Src kinase, the first tyrosine kinase discovered, has been shown to play critical roles in a variety of cellular processes,
including cell motility/migration, mechanotranduction, and cancer development. Based on fluorescent resonance energy
transfer (FRET), we have developed and characterized a genetically encoded single-molecule Src biosensor, which
enables the imaging and quantification of temporal-spatial activation of Src in live cells. In this paper, we summarize the
application of this biosensor to study a variety of cellular functions. First, we introduced a local mechanical stimulation
by applying laser-tweezer-induced traction on fibronectin-coated beads adhered to the cells. Using a membrane-anchored
Src biosensor, we observed a wave propagation of Src activation in a direction opposite to the applied force. This Src
reporter was also applied to visualize the interplays between cell-cell and cell-ECM adhesions. The results indicate that
integrin-ligation can induce Src activation around cell-cell junctions and cause the disruption of adherens junctions.
Lastly, the flow-induced dynamic Src activation at subcellular levels was visualized by the FRET biosensor
simultaneously with actin-fused mCherry, a red fluorescence protein. Our results indicate that shear stress induced a
moderate up-regulation of Src activation in the whole cell, but a significant translocation of active Src from perinuclear
regions toward cell periphery. In summary, our novel Src biosensor has made it possible to monitor key signaling
transduction cascades involving Src in live cells with temporal-spatial characterization in mechanobiology.