ausblenden:
Schlagwörter:
Desmoplakin, Tension Sensor, Mechanobiology, Mechanotransduction, Mechanosensing, FRET, FLIM, Molecular Force, Cellular Force, Force Sensor, Desmosome, Cell adhesion, Cell-Cell, Cell Contact, Adhesion Site, Keratinocytes, Skin, Biosensor, Kraftsensor, Zellkontakt, Adhäsionsverbindung
Zusammenfassung:
Desmosomes are cell–cell adhesion sites especially important in heart and skin tissues. Both tissues are exposed to mechanical stress and desmosomes are essential for stable cell–cell adhesion but whether and how forces act on desmosomes was unclear. Here, a desmoplakin tension sensor was developed reporting on molecular forces experienced by desmoplakin, which is essential for the connection to the intermediate filament cytoskeleton. Tension measured with the desmoplakin tension sensor can therefore also serve as a proxy for forces transduced across desmosomes towards the intermediate filament cytoskeleton. Fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurements of the desmoplakin tension sensor revealed the absence of desmoplakin forces during the formation of desmomes in keratinocytes. Forces are experienced by desmoplakin, however, on very soft substrates, where the substrate stiffness is in the range of the intermediate filament stiffness. Furthermore, desmosomes are transiently loaded in response to external mechanical stress. The stress-induced loading depends on the magnitude and orientation of the applied tissue deformation. These observations suggest that desmosomes act as stress absorbers and evolved in mammalian tissues to complement adherens junctions especially in more extreme situations.
Next to the development and experiments with the desmoplakin tension sensor, the fluorescence lifetime analysis and merge software (FLAMES) was developed. The software provides an automated data analysis pipeline for FRET-based tension sensor experiments measured with FLIM. FLAMES thereby improves the estimation of lifetimes from photon count curves for the signal of interest. Moreover, FLAMES also allows the determination of the relative amount of molecules under tension, which provides a new way for the analysis of tension sensor experiments.
