Osmotic Gradients in Epithelial Acini Increase Mechanical Tension across E-cadherin, Drive Morphogenesis, and Maintain Homeostasis.

Epithelial cells spontaneously form acini (also known as cysts or spheroids) with a single, fluid-filled central lumen when grown in 3D matrices. The size of the lumen is dependent on apical secretion of chloride ions, most notably by the CFTR channel, which has been suggested to establish pressure in the lumen due to water influx. To study the cellular biomechanics of acini morphogenesis and homeostasis, we used MDCK-2 cells. Using FRET-force biosensors for E-cadherin, we observed significant increases in the average tension per molecule for each protein in mature 3D acini as compared to 2D monolayers. Increases in CFTR activity resulted in increased E-cadherin forces, indicating that ionic gradients affect cellular tension. Direct measurements of pressure revealed that mature acini experience significant internal hydrostatic pressure (37 ± 10.9 Pa). Changes in CFTR activity resulted in pressure and/or volume changes, both of which affect E-cadherin tension. Increases in CFTR chloride secretion also induced YAP signaling and cellular proliferation. In order to recapitulate disruption of acinar homeostasis, we induced epithelial-to-mesenchymal transition (EMT). During the initial stages of EMT, there was a gradual decrease in E-cadherin force and lumen pressure that correlated with lumen infilling. Strikingly, increasing CFTR activity was sufficient to block EMT. Our results show that ion secretion is an important regulator of morphogenesis and homeostasis in epithelial acini. Furthermore, this work demonstrates that, for closed 3D cellular systems, ion gradients can generate osmotic pressure or volume changes, both of which result in increased cellular tension.

Spatial Proliferation of Epithelial Cells Is Regulated by E-Cadherin Force.

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.