Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors

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.

[1]  Giulio Superti-Furga,et al.  RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane. , 2004, Developmental cell.

[2]  M. Ginsberg.  Faculty Opinions recommendation of VEGF controls endothelial-cell permeability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin. , 2006 .

[3]  P. Schwartzberg,et al.  Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability. , 1999, Molecular cell.

[4]  R. Juliano,et al.  Phorbol ester modulation of integrin-mediated cell adhesion: a postreceptor event , 1989, The Journal of cell biology.

[5]  Frits Michiels,et al.  Matrix-dependent Tiam1/Rac Signaling in Epithelial Cells Promotes Either Cell–Cell Adhesion or Cell Migration and Is Regulated by Phosphatidylinositol 3-Kinase , 1998, The Journal of cell biology.

[6]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[7]  K. Hahn,et al.  Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. , 2002, Nature Reviews Molecular Cell Biology.

[8]  Y. Takai,et al.  Interaction and localization of Necl‐5 and PDGF receptor β at the leading edges of moving NIH3T3 cells: Implications for directional cell movement , 2008, Genes to cells : devoted to molecular & cellular mechanisms.

[9]  Jing Xu,et al.  Visualization of Polarized Membrane Type 1 Matrix Metalloproteinase Activity in Live Cells by Fluorescence Resonance Energy Transfer Imaging* , 2008, Journal of Biological Chemistry.

[10]  天野 恭志.  Interaction and localization of Necl-5 and PDGF receptor β at the leading edges of moving NIH3T3 cells : Implications for directional cell movement , 2009 .

[11]  J. Cambier,et al.  Activation of phosphatidylinositol-3' kinase by Src-family kinase SH3 binding to the p85 subunit. , 1994, Science.

[12]  D. Schlaepfer,et al.  Integrin-regulated FAK-Src signaling in normal and cancer cells. , 2006, Current opinion in cell biology.

[13]  G. Martin,et al.  Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function , 2004, The Journal of cell biology.

[14]  Richard G. W. Anderson,et al.  Integrins Regulate Rac Targeting by Internalization of Membrane Domains , 2004, Science.

[15]  G. Borisy,et al.  Cell Migration: Integrating Signals from Front to Back , 2003, Science.

[16]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[17]  Elliot L. Botvinick,et al.  Visualizing the mechanical activation of Src , 2005, Nature.

[18]  R. Tsien,et al.  Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells , 2002, Science.

[19]  Kristiina Vuori,et al.  GEF what? Dock180 and related proteins help Rac to polarize cells in new ways. , 2007, Trends in cell biology.

[20]  V. Brunton,et al.  Translocation of Src kinase to the cell periphery is mediated by the actin cytoskeleton under the control of the Rho family of small G proteins , 1996, The Journal of cell biology.

[21]  K. Hahn,et al.  Localized Rac activation dynamics visualized in living cells. , 2000, Science.

[22]  K. Hahn,et al.  Spatiotemporal dynamics of RhoA activity in migrating cells , 2006, Nature.

[23]  H. Erickson,et al.  An experimental study of GFP‐based FRET, with application to intrinsically unstructured proteins , 2007, Protein science : a publication of the Protein Society.

[24]  Sheila M. Thomas,et al.  Cellular functions regulated by Src family kinases. , 1997, Annual review of cell and developmental biology.

[25]  Shu Chien,et al.  Fluorescence proteins, live-cell imaging, and mechanobiology: seeing is believing. , 2008, Annual review of biomedical engineering.

[26]  A. Miyawaki,et al.  Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Patrick S Daugherty,et al.  Evolutionary optimization of fluorescent proteins for intracellular FRET , 2005, Nature Biotechnology.

[28]  M. Mareel,et al.  Clinical, cellular, and molecular aspects of cancer invasion. , 2003, Physiological reviews.

[29]  D. Piston,et al.  Fluorescent protein FRET: the good, the bad and the ugly. , 2007, Trends in biochemical sciences.

[30]  M. Matsuda,et al.  Activation of Rac and Cdc42 Video Imaged by Fluorescent Resonance Energy Transfer-Based Single-Molecule Probes in the Membrane of Living Cells , 2002, Molecular and Cellular Biology.

[31]  C. Waterman-Storer,et al.  Conserved microtubule–actin interactions in cell movement and morphogenesis , 2003, Nature Cell Biology.

[32]  Maria Julia Marinissen,et al.  Regulation of c-myc expression by PDGF through Rho GTPases , 2001, Nature Cell Biology.

[33]  K. Vuori,et al.  A novel and evolutionarily conserved PtdIns(3,4,5)P3-binding domain is necessary for DOCK180 signalling , 2005, Nature Cell Biology.

[34]  N. Ferrara.  Role of vascular endothelial growth factor in regulation of physiological angiogenesis. , 2001, American journal of physiology. Cell physiology.

[35]  Kenneth M. Yamada,et al.  Polymerizing Actin Fibers Position Integrins Primed to Probe for Adhesion Sites , 2007, Science.

[36]  G. Bokoch,et al.  Mechanism of human dermal fibroblast migration driven by type I collagen and platelet-derived growth factor-BB. , 2003, Molecular biology of the cell.

[37]  M. White,et al.  Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. , 1998, Science.

[38]  M. Schwartz,et al.  Integrin signalling in directed cell migration , 2006, Biology of the cell.

[39]  A. Hall,et al.  Rho GTPases and the control of cell behaviour. , 2005, Biochemical Society transactions.

[40]  R. Tsien,et al.  Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin , 1997, Nature.