Associate Professor Old Dominion University Norfolk, Virginia, United States
Introduction: Multi-cellular tissues like epithelia are subject to stretch during the course of normal physiological function. Occasional external stretch beyond a certain level is also known to cause tissue injury. Thus, it is essential to understand how epithelial tissues respond to external stretch and what factors affect their response to stretch. Of particular importance is the role of cell-cell contacts in resisting or rupturing in response to external stretch. Furthermore, how the level of cell contractility affects this response is not clear. The objective of our study is to test the effect of reduced contractility of the integrity of epithelial islands when challenged by external stretch.
Materials and
Methods: Madin-Darby Canine Kidney (MDCK) II cells were cultured in DMEM with 10% FBS atop collagen I-coated silicone sheets (Speciality Manufacturing, Saginaw, MI) overnight. To reduce contractility, blebbistatin was used at concentrations of 1 or 50 µM for 1 h. Then, a custom-built biaxial cell stretcher was used to apply a linear strain of over 35%. Phase and fluorescence images (corresponding to plasma membrane staining with CellBrite, Biotium, Fremont, CA) were obtained with a Leica DMi8 epifluorescence microscope. Fluorescence imaging of the plasma membrane stained by CellBrite enabled the clear identification of cell-cell contact rupture. Ruptured cell-cell contacts were identified by at least one localized gap between adjacent cells within a cell island. For statistical analysis, t-tests or ANOVA was used as appropriate.
Results, Conclusions, and Discussions: In order to understand how a moderate decrease in contractility affects multi-cellular integrity upon stretch, we wanted to first determine initial conditions that decrease the contractility but still largely maintain cell-cell contact integrity in the absence of stretch. We treated MDCK cell islands with different concentrations of blebbistatin (Fig. 1) and found that, a concentration of 50 µM blebbistatin led to the rupture of about 40% of the cell-cell contacts (Fig. 2). However, a low concentration of 1 µM blebbistatin corresponded to a state where nearly 90% of the cell-cell contacts’ integrity was maintained, with about 10% rupturing similar to the control case due to normal contact turnover (Fig. 1, 2).
We then subjected control and 1 µM blebbistatin-treated epithelial islands to external stretch. Stretch corresponding to a linear strain of over 35% showed that both islands showed increase in cell-cell contact rupture to a similar extent (Fig. 3). This showed that the expected decrease in cell-cell adhesion strength as well as the expected decrease in inter-cellular forces due to lower contractility led to a similar extent of decrease in multi-cellular integrity, compared to the control case. Thus, a moderate decrease in contractility does not seem to adversely affect multi-cellular integrity when subject to large stretch. Our results suggest that the balance of cell-cell adhesion strength on the one hand, and active inter-cellular forces and passive forces on the other hand in response to stretch may preserve multi-cellular integrity at even lower than normal levels of contractility.
