Highlights d E-cadherin experiences higher mechanical tension in acini compared with monolayers d E-cadherin tension is positively correlated to CFTR activity d Epithelial acini have significant lumen pressure due, in part, to CFTR activity d Increased CFTR activity increases cell proliferation and blocks EMT progression
Cell-cell junctions are critical structures in a number of tissues for mechanically coupling cells together, cell-to-cell signaling, and establishing a barrier. In many tissues, desmosomes are an important component of cell-cell junctions. Loss or impairment of desmosomes presents with clinical phenotypes in the heart and skin as cardiac arrhythmias and skin blistering, respectively. Because heart and skin are tissues that are subject to large mechanical stresses, we hypothesized that desmosomes, similar to adherens junctions, would also experience significant tensile loading. To directly measure mechanical forces across desmosomes, we developed and validated a desmoglein-2 (DSG-2) force sensor, using the existing TSmod Förster resonance energy transfer (FRET) force biosensor. When expressed in human cardiomyocytes, the force sensor reported high tensile loading of DSG-2 during contraction. Additionally, when expressed in Madin-Darby canine kidney (MDCK) epithelial or epidermal (A431) monolayers, the sensor also reported tensile loading. Finally, we observed higher DSG-2 forces in 3D MDCK acini when compared to 2D monolayers. Taken together, our results show that desmosomes experience low levels of mechanical tension in resting cells, with significantly higher forces during active loading.
The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a structure consisting of nesprin, SUN, and lamin proteins. A principal function of the LINC complex is anchoring the nucleus to the actin, microtubule, and intermediate filament cytoskeletons. The LINC complex is present in nearly all cell types, including endothelial cells. Endothelial cells line the inner most surfaces of blood vessels, and are critical for blood vessel barrier function. In addition, endothelial cells have specialized functions, including adaptation to the mechanical forces of blood flow. Previous studies have shown that depletion of individual nesprin isoforms results in impaired endothelial cell function. To further investigate the role of the LINC complex in endothelial cells we utilized dominant negative KASH (DN-KASH), a dominant negative protein which displaces endogenous nesprins from the nuclear envelope and disrupts nuclear-cytoskeletal connections. Endothelial cells expressing DN-KASH had altered cell-cell adhesion and barrier function, as well as altered cell-matrix adhesion and focal adhesion dynamics. In addition, cells expressing DN-KASH failed to properly adapt to shear stress or cyclic stretch. DN-KASH expressing cells exhibited impaired collective cell migration in wound healing and angiogenesis assays. Our results demonstrate the importance of an intact LINC complex in endothelial cell function and homeostasis. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]
Cell proliferation and contact inhibition play a major role in maintaining epithelial cell homeostasis. Prior experiments have shown that externally applied forces, such as stretch, result in increased proliferation in an E-cadherin force-dependent manner. In this study, the spatial regulation of cell proliferation in large epithelial colonies was examined. Surprisingly, cells at the center of the colony still had increased proliferation as compared to cells in confluent monolayers. E-cadherin forces were found to be elevated for both cells at the edge and center of these larger colonies when compared to confluent monolayers. To determine if high levels of E-cadherin force were necessary to induce proliferation at the center of the colony, a lower-force mutant of E-cadherin was developed. Cells with lower E-cadherin force had significantly reduced proliferation for cells at the center of the colony but minimal differences for cells at the edges of the colony. Similarly, increasing substrate stiffness was found to increase E-cadherin force and increase the proliferation rate across the colony. Taken together, these results show that forces through cell-cell junctions regulate proliferation across large groups of epithelial cells. In addition, an important finding of this study is that junction forces are dynamic and modulate cellular function even in the absence of externally applied loads.
Fluorescence microscopy of FRET-based biosensors allow nanoscale interactions to be probed in living cells. This paper describes a novel approach to spectrally resolved fluorescence microscopy, termed sensorFRET, that enables quantitative measurement of FRET efficiency. This approach is an improvement on existing methods (FLIM, sRET, luxFRET, pFRET), as it does not require single fluorophore standards to be measured with every experiment and the acquisition is intensity independent, allowing the laser power to be optimized for varying levels of fluorophore expression. Additionally, it was found that all spectral based methods, including sensorFRET, fail at specific fluorophore-excitation wavelength combinations. These combinations can be determined a priori using sensorFRET, whereas other methods would give no indication of inaccuracies. This method was thoroughly validated and compared to existing methods using simulated spectra, Fluorescein and TAMRA dye mixtures as a zero FRET control, and Cerulean-Venus FRET standards as positive FRET controls. Simulations also provided a means of quantifying the uncertainty in each measurement by relating the fit residual of noisy spectra to the standard deviation of the measured FRET efficiency. As an example application, Teal-Venus force sensitive biosensors integrated into E-cadherin were used to resolve piconewton scale forces along different parts of an individual cell junction.
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