Real-time detection of α1A-AR movement stimulated by phenylephrine in single living cells
AbstractAim:To investigate the movement of α1A-adrenergic receptors(α1A-AR) stimulated by agonist, phenylephrine (PE), and the dynamics of receptor movement in real time in single living cells with millisecond resolution.Methods:We labeled α1A-AR using the monoclonal, anti-FLAG (a kind of tag) antibody and Cy3-conjugated goat anti-mouse IgG and recorded the trajectory of their transport process in living HEK293A cells stimulated by agonist, PE, and then analyzed their dynamic properties.Results:The specific detection of α1A-AR on the surface of living HEK293A-α1A cells was achieved. α1A-AR internalize under the stimulation of PE. After the cells were stimulated with PE for 20 min, apparent colocalization was found between α1A-AR and F-actins. After 40 min stimulation of PE, trajectories of approximate linear motion in HEK293A-α1A cells were recorded, and their velocity was calculated.Conclusion:The specific labeling method on the living cell surface provides a convenient means of real-time detection of the behavior of surface receptors. By this method we were able to specifically detect α1A-AR and record the behavior of individual particles of receptors with 50 ms exposure time in real time in single living cells.