Epithelial cells exert differential traction stress in response to substrate stiffness.

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of studies, the effects that environmental changes have on this form of movement are poorly understood. To examine the role of substrate compliancy on multi-layered epithelial sheet migration, we performed traction force and confocal microscopy to determine differences in traction forces and to examine focal adhesions on synthetic and biological substrates. The leading edges of corneal epithelial sheets undergo retraction or contraction prior to migration, and alterations in the sheet's stiffness are affected by the amount of force exerted by cells at the leading edge. On substrates of 30 kPa, cells exhibited greater and more rapid movement than on substrates of 8 kPa, which are similar to that of the corneal basement membrane. Vinculin and its phosphorylated residue Y1065 were prominent along the basal surface of migrating cells, while Y822 was prominent between neighboring cells along the leading edge. Vinculin localization was diffuse on a substrate where the basement membrane was removed. Furthermore, when cells were cultured on fibronectin-coated acrylamide substrates of 8 and 50 kPa and then wounded, there was an injury-induced phosphorylation of Y1065 and substrate dependent changes in the number and size of vinculin containing focal adhesions. These results demonstrate that changes in substrate stiffness affected traction forces and vinculin dynamics, which potentially could contribute to the delayed healing response associated with certain corneal pathologies.

Dependence of Tensional Homeostasis on Cell Type and on Cell-Cell Interactions.

INTRODUCTION: The ability to maintain a homeostatic level of cell tension is essential for many physiological processes. Our group has recently reported that multicellularity is required for tensional homeostasis in endothelial cells. However, other studies have shown that isolated fibroblasts also maintain constant tension over short time scales without the need of cell-cell contacts. Therefore, in this study, our aim was to determine how different cell types regulate tension as isolated cells or in small clustered groupings and to investigate the role of cell-cell adhesion molecules, such as E-cadherin, in this system. METHODS: Micropattern traction force microscopy was used to determine how bovine aortic endothelial cells, bovine vascular smooth muscle cells, mouse embryonic fibroblasts, and human gastric adenocarcinoma cells, with or without cell-cell interactions due to E-cadherin, maintain tensional homeostasis over time. Tension temporal fluctuations in single cells and cell clusters were evaluated. RESULTS: We found that only endothelial cells require clustering for tensional homeostasis. The same was not verified in fibroblasts or vascular smooth muscle cells. Of relevance, in adenocarcinoma cells, we verified that tensional homeostasis was dependent on the competence of the adhesion molecule E-cadherin at both the single cells and multicellular levels. CONCLUSION: These findings indicate that cell-cell contacts may be critical for tensional homeostasis and, potentially, for barrier function of the endothelium. Furthermore, the cell-cell adhesion molecule E-cadherin is an important regulator of tensional homeostasis, even in the absence of cadherin engagement with neighboring cells, which demonstrates its relevance not only as a structural molecule but also as a signaling moiety.

Fibronectin, the extracellular glue.

Fibronectin is an extracellular matrix protein that is present during periods of change within tissues. It is upregulated and necessary in a number of developmental contexts, and it is also present during pathological progression of tissues and during wound healing. Thus, it has been studied in a broad number of contexts from basic science to pathology. One of the unique features of fibronectin is its ability to specifically bind a large number of molecules including other components of the extracellular matrix, signaling molecules, and cell adhesion molecules. Cellular interactions with fibronectin lead to bidirectional crosstalk that directs cell function and also leads to cell-dependent changes in the extracellular matrix. Interestingly, fibronectin exists in a functional form composed of fibers that are nm to µm in diameter that is highly interwoven, and fibronectin molecules that constitute this material have a labile molecular conformation that can be altered through binding of allosteric partners and strain resulting from application of cell contractile forces. This review focuses on summarizing the many binding partners for fibronectin such as ECM proteins, growth factors, and synthetic binding partners with a particular interest in binding partners whose adhesiveness is impacted by the molecular conformation of the fibronectin fibers.

Tensional homeostasis in endothelial cells is a multicellular phenomenon.

Mammalian cells of various types exhibit the remarkable ability to adapt to externally applied mechanical stresses and strains. Because of this adaptation, cells can maintain their endogenous mechanical tension at a preferred (homeostatic) level, which is essential for normal physiological functions of cells and tissues and provides protection against various diseases, including atherosclerosis and cancer. Conventional wisdom is that the cell possesses the ability to maintain tensional homeostasis on its own. Recent findings showed, however, that isolated cells cannot maintain tensional homeostasis. Here we studied the effect of multicellular interactions on tensional homeostasis by measuring traction forces in isolated bovine aortic endothelial cells and in confluent and nonconfluent cell clusters of different sizes. We found that, in isolated cells, the traction field exhibited a highly dynamic and erratic behavior. However, in cell clusters, dynamic fluctuations of the traction field became attenuated with increasing cluster size, at a rate that was faster in nonconfluent than confluent clusters. The driving mechanism of attenuation of traction field fluctuations was statistical averaging of the noise, and the impeding mechanism was nonuniform stress distribution in the clusters, which resulted from intercellular force transmission, known as a "global tug-of-war." These results show that isolated cells could not maintain tensional homeostasis, which confirms previous findings, and that tensional homeostasis is a multicellular phenomenon, which is a novel finding.