Metastasis is the main cause of death in cancer patients. While multiple steps are involved in metastasis in different types of cancer, the degradation of basal membrane is considered to constitute the crucial step. Recent evidence indicates that both Src kinase and a membrane-anchored metalloproteinase, MT1-MMP, play important roles in cancer invasion and metastasis. While it is clear that cells coordinate the subcellular localization of signaling molecules, it remains unclear as to the subcellular molecular hierarchy regulating cancer invasion. Fluorescence resonance energy transfer (FRET) technology coupled with genetically encoded biosensors provide powerful tools for visualizing active molecular events with high spatiotemporal resolutions in live cells. Utilizing a high- efficiency FRET pair ECFP and YPet, we have developed two FRET biosensors capable of detecting the spatiotemporal Src and MT1-MMP activities in live cells. However, only one type of molecule can be visualized in the same live cells utilizing the ECFP/YPet- based biosensors. Recently, two fluorescence proteins mOrange2 and mCherry were shown to be spectrally distinguishable from ECFP and YPet. Hence, mOrange2 and mCherry may serve as the donor and acceptor for a second FRET pair. However, there is a substantial overlap between the excitation spectra of mOrang2 and mCherry. In this proposal, we will apply and integrate a real time frequency-domain fluorescence lifetime microscopy (FLIM) technology with our novel FRET biosensors to eliminate the crosstalk between fluorescence proteins. Two specific aims are proposed here: (1) Apply FLIM to characterize and visualize the ECFP/YPet-based Src biosensor and mOrange2/ mCherry - based MT1-MMP biosensor in vitro and in live mammalian cells. (2) Utilize FLIM to visualize the spatiotemporal activities of Src and MT1-MMP in a simultaneous fashion within live cancer cells and characterize the regulatory interplay between these activities during invasion. The success of this proposal will provide new insights into the molecular mechanism regulating the invasive properties of cancer cells. With the integration of advanced optical systems such as two photon fluorescence microscope, further studies will allow us to apply these biosensors in vivo. The newly developed biosensors will also provide functional readouts for detecting changes in cancer cell motility/invasion as well as the efficacy of therapeutic inhibitors. PUBLIC HEALTH RELEVANCE: It is of great importance to visualize multiple types of active molecular events in live cells during cancer cell invasion. However, only one type of molecular event can be visualized in the same cell in most cases at the current stage. This proposal will integrate novel FRET biosensors and FLIM to allow a visualization of two molecular events simultaneous in the same live cell during cancer invasion. The results should shed new light and advance our systematic understanding of the molecular mechanism involved in cancer invasion, and provide a rational basis for the diagnostic analysis and therapeutic treatment for cancer diseases.