The impact of mechanical stretch on the stability of desmosomes in epithelia

Abstract

The mechanoresponse of epithelia is a well-known and essential process. Both cytoskeleton and cell adhesion sites react to mechanical stimuli, e.g. mechanical stretch. Cell adhesions like desmosomes act as connection sites in epithelia, they mediate cell-cell adhesion by providing mechanical strength and promoting an intercellular network by connecting the intermediate filaments between adjacent cells. Desmosomes are highly dynamic structures, which need to assemble and disassemble in order to adapt to an everchanging mechanical environment. This dynamic is put into excecution by protein exchange between the desmosomal structure and a pool of freely diffusing molecules in the cytoplasma. All in all, desmosomes are important sites of epithelial mechanoresponse, however, the research on desmosomes is predominantly based on experiments performed on static conditions. Therefore, little is known about the behaviour of desmosomal proteins, including their exchange kinetics upon mechanical stretch. <br/> In order to address this knowledge gap, an experimental approach to analyse the responses of several desmosomal proteins to mechanical stretch was carried out within the scope of this thesis. <br/> A monolayered epithelial system of MDCK (Madin-Darby Canine Kidney) cell lines stably expressing a fluorescently tagged desmosomal fusion protein was probed with FRAP (Fluorescence Recovery After Photobleaching) to analyse protein exchange kinetics of the desmosomal proteins DP (desmoplakin), PG (plakoglobin) and Dsc (desmocollin). <br/> An experimental set-up with sealable silicone chambers was developped for cell stretching. The application of mechanical stretch was performed with a computer-controlled apparatus, the cell stretcher. Uniaxial, cyclic stretch with an 80 mHz (Millihertz) frequency was applied for a duration of 2 hours (h) to the analysed samples in live cell conditions. <br/> The exchange kinetics of the desmosomal proteins DP, PG and Dsc were analysed at different time points after stretch in order to test the impact of mechanical stretch on the stability of desmosomes. The recovery curves displayed a biphasic ascent, indicating the presence of two distinct kinetic processes, free diffusion and exchange kinetics at the desmosomal sites. <br/> To decide whether an exponential fit could be applied to the recovery curves, a straight was drawn between the first and the last point of the exponential fit while excluding the offset. If the maximum distance between this straight and the exponential fit was exceeding the standard deviation of the noise from the recovery curves this recovery curve was fitted with an exponential fit, otherwise, it was fitted with a linear fit. <br/> This analysis has shown that, in the absence of mechanical stretch, the analysed desmosomal proteins are stable components of the desmosomal structure. Upon exposure to uniaxial, cyclic stretch PG and Dsc exhibit a reversible mechanoresponse, which expresses as an increase of their exchanging fraction. This mechanoresponse is not detectable 24 h after the stretching event. <br/> In essence, this work analysed the exchange kinetic of desmosomal proteins under the influence of mechanical stretch.