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Proceedings Paper

Molecular signaling in live cells studied by FRET
Author(s): Shu Chien; Yingxiao Wang
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Paper Abstract

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) enables visualization of signaling events in live cells with high spatiotemporal resolution. We have used FRET to assess temporal and spatial characteristics for signaling molecules, including tyrosine kinases Src and FAK, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. Activations of Src and Rac by platelet derived growth factor (PDGF) led to distinct subcellular patterns during cell migration on micropatterned surface, and these two enzymes interact with each other to form a feedback loop with differential regulations at different subcellular locations. We have developed FRET biosensors to monitor FAK activities at rafts vs. non-raft regions of plasma membrane in live cells. In response to cell adhesion on matrix proteins or stimulation by PDGF, the raft-targeting FAK biosensor showed a stronger FRET response than that at non-rafts. The FAK activation at rafts induced by PDGF is mediated by Src. In contrast, the FAK activation at rafts induced by adhesion is independent of Src activity, but rather is essential for Src activation. Thus, Src is upstream to FAK in response to chemical stimulation (PDGF), but FAK is upstream to Src in response to mechanical stimulation (adhesion). A novel biosensor has been developed to dynamically visualize the activity of membrane type-1-matrix metalloproteinase (MT1-MMP), which proteolytically remodels the extracellular matrix. Epidermal growth factor (EGF) directed active MT1-MMP to the leading edge of migrating live cancer cells with local accumulation of EGF receptor via a process dependent on an intact cytoskeletal network. In summary, FRET-based biosensors enable the elucidation of molecular processes and hierarchies underlying spatiotemporal regulation of biological and pathological processes, thus advancing our knowledge on how cells perceive mechanical/chemical cues in space and time to coordinate molecular/cellular functions.

Paper Details

Date Published: 13 March 2012
PDF: 6 pages
Proc. SPIE 8329, Tenth International Conference on Photonics and Imaging in Biology and Medicine (PIBM 2011), 832902 (13 March 2012); doi: 10.1117/12.919126
Show Author Affiliations
Shu Chien, Univ. of California, San Diego (United States)
Yingxiao Wang, Univ. of Illinois at Urbana-Champaign (United States)

Published in SPIE Proceedings Vol. 8329:
Tenth International Conference on Photonics and Imaging in Biology and Medicine (PIBM 2011)
Qingming Luo; Lihong V. Wang; Valery V. Tuchin, Editor(s)

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