| | "Molecular Engineering and Live Cell Imaging" | |
|
|
| |
| |
| 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, Rac1, MT1-MMP, and Calcium. 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. A Rac biosensor further revealed that the Rac activity in cells constrained on micropatterned extracellular-matrix surface is polarized with higher activity concentrated at the leading edge of migrating cells upon PDGF stimulation, whereas Src activities in these cells displayed global activation patterns without obvious polarity. 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 an intact cytoskeletal network. Most recently, our calcium biosensor revealed that there is a spontaneous Ca2+ oscillation in human mesenchymal stem cells (HMSCs) both inside the cytoplasm and endoplasmic reticulum (ER). The substrate stiffness where HMSCs are cultured can significantly affect this Ca2+ oscillation, in a fashion dependent on the RhoA signaling pathway. 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. |
| |
| |
|
|
