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Biosensors - Activities

Objectives

  • to generate and validate new biosensors.
  • to develop novel strategies for probing phosphorylation events in complex cellular pathways including environment sensitive dyes, and chelation enhanced fluorophores.
  • to generate and foster the production of activation specific antibodies.
  • to develop signaling platforms for high throughput analyses of activation.
  • to support the use of these technologies and assist in the development of new probes.

Assay & Probe Development | Technology Development | Other Migration-related Biosensors

Assay & Probe Development

Target Probe Type Status / Publication Contact (Collaborators)
Abl - (SH2 domain) Environment sensitive fluorophore Completed - Vázquez et al., 2005 (CMC Update Article) Imperiali
Abl kinase Chelation-enhanced fluorophore Completed - Shults and Imperiali, 2003 (CMC Update Article) Imperiali
Akt / PKA Chelation-enhanced fluorophore Completed - assay of pure enzymes and cell lysates, live cell phase in progress Imperiali (Jacobson)
Arf FRET - dual chain Completed - Hall et al., 2008 (CMC Update Article) Schwartz (Sligar, Casanova)
Cdc42 Affinity reagent for endogenous Cdc42 - (environmentally sensitive dye) Completed - Nalbant et al., 2004 (CMC Update Article); Tan et al., 2004; Hodgson et al., 2006 (CMC Update Article); El-Sibai et al., 2007 (CMC Update Article); Frantz et al., 2007 (CMC Update Article) Hahn
Cdc42 Affinity reagent for endogenous Cdc42 - (genetically encode FRET) Completed - El-Sibai et al., 2008 Hahn
Cdc42 FRET - dual chain Completed - Tzima et al., 2003 Schwartz
Erk Chelation-enhanced fluorophore Completed - in vitro semi-synthesis of ERK chemosensor, which integrates the Ets docking domain and demonstrates excellent activity with ERK. Imperiali
Erk Chelation-enhanced fluorophore Completed - selectivity studies of chemosensor in unfractionated cell lysates in three different cell lines (HeLa, NIH3T3 and HT29). Imperiali
Erk Chelation-enhanced fluorophore - (Test SOX-based Erk assay in living cells) Completed - collaborative studies investigating utility of ERK activity in live cells. Imperiali (Schwartz and Danuser)
Erk - (Erk2) Affinity reagent for endogenous Erk2 - (genetically encode FRET or environmentally sensitive dye) Completed - Hodgson et al., 2008 (CMC Update Article); Macnevin et al., 2013 (CMC Update Article) Hahn
FAK FRET - single chain Completed - Cai et al., 2008 (CMC Update Article) Hahn
Glycoprotein 120 - (GP120) Affinity reagent for endogenous GP120 - (environmentally sensitive dye) Completed - Macnevin et al., 2013 (CMC Update Article) Hahn
MLC Environment sensitive fluorophore - (Make protein for caged myosin light chain (MLC) by in vitro semi-synthesis) Completed - Goguen et al., 2011 (CMC Update Article) Imperiali (Chisholm, Horwitz, Jacobson, Schwartz)
PAK Generically encoded caged PAK inhibitor Completed Hahn
paxillin - (peptide) Environment sensitive fluorophore Completed - TEE (DANA) PpYSYPTGN and EEE(DANA)VpYSFPN - DANA based fluorophores completed (used as basis for design of Pax Tyr31 and Tyr118, respectively) Vogel and Imperiali, 2007 (CMC Update Article) Imperiali
paxillin - (Tyr31) semisynthetic mutants Completed - Vogel and Imperiali, 2007 (CMC Update Article) Imperiali
paxillin - (Tyr31) semisynthetic mutants Completed Imperiali
paxillin - (Tyr31) Environment sensitive fluorophore Completed Imperiali
Peptide - (any) FRET - single chain Completed - Bark and Hahn, 2000 Hahn
PKA Chelation-enhanced fluorophore Completed - Shults and Imperiali, 2003 (CMC Update Article); Shults et al., 2005 (CMC Update Articles: 1, 2) Imperiali
Prkc - (PKC) Chelation-enhanced fluorophore Completed - Shults and Imperiali, 2003 (CMC Update Article); Shults et al., 2005 (CMC Update Articles: 1, 2) Imperiali
Protein - (any) Environment sensitive fluorophore Completed - Macnevin et al., 2013 (CMC Update Article) Hahn
rac FRET - dual chain Completed - Chamberlain et al., 2000; Kraynov et al., 2000; Gardiner et al., 2002 Hahn
rac Generically encoded caged Rac Completed - Wang et al., 2010 Hahn
rac - (Rac1) three-segment ligation and two-segment - (Make Rac by in vitro semi-synthesis for caged Rho GTPase biosensor) Completed Imperiali
rac - (Rac1) three-segment ligation - (Make Rac amino acids for caged Rho GTPase biosensor) Completed Imperiali (Schwartz)
rac - (Rac1) FRET - single chain Completed Hahn
rho - (RhoA) FRET - single chain - (design for intact GDI regulation) Completed - Pertz et al., 2006 (CMC Update Article); Van Keymeulen et al., 2006; Wong et al., 2006; Birkenfeld et al., 2007 (CMC Update Article) Hahn
S100A4 - (Metastasis factor) environmentally sensitive fluorophore Completed - Garrett et al., 2008 Hahn
Src Affinity reagent for endogenous Src - (genetically encode FRET) Completed Hahn
Src Chelation-enhanced fluorophore Completed - Sox based chelation-enhanced fluorophore Imperiali
Src Affinity reagent for endogenous Src - (environmentally sensitive dye) Completed - Gulyani et al., 2011 (CMC Update Article) Hahn
STK11 - (LKB-1) FRET - dual chain Completed - Sebbagh et al., 2009 Schwartz
Tension Mechanical tension sensor - (elastic domain based sensors) Completed - Vinculin tension sensors have been generated using the elastic domain based sensor modules. Initial in vivo imaging results are promising. Grashoff et al., 2010 (CMC Update Article) Schwartz (Sligar)
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Technology Development

Technology Developments Component(s) Status / Publication Contact (Collaborators)
High Throughput/High Content Microscopy Equipment / Software Completed - Price et al., 2002; Shen et al., 2006; Shen et al., 2006; Shen et al., 2008 (CMC Update Article) Hahn
Incorporation of environment sensitive fluorophores into peptides Environment sensitive fluorophones 6-DMN and DAPA Completed - 3 articles describing the synthesis and peptide incorporation of two new environment sensitive fluorophores are published. Procedures are extensively detailed and enable the synthesis of any peptide incorporating the new fluorophores Sainlos and Imperiali, 2007 (CMC Update Article); Sainlos and Imperiali, 2007 (CMC Update Article); Sainlos and Imperiali, 2007 (CMC Update Article) Imperiali
Living Cell Dyes Chemistry Completed - Toutchkine et al., 2002; Toutchkine et al., 2003; Toutchkine et al., 2007 (CMC Update Article); Toutchkine et al., 2007 (CMC Update Article); Toutchkine et al., 2007 (CMC Update Article) Hahn
Phosphorylation Signaling Chip Antibody Pairs/Microarray Format Completed - available with Biosource International Parsons (Geiger, Lauffenburger, Schaefer)
Sox-based kinase assays for a wide range of kinases Chelation-enhanced fluorophore SOX Completed - Biosource/Invitrogen now carries sox-based kinase assays (OMNIA) for a wide range of kinases and custon assays can be developed to suit customer needs Shults et al., 2006, examples of this assay in use: Papp et al., 2007, Ahn et al., 2007 Imperiali
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Incomplete List of Other Migration-related Biosensors

Class Target Type Publications
Adhesion Vinculin Intramolecular FRET between FPs - (Insertion of a FRET pair in vinculin) Chen et al., 2005
Adhesion α4 Intermolecular FRET between fluorescent dyes - (LDV-FITC; octadecyl rhodamine B) Chigaev et al., 2003
Adhesion αIIb, β3 Intermolecular FRET between fluorescent dyes - (Dye-Fab fragments of two different antibodies) Coutinho et al., 2007
Adhesion αL, β2 Intermolecular FRET between FPs - (αL-mCFP; αL-mYFP) Kim et al., 2003, Kim and Lee, 2004
Cell cycle Cdt1 / Geminin Probe redistribution - (FP-tagged Cdt1 and Geminin) Sakaue-Sawano et al., 2008
Cytoskeletal Myosin II Intermolecular FRET between fluorescent dyes - (Dye-RLC and -HC of Myosin II) Post et al., 1994, Post et al., 1995
Cytoskeletal N-WASP Intramolecular FRET between FPs - (YFP- N-WASP-CFP (Stinger) or CFP- N-WASP-YFP) Lorenz et al., 2004, Ward et al., 2004
Cytoskeletal Profilin Probe redistribution - (Dye-actin with increased affinity to profilin) Giuliano and Taylor, 1994
GTPase RhoA Intramolecular FRET between FPs - (YFP-RBD (PKN)-RhoA-CFP-Farnesyl moiety (Raichu)) Yoshizaki et al., 2003
GTPases Arf6 Intermolecular FRET between FPs - (Arf6-CFP; YFP-GGA3 effector domain) Hall et al., 2008
GTPases Cdc42 Environment-sensing fluorescent dye - (Dye-CBD from WASP (MeroCBD)) Nalbant et al., 2004
GTPases Cdc42 Intermolecular FRET between FP and fluorescent dye - (GFP-Cdc42; Alexa-PBD) Tzima et al., 2003
GTPases Cdc42 / Rac Intramolecular FRET between FPs - (EBFP-PBD of PAK-EGFP) Graham et al., 2001
GTPases heterotrimeric G-proteins Intermolecular FRET between FPs - (alpha-CFP; beta-YFP) Janetopoulos et al., 2001
GTPases Rac1 Intermolecular FRET between FP and fluorescent dye - (GFP-Rac1; Alexa546-PBD from PAK (FLAIR)) Kraynov et al., 2000
GTPases Rac1 / Cdc42 Intramolecular FRET between FPs - (YFP-PBD (PAK)-Rac1/Cdc42-CFP-Farnesyl moiety (Raichu)) Itoh et al., 2002
GTPases Ran Intramolecular FRET between FPs - (YFP-RBD (Yrb1)-CFP; YFP-IBB (importin alpha)-CFP) Kalab et al., 2002
GTPases Ras / Rap1 Intramolecular FRET between FPs - (YFP-Ras/Rap1-RBD (Raf)-CFP-Farnesyl moiety (Raichu)) Mochizuki et al., 2001
GTPases RhoA Intramolecular FRET between FPs - (RBD (rhotekin)-CFP-YFP-RhoA) Pertz et al., 2006
Ions Calcium Intramolecular FRET between FPs - (CFP-CaM-CaMBP(CKK)-YFP) Truong et al., 2001
Ions Calcium circular permutation - (EGFP with EF-hand insertion) Zou et al., 2007
Ions Calcium Circular permutation - (CaM inserted in EYFP) Baird et al., 1999
Ions Calcium Intramolecular FRET between FPs - (CFP-CaM-M13-YFP) Miyawaki et al., 1997
Ions Calcium Circular permutation of FP fusion - (M13-cpEGFP-CaM) Nakai et al., 2001
Ions Calcium Intramolecular FRET between FPs - (CFP-Troponin C-YFP) Heim and Griesbeck, 2004
Kinases Akt - PKB Intramolecular FRET between FPs - (CFP-14-3-3-substrate-YFP) Sasaki et al., 2003
Kinases Akt - PKB Intramolecular FRET between FPs - (CFP-FHA2-Akt substrate-YFP) Kunkel et al., 2005
Kinases CaMKII Intramolecular FRET between FPs - (YFP-CaMKII-CFP) Takao et al., 2005
Kinases EGFR Intermolecular FRET using FLIM - (GFP-EGFR; dye-pTyr antibody) Wouters and Bastiaens, 1999
Kinases Erk Intramolecular FRET between FPs - (CFP-Erk-YFP) Sato et al., 2007
Kinases FAK Intermolecular FRET between FPs - (CFP-FAK; dSH2 (Src)-YFP) Cai et al., 2008
Kinases FAK Intramolecular FRET between FPs - (Insertion of a FRET pair in FAK) Cai et al., 2008
Kinases MK2 Intramolecular FRET between FPs - (CFP-MK2-YFP) Neininger et al., 2001
Kinases MLCK Intramolecular FRET between FPs - (MLCK-GFP-CaMBD-BFP (MLCK-FIP)) Chew et al., 2002
Kinases phospho-CrkII Intramolecular FRET between FPs - (CFP-CrkII-YFP) Kurokawa et al., 2001
Kinases PKA Intramolecular FRET between FPs - (CFP-KID of CREB-YFP) Nagai et al., 2000
Kinases PKA Intramolecular FRET between FPs - (CFP-14-3-3tau-PKA substrate-YFP) Zhang et al., 2001
Kinases PKC Environment-sensing fluorescent dye - (Caged dye-substrate) Dai et al., 2007
Kinases PKC Intermolecular FRET using FLIM - (FP-PKC; dye-phospho antibody) Ng et al., 1999
Kinases PKC Intramolecular FRET between FPs - (CFP-FHA2-PKC substrate-YFP) Violin et al., 2003
Kinases PKD Intramolecular FRET between FPs - (CFP-FHA2-PKD substrate-YFP) Kunkel et al., 2007
Kinases PTK Environment-sensing fluorescent dye - (caged dye-substrate) Wang et al., 2006
Kinases pTyr Probe redistribution - (YFP-dSH2 (Src)) Kirchner et al., 2003
Kinases pTyr Intramolecular FRET between FPs - (CFP-SH2-Tyr substrate-YFP) Ting et al., 2001
Kinases Src Intramolecular FRET between FPs - (CFP-SH2-Tyr substrate-YFP) Ting et al., 2001, Wang et al., 2005
Membrane Lipid rafts Intermolecular FRET between FPs - (MyrPalm/GerGer/PalmPalm/Caveolin-mCFP/mYFP) Zacharias et al., 2002
Membrane PI3P Probe redistribution - (GFP-RING FYVE domains) Burd and Emr, 1998
Membrane PIP - (4) Probe redistribution - (GFP-PH of OSBP) Levine and Munro, 2002
Membrane PIP2 - (4,5) Probe redistribution - (GFP-PH of PLCdelta1) Stauffer et al., 1998, Várnai and Balla, 1998
Membrane PIP3 - (3,4,5) Probe redistribution - (PH (ARNO or GRP1)-GFP) Oatey et al., 1999
Membrane PIP3 - (3,4,5) Probe redistribution - (PH (BtK)-GFP) Várnai et al., 1999
Membrane PIP3 - (3,4,5) Intramolecular FRET between FPs - (YFP-binding domain of IP3R1-CFP) Matsu-ura et al., 2006
Membrane PIP3 / PIP2 Probe redistribution - (PH (PKB or GRP1)-GFP) Gray et al., 1999
Others Calmodulin Environment-sensing fluorescent dye - (Dye-Calmodulin (MeroCaM)) Hahn et al., 1990, Hahn et al., 1992
Others S100A4 Environment-sensing fluorescent dye - (Mero-S100A4) Garrett et al., 2008
Others Voltage Intramolecular FRET between FPs - (CFP-YFP-Ci-VSP) Dimitrov et al., 2007
Proteases Calpain Intramolecular FRET between FPs - (YFP-Calpain cleavage site (alpha-spectrin)-CFP) Vanderklish et al., 2000
Proteases Caspase-3 Intramolecular FRET between FPs - (CFP-Caspase-3 substrate-Venus (improvement)) Takemoto et al., 2003
Proteases Caspase-3 Intramolecular FRET between FPs - (GFP-Caspase-3 substrate-BFP) Xu et al., 1998
Small molecules cAMP Intermolecular FRET between FPs - (PKA-GFP; PKA RII-EBFP) Zaccolo et al., 2000
Small molecules cAMP Intramolecular FRET between FPs - (CFP-Epac1-YFP) DiPilato et al., 2004
Small molecules cGMP Intramolecular FRET between FPs - (CFP-cGPK-YFP) Honda et al., 2001
Small molecules glutamate Intramolecular FRET between FPs - (CFP-ybeJ-YFP) Okumoto et al., 2005
Small molecules maltose Intramolecular FRET between FPs - (CFP-MBP-YFP) Fehr et al., 2002
Small molecules phosphate Intramolecular FRET between FPs - (CFP-PiBP-YFP) Gu et al., 2006
Small molecules Tryptophan Intramolecular FRET between FPs - (CFP-TrpR-YFP) Kaper et al., 2007
Transcription factors bZIP and Rel BiFC - (bZIP and Rel-fragments of YFP) Hu et al., 2002
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