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There is now a CONTENT FREEZE for Mercury while we switch to a new platform. It began on Friday, March 10 at 6pm and will end on Wednesday, March 15 at noon. No new content can be created during this time, but all material in the system as of the beginning of the freeze will be migrated to the new platform, including users and groups. Functionally the new site is identical to the old one. webteam@gatech.edu
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Abstract:
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. A Rac biosensor 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. Our calcium biosensor also allowed the revelation 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 seeded can significantly affect this Ca2+ oscillation, in a fashion dependent on the RhoA signaling pathway. We have further developed a FAK FRET biosensor and targeted it into lipid rafts or non-rafts of plasma membrane by lipid modifications. Upon cell adhesion on extracellular matrix proteins or stimulation by platelet-derived growth factor, the raft-targeting FAK biosensor showed a surprisingly stronger FRET response than that at non-rafts, suggesting that the FAK activation mainly occurs at lipid rafts. Further experiments revealed that the PDGF-induced FAK activation at rafts is mediated by the kinase activity of Src, whereas FAK activation induced by adhesion is independent of, and in fact essential for the Src activation. These results suggest that FAK is activated at rafts with distinct activation mechanisms in response to different physiological stimuli. In summary, our novel FRET biosensors in combination with tools in nano-biotechnology and bio-photonics have made it possible to monitor key signaling cascades in live cells with subcellular and dynamic characterization when cells interact with their physical/chemical microenvironment.
