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Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) enables
visualization of signaling events in live cells with high spatiotemporal resolution. We have used
FRET to assess temporal and spatial characteristics for signaling molecules, including tyrosine
kinases Src and FAK, small GTPase Rac, calcium, and a membrane-bound matrix
metalloproteinase MT1-MMP. Activations of Src and Rac by platelet derived growth factor
(PDGF) led to distinct subcellular patterns during cell migration on micropatterned surface, and
these two enzymes interact with each other to form a feedback loop with differential regulations
at different subcellular locations. We have developed FRET biosensors to monitor FAK
activities at rafts vs. non-raft regions of plasma membrane in live cells. In response to cell
adhesion on matrix proteins or stimulation by PDGF, the raft-targeting FAK biosensor showed a
stronger FRET response than that at non-rafts. The FAK activation at rafts induced by PDGF is
mediated by Src. In contrast, the FAK activation at rafts induced by adhesion is independent of
Src activity, but rather is essential for Src activation. Thus, Src is upstream to FAK in response to
chemical stimulation (PDGF), but FAK is upstream to Src in response to mechanical stimulation
(adhesion). A novel biosensor has been developed to dynamically visualize the activity of
membrane type-1-matrix metalloproteinase (MT1-MMP), which proteolytically remodels the
extracellular matrix. Epidermal growth factor (EGF) directed active MT1-MMP to the leading
edge of migrating live cancer cells with local accumulation of EGF receptor via a process
dependent on an intact cytoskeletal network. In summary, FRET-based biosensors enable the
elucidation of molecular processes and hierarchies underlying spatiotemporal regulation of
biological and pathological processes, thus advancing our knowledge on how cells perceive
mechanical/chemical cues in space and time to coordinate molecular/cellular functions.
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