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Keynote Address:
Seeing is Believing: Molecular Imaging, NanoBiotechnology, and MechanoBiology in Live Cells Yingxiao Wang University of Illinois at Urbana-Champaign |
| Signaling molecules and their activities are well coordinated in space and time to regulate cellular functions in response to mechanical and chemical microenvironment. Based on fluorescent resonance energy transfer (FRET), we have developed several genetically encoded biosensors for detecting the spatiotemporal activities of signaling molecules, including Src, MT1-MMP, Rac, and FAK. With a Src biosensor, local mechanical stimulation induced by laser-tweezer-traction can be observed to cause a directional wave propagation of Src activation along the plasma membrane. Different Src activities can also be observed at different subcompartments when the Src biosensor is tethered on plasma membrane in or outside of lipid raft. Src activities in cells constrained to grow on micropatterned extracellular matrix surface displayed distinctive dynamic patterns comparing to those on none-patterned surface. With a MT1-MMP biosensor, epidermal growth factor (EGF) can be observed to induce significant FRET changes in live cancer cells expressing MT1-MMP, but not in MT1-MMP-deficient cells. Active MT1-MMP was directed to the leading edge of migrating cells along micropatterned fibronectin stripes, via a process dependent upon Rac and an intact cytoskeletal network. A Rac biosensor further revealed that the Rac activity is polarized with higher activity concentrated at the leading edge of migrating cells. With a FAK biosensor, a hot zone with high FAK activities at the base of lamellipodium in spreading cells can be observed, suggesting an important role of focal adhesion turnover in cell motility. In summary, our novel FRET biosensors in combination with tools in nanobiotechnology and biophotonics have made it possible to monitor key signaling cascades in live cells with spatiotemporal characterization and to elucidate the underlying molecular mechanisms in Mechanobiology. |