Determination of Hierarchical Relationship of Src and Rac at Subcellular Locations With FRET Biosensors

Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14353-8. doi: 10.1073/pnas.0807537105. Epub 2008 Sep 17.

Abstract

Genetically encoded biosensors based on FRET have enabled the visualization of signaling events in live cells with high spatiotemporal resolution. However, the limited sensitivity of these biosensors has hindered their broad application in biological studies. We have paired enhanced CFP (ECFP) with YPet, a variant of YFP. This ECFP/YPet FRET pair markedly enhanced the sensitivity of biosensors (several folds enhancement without the need of tailored optimization for each individual biosensor) for a variety of signaling molecules, including tyrosine kinase Src, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. The application of these improved biosensors revealed that the activations of Src and Rac by PDGF displayed distinct subcellular patterns during directional cell migration on micropatterned surface. The activity of Rac is highly polarized and concentrated at the leading edge, whereas Src activity is relatively uniform. These FRET biosensors also led to the discovery that Src and Rac mutually regulate each other. Our findings indicate that molecules within the same signaling feedback loop can be differentially regulated at different subcellular locations. In summary, ECFP/YPet may serve as a general FRET pair for the development of highly sensitive biosensors to allow the determination of molecular hierarchies at subcellular locations in live cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques*
  • Cell Line
  • Fibroblasts / cytology
  • Fluorescence Resonance Energy Transfer*
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Intracellular Space / enzymology*
  • Luminescent Proteins / metabolism*
  • Mice
  • Platelet-Derived Growth Factor / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Sensitivity and Specificity
  • Vascular Endothelial Growth Factor A / metabolism
  • src-Family Kinases / metabolism*

Substances

  • Cyan Fluorescent Protein
  • Luminescent Proteins
  • Platelet-Derived Growth Factor
  • Vascular Endothelial Growth Factor A
  • Green Fluorescent Proteins
  • src-Family Kinases
  • Proto-Oncogene Proteins c-akt