variants and plakophilins isoforms modulate desmosome force propagation. Finally, we will use our recently developed tension sensor multiplexing approach to investigate the mechanical loading of desmosomes and adherens junctions simultaneously. Altogether, the expected results should help explain how the distinct intercellular junctions allow the construction of epithelia that are both dynamic and physically robust, two seemingly contradictory properties that are nonetheless essential for mammalian life.