Sustained CA2+ mobilizations: a quantitative approach to predict their importance in cell-cell communication
Lee, Yoonjoo Katherine
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Epithelial wound healing requires the coordination of cells to migrate as a unit over the basement membrane after injury. An excellent model tissue is the corneal epithelium, which is an avascular stratified squamous tissue that responds to growth factors and nucleotides when the epithelial barrier is damaged. One signal that has a ubiquitous response in epithelial wound healing is the cellular release of the nucleotide ATP, which may occur because of mechanics forces and/or change in cell shape. Within milliseconds to seconds after injury, extracellular ATP binds to purinoreceptors and triggers a transient Ca2+ wave, which is used by cells to transduce mechanical signals into chemical signals and alter signaling pathways. To understand the process of this coordinated movement, it is critical to study the dynamics of cell-cell communication. In this study we developed a novel method to identify and characterize the degree of cell-cell communication that occurs through sustained Ca2+ mobilizations after injury, which are concentrated along the epithelial wound edge and reduced in cells distal to the injury. Using MATLAB analyses, we generated profiles of the sustained Ca2+ mobilizations, and demonstrated that the Ca2+ response was replicated in ex vivo organ culture models. The sustained Ca2+ mobilizations were present also after stimulation with either BzATP or UTP, which are agonists of P2X7 and P2Y2 respectively. The probability that cells would communicate was greater in response to BzATP compared to UTP. The specificity of these ligands was demonstrated using competitive inhibitors of P2Y2 and P2X7 receptors, AR-C 118925XX and A438079, respectively. An inhibitor of pannexin-1, 10Panx, attenuated both wound closure and BzATP agonist-initiated response. These sustained mobilizations are correlated with changes in cellular morphology and motility, which were prominent in cells at the leading edge of the wound during cell migration. Together, our results demonstrate that the sustained Ca2+ mobilizations mediated by purinoreceptors and pannexins are a vital component in regulating the long-term response to injury, as studied in organ culture.
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