In recent years reactive oxygen species (ROS) and by extension changes in the intracellular reductive/oxidative (redox) balance have come into focus as major regulators of key cellular functions in both physiological and pathological settings. Traditionally viewed as mediators of cell damage by exogenous noxae, oxygen intermediates have been also recognized of signaling roles downstream of cytokine and mitogen receptors, activated oncogenes, nutrient sensors and pro-apoptotic stimuli, when endogenously generated by a number of intracellular biochemical sources. The signaling properties of ROS are largely due to the reversible oxidation of redox-sensitive target proteins, and especially of protein tyrosine phosphatases, whose activity is dependent on the redox state of a low pKa active site cysteine. Cell spreading, adhesion and migration requires ROS generation and interaction with protein tyrosine phosphatases downstream of adhesion molecules. We have taken advantage of a redox-sensitive mutant of the Yellow Fluorescent protein (rxYFP), employed ratiometrically, to draw real-time redox maps of adhering and migrating cells. A quantitative analysis of redox maps allows to disclose a peculiar spatial organization of the redox environment, providing evidence that intracellular ROS are generated after integrin engagement and that these oxidant intermediates are necessary for integrin signaling during cell spreading, adhesion and migration. Taken together these observation support the application of rxYFP in the subcellular mapping of physiological dynamic redox phenomena involved in signal transduction.
- CELL ADHESION