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.
Biosensors designed on the principle of fluorescent resonance energy transfer (FRET) have been
widely applied to visualize signaling cascades in live cells with high spatiotemporal resolution. In
this paper, we review the work in our lab related to the application of FRET biosensors in studying
molecular events in live cells, and our work using computational analysis methods to explore
complex biological information implicated in FRET images. Membrane-tethered Src biosensors
were used to visualize the dynamics of Src activity in subcellular microdomains. We have developed
a finite element (FE) method to analyze the movement of biosensors. Based on fluorescence
recovery after photobleaching (FRAP) experiments, the estimation and subtraction of biosensor
diffusion revealed a high Src activity at cell periphery upon growth factor stimulation. In addition to
Src, a RhoA biosensor was used to study the subcellular feature of RhoA activity in migrating HeLa
cells. We have developed an image registration method to automatically track and quantify the
FRET signals within user-defined subcellular regions, and classify the dynamics of subcellular
pixels according FRET signals. The results revealed that the RhoA activity is polarized in the
migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration.
Therefore, FRET biosensors integrated with computational analysis provide powerful tools to
precisely decode the complex dynamics of signaling transduction regulated in subcellular locations
of live cells.
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.
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