How ARVC-Related Mutations Destabilize Desmoplakin: An MD Study

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial heart disease linked to mutations in several desmosomal proteins, but the specific effects of these mutations on the molecular level are poorly understood. Among the many documented ARVC-related genetic variants, a striking hotspot of nine mutations has been identified in the plakin domain of desmoplakin. This hotspot can be found at the meeting point of three different subdomains of desmoplakin: two spectrin repeats and a Src homology 3 domain. We set out to understand the effect of these mutations. We determine, using molecular dynamics simulations, how these mutations affect the mechanics of this interface, performing two different classes of simulations. First, we sample the dynamics of the plakin domain, in particular the tendency of the interdomain hinge to buckle, and then we apply an external force onto the constructs and determine the force necessary to break them. We find that surface-exposed mutations are not affecting the dynamics to a very large degree but that most buried mutations make the junction more flexible and decrease the rupture forces observed. Our data suggest that buried ARVC mutations destabilize desmoplakin and thereby impair desmosome integrity under tension.     

Cells Under Stress: An Inertial-Shear Microfluidic Determination of Cell Behavior

The deformability of a cell is the direct result of a complex interplay between the different constituent elements at the subcellular level, coupling a wide range of mechanical responses at different length scales. Changes to the structure of these components can also alter cell phenotype, which points to the critical importance of cell mechanoresponse for diagnostic applications. The response to mechanical stress depends strongly on the forces experienced by the cell. Here, we use cell deformability in both shear-dominant and inertia-dominant microfluidic flow regimes to probe different aspects of the cell structure. In the inertial regime, we follow cellular response from (visco-)elastic through plastic deformation to cell structural failure and show a significant drop in cell viability for shear stresses >11.8 kN/m². Comparatively, a shear-dominant regime requires lower applied stresses to achieve higher cell strains. From this regime, deformation traces as a function of time contain a rich source of information including maximal strain, elastic modulus, and cell relaxation times and thus provide a number of markers for distinguishing cell types and potential disease progression. These results emphasize the benefit of multiple parameter determination for improving detection and will ultimately lead to improved accuracy for diagnosis. We present results for leukemia cells (HL60) as a model circulatory cell as well as for a colorectal cancer cell line, SW480, derived from primary adenocarcinoma (Dukes stage B). SW480 were also treated with the actin-disrupting drug latrunculin A to test the sensitivity of flow regimes to the cytoskeleton. We show that the shear regime is more sensitive to cytoskeletal changes and that large strains in the inertial regime cannot resolve changes to the actin cytoskeleton.