Molecular Engineering and Live Cell Imaging for Studying Cell-Environment Interactions

Abstract:

Signaling molecules and their activities are well coordinated inspace and time to regulate cellular functions in response to mechanicaland chemical microenvironment. Based on fluorescent resonance energytransfer (FRET), we have developed several genetically encodedbiosensors for detecting the spatiotemporal activities of signalingmolecules, including Src, Rac1, MT1-MMP, and Calcium. A Rac biosensorrevealed that the Rac activity in cells constrained on micropatternedextracellular-matrix surface is polarized with higher activityconcentrated at the leading edge of migrating cells upon PDGFstimulation, whereas Src activities in these cells displayed globalactivation patterns without obvious polarity. Our calcium biosensoralso allowed the revelation that there is a spontaneous Ca2+oscillation in human mesenchymal stem cells (HMSCs) both inside thecytoplasm and endoplasmic reticulum (ER). The substrate stiffness whereHMSCs are seeded can significantly affect this Ca2+ oscillation, in afashion dependent on the RhoA signaling pathway. We have furtherdeveloped a FAK FRET biosensor and targeted it into lipid rafts ornon-rafts of plasma membrane by lipid modifications. Upon cell adhesionon extracellular matrix proteins or stimulation by platelet-derivedgrowth factor, the raft-targeting FAK biosensor showed a surprisinglystronger FRET response than that at non-rafts, suggesting that the FAKactivation mainly occurs at lipid rafts. Further experiments revealedthat the PDGF-induced FAK activation at rafts is mediated by the kinaseactivity of Src, whereas FAK activation induced by adhesion isindependent of, and in fact essential for the Src activation. Theseresults suggest that FAK is activated at rafts with distinct activationmechanisms in response to different physiological stimuli. In summary,our novel FRET biosensors in combination with tools innano-biotechnology and bio-photonics have made it possible to monitorkey signaling cascades in live cells with subcellular and dynamiccharacterization when cells interact with their physical/chemicalmicroenvironment.