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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.

Collaboration and Release Policy

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

Assay & Probe Development

Target Probe Type Status / Publication Contact (Collaborators)
Abl kinase Chelation-enhanced fluorophore Data available - Shults and Imperiali 2003 Imperiali
Abl (SH2 domain) Environment sensitive fluorophore Data available - Vazquez et al., 2005 Imperiali
Arf FRET - dual chain Data available - Hall, et al., 2007. Schwartz (Sligar, Casanova)
Cdc42 Affinity reagent for endogenous Cdc42; environmentally sensitive dye Data available - Frantz et al., 2007; El-Sibai et al., 2007; Hodgson et al., 2006, Tan et al., 2004, Nalbant et al., 2004 Hahn
Cdc42 FRET - dual chain Data available - Tzima et al., 2003 Schwartz
paxillin (peptide) Environment sensitive fluorophore Data available - TEE (DANA) PpYSYPTGN and EEE(DANA)VpYSFPN - DANA based fluorophores completed (used as basis for design of Pax Tyr31 and Tyr118, respectively) see Vogel & Imperiali, 2007 Imperiali
paxillin Tyr31 semisynthetic mutants Data available - Vogel & Imperiali, 2007 Imperiali
Prkc PKC Chelation-enhanced fluorophore Data available - Shults and Imperiali 2003 , Shults et al., 2005 Imperiali
rac FRET - dual chain Data available - Gardiner et al., 2002;Kraynov et al., 2000;Chamberlain et al., 2000 Hahn
rho (RhoA) FRET - single chain design for intact GDI regulation Data available - Birkenfeld et al., 2007;Van Keymeulen et al., 2006;Wong et al., 2006;Pertz et al., 2006 Hahn
FAK FRET - single chain Data available - Cai et al., 2007 Hahn
Peptide (any) FRET - single chain Data available - Bark & Hahn, 2000 Hahn
PKA Chelation-enhanced fluorophore Data available - Shults and Imperiali 2003 , Shults et al., 2005 Imperiali
S100A4 Metastasis factor environmentally sensitive fluorophore Data available - Garrett et al., 2008 Hahn
Akt & PKA Chelation-enhanced fluorophore Completed - assay of pure enzymes and cell lysates, live cell phase in progress Imperiali (Jacobson)
Cdc42 Affinity reagent for endogenous Cdc42; genetically encode FRET In progress Hahn
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) In progress - 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 In progress Hahn
paxillin Tyr118 Environment sensitive fluorophore SUSPENDED - Intact paxillin via unnatural amino acid mutagenesis DANA & 6-DMN based environment sensitive fluorophore Imperiali
paxillin Tyr31 semisynthetic mutants In progress - Chemistry completed, mutants currently under investigation in vitro. Imperiali
paxillin Tyr31 Environment sensitive fluorophore In progress - Intact paxillin via native ligation DANA & 6-DMN based environment sensitive fluorophore Imperiali
rac Generically encoded caged Rac In progress Hahn
rac (Rac1) three-segment ligation (Make Rac amino acids for caged Rho GTPase biosensor) Completed caged asparagine synthesis and peptide incorporation. Decaging demonstrated. Residue now awaits completion of ligation feasibility studies. Imperiali (Schwartz)
rac (Rac1) FRET - single chain In progress Hahn
rac (Rac1) three-segment ligation and two-segment (Make Rac by in vitro semi-synthesis for caged Rho GTPase biosensor) In progress - two and three segment ligations are attempted. Two-segment strategy more promisiing. Expression of 42-192-residue segment completed, as is synthesis of the 1-41 segment. Currently developing ligation methodology with denatured precursors to avoid solubility problems. Imperiali
Src Chelation-enhanced fluorophore Completed - Sox based chelation-enhanced fluorophore Imperiali
Src Affinity reagent for endogenous Src; genetically encode FRET In progress Hahn
Src Affinity reagent for endogenous Src; environmentally sensitive dye In progress - screening of phage display library to find Src binder Hahn
STK11 (LKB-1) FRET - dual chain Completed - Manuscript in preparation Schwartz
Glycoprotein 120 (GP120) Affinity reagent for endogenous GP120; environmentally sensitive dye In progress Hahn
MLC Environment sensitive fluorophore Make caged MLC variants at positions 18 and 19 and test in vitro) In progress - Currently focused on the semisynthesis of variants of MLC that show a null phenotype prior to uncaging. Many variants have been prepared however, to date all show residual activity prior to uncaging. Efforts ongoing to identify ideal caged variants. Imperiali (Chisholm, Horwitz, Jacobson, Schwartz)
MLC Environment sensitive fluorophore (Make protein for caged myosin light chain (MLC) by in vitro semi-synthesis) In progress - Semisynthesis of MLC completed and sucessful with a wide variety of MLC mutants. Currently making a variant of MLC that can be used to signal dynamic interactions of MLC with protein partners and intramolecular conformational changes. Imperiali (Chisholm, Horwitz, Jacobson, Schwartz)
PAK Generically encoded caged PAK inhibitor In progress Hahn
Protein (any) Environment sensitive fluorophore Completed - publication in progress; Gremyachinskiy and Hahn, 2007. A combinatorial approach to merocyanines for live cell imaging; a toolchest of dyes for different biosensor designs. Submitted. Hahn
Tension Mechanical tension sensor(titin-based FRET sensor) Currently refining and testing vinculin sensors based on elastic domains Schwartz (Sligar, Stossel)
Tension Mechanical tension sensor (elastic domain based sensors) In progress - Elastic domain based sensors - currently expressing and purifying the elastic domain sensors and developing the surface attachment protocols for single molecule force vs. FRET measurements. Schwartz (Sligar)
Tension Mechanical tension sensor(leucine zipper based sensors) In progress - Filamin A based sensors: A re-design of the leucine zipper interface is being considered since current sensors do not display an effective dynamic range in vivo. Schwartz (Sligar, Stossel, Ha)
Tension Mechanical tension sensor(elastic domain based sensors) In progress - Vinculin tension sensors have been generated using the elastic domain based sensor modules. Initial in vivo imaging results are promising. Schwartz (Sligar)
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Technology Development

Technology Developments Component(s) Status / Publication Contact (Collaborators)
Sox-based kinase assays for a wide range of kinases Chelation-enhanced fluorophore SOX Data available - 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; Papp et al., 2007; Ahn et al., 2007 Imperiali
Incorporation of environment sensitive fluorophores into peptides Environment sensitive fluorophones 6-DMN and DAPA Data available - 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 & Imperiali, 2007; Sainlos & Imperiali, 2007; Sainlos & Imperiali, 2007. Imperiali
High Throughput/High Content Microscopy Equipment / Software Data available - Shen et al, 2008; Shen et al., 2006; Shen et al., 2006; Price et al., 2002 Hahn
Living Cell Dyes Chemistry Data available - Toutchkine et al., 2007; Toutchkine et al., 2007, CMC Update, July 2007; Toutchkine et al., 2007, CMC Update, July 2007;Toutchkine, et al., 2003; Toutchkine et al., 2002 Hahn
Phosphorylation Signaling Chip Antibody Pairs/Microarray Format Completed - available with Biosource International Parsons, Geiger, Lauffenburger (Schaefer)
Imaging of multiple phosphorlated adhesion proteins Direct and indirect labeling of phospho-specific antibodies. In progress Parsons
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Incomplete List of Other Migration-related Biosensors

Class Target Probe Type Probe Design Reference
Adhesion Vinculin Intramolecular FRET between FPs Insertion of a FRET pair in vinculin Chen, H. et al, 2005
Adhesion α4 Intermolecular FRET between fluorescent dyes LDV-FITC; octadecyl rhodamine B Chigaev, A. et al, 2003
Adhesion αIIb / β3 Intermolecular FRET between fluorescent dyes Dye-Fab fragments of two different antibodies Coutinho, A. et al, 2007
Adhesion αL / β2 Intermolecular FRET between FPs αL-mCFP; αL-mYFP Kim, M. et al, 2004, Kim, M. et al, 2003
Cell cycle Cdt1 and Geminin Probe redistribution FP-tagged Cdt1 and Geminin Sakaue-Sawano, A. et al, 2008
Cytoskeletal Myosin II Intermolecular FRET between fluorescent dyes Dye-RLC and -HC of Myosin II Post, P. L. et al, 1994, Post, P. L. et al, 1995
Cytoskeletal N-WASP Intramolecular FRET between FPs YFP- N-WASP-CFP (Stinger) or CFP- N-WASP-YFP Ward, M. E. et al, 2004, Lorenz, M. et al, 2004
Cytoskeletal Profilin Probe redistribution Dye-actin with increased affinity to profilin Giuliano, K. A. and D. L. Taylor, 1994
GTPase RhoA Intramolecular FRET between FPs YFP-RBD (PKN)-RhoA-CFP-Farnesyl moiety (Raichu) Yoshizaki, H. et al, 2003
GTPases Arf6 Intermolecular FRET between FPs Arf6-CFP; YFP-GGA3 effector domain Hall, B. et al, 2008
GTPases Cdc42 Environment-sensing fluorescent dye Dye-CBD from WASP (MeroCBD) Nalbant, P., L. Hodgson et al, 2004
GTPases Cdc42 Intermolecular FRET between FP and fluorescent dye GFP-Cdc42; Alexa-PBD Tzima, E. et al, 2003
GTPases Cdc42/Rac Intramolecular FRET between FPs EBFP-PBD of PAK-EGFP Graham, D. L. et al, 2001
GTPases heterotrimeric G-proteins Intermolecular FRET between FPs alpha-CFP; beta-YFP Janetopoulos, C. et al, 2001
GTPases Rac1 Intermolecular FRET between FP and fluorescent dye GFP-Rac1; Alexa546-PBD from PAK (FLAIR) Kraynov, V. S. et al, 2000
GTPases Rac1/Cdc42 Intramolecular FRET between FPs YFP-PBD (PAK)-Rac1/Cdc42-CFP-Farnesyl moiety (Raichu) Itoh, R. E. et al, 2002
GTPases Ran Intramolecular FRET between FPs YFP-RBD (Yrb1)-CFP; YFP-IBB (importin alpha)-CFP Kalab, P. et al, 2002
GTPases Ras/Rap1 Intramolecular FRET between FPs YFP-Ras/Rap1-RBD (Raf)-CFP-Farnesyl moiety (Raichu) Mochizuki, N. et al, 2001
GTPases RhoA Intramolecular FRET between FPs RBD (rhotekin)-CFP-YFP-RhoA Pertz, O. et al, 2006
Ions Calcium Circular permutation CaM inserted in EYFP Baird, G. S. et al, 1999
Ions Calcium Intramolecular FRET between FPs CFP-CaM-M13-YFP Miyawaki, A. et al, 1997
Ions Calcium Circular permutation of FP fusion M13-cpEGFP-CaM Nakai, J. et al, 2001
Ions Calcium Intramolecular FRET between FPs CFP-CaM-CaMBP(CKK)-YFP Truong, K. et al, 2001
Ions Calcium Intramolecular FRET between FPs CFP-Troponin C-YFP Heim, N. et al, 2004
Ions Calcium circular permutation EGFP with EF-hand insertion Zou, J. et al, 2007
Kinases Akt/PKB Intramolecular FRET between FPs CFP-14-3-3-substrate-YFP Sasaki, K. et al, 2003
Kinases Akt/PKB Intramolecular FRET between FPs CFP-FHA2-Akt substrate-YFP Kunkel, M. T. et al, 2005
Kinases CaMKII Intramolecular FRET between FPs YFP-CaMKII-CFP Takao, K. et al, 2005
Kinases EGFR Intermolecular FRET using FLIM GFP-EGFR; dye-pTyr antibody Wouters, F. S. and P. I. Bastiaens, 1999
Kinases Erk Intramolecular FRET between FPs CFP-Erk-YFP Sato, M. et al, 2007
Kinases FAK Intermolecular FRET between FPs CFP-FAK; dSH2 (Src)-YFP Cai, X. et al, 2008
Kinases FAK Intramolecular FRET between FPs Insertion of a FRET pair in FAK Cai, X. et al, 2008
Kinases MK2 Intramolecular FRET between FPs CFP-MK2-YFP Neininger, A. et al, 2001
Kinases MLCK Intramolecular FRET between FPs MLCK-GFP-CaMBD-BFP (MLCK-FIP) Chew, T. L. et al, 2002
Kinases phospho-CrkII Intramolecular FRET between FPs CFP-CrkII-YFP Kurokawa, K. et al, 2001
Kinases PKA Intramolecular FRET between FPs CFP-KID of CREB-YFP Nagai, Y. et al, 2000
Kinases PKA Intramolecular FRET between FPs CFP-14-3-3tau-PKA substrate-YFP Zhang, J. et al, 2001
Kinases PKC Intermolecular FRET using FLIM FP-PKC; dye-phospho antibody Ng, T. et al, 1999
Kinases PKC Intramolecular FRET between FPs CFP-FHA2-PKC substrate-YFP Violin, J. D. et al, 2003
Kinases PKC Environment-sensing fluorescent dye Caged dye-substrate Dai, Z. et al, 2007
Kinases PKD Intramolecular FRET between FPs CFP-FHA2-PKD substrate-YFP Kunkel, M. T. et al, 2007
Kinases PTK Environment-sensing fluorescent dye caged dye-substrate Wang, Q. et al, 2006
Kinases pTyr Probe redistribution YFP-dSH2 (Src) Kirchner, J. et al, 2003
Kinases pTyr Intramolecular FRET between FPs CFP-SH2-Tyr substrate-YFP Ting, A. Y. et al, 2001
Kinases Src Intramolecular FRET between FPs CFP-SH2-Tyr substrate-YFP Ting, A. Y. et al, 2001, Wang, Y. et al, 2005
Membrane Lipid rafts Intermolecular FRET between FPs MyrPalm/GerGer/PalmPalm/Caveolin-mCFP/mYFP Zacharias, D. A. et al, 2002
Membrane PI(3,4,5)P3 Probe redistribution PH (ARNO or GRP1)-GFP Oatey, P. B. et al, 1999
Membrane PI(3,4,5)P3 Probe redistribution PH (BtK)-GFP Varnai, P. et al, 1999
Membrane PI(3,4,5)P3 Intramolecular FRET between FPs YFP-binding domain of IP3R1-CFP Matsu-ura, T. et al, 2006
Membrane PI(4)P Probe redistribution GFP-PH of OSBP Levine, T. P. and S. Munro, 2002
Membrane PI(4,5)P2 Probe redistribution GFP-PH of PLCdelta1 Stauffer, T. P. et al, 1998, Varnai, P. and T. Balla, 1998
Membrane PI3P Probe redistribution GFP-RING FYVE domains Burd, C. G. and S. D. Emr, 1998
Membrane PIP3/PIP2 Probe redistribution PH (PKB or GRP1)-GFP Gray, A. et al, 1999
Others Calmodulin Environment-sensing fluorescent dye Dye-Calmodulin (MeroCaM) Hahn, K. M. et al, 1990, Hahn, K. et al, 1992
Others S100A4 Environment-sensing fluorescent dye Mero-S100A4 Garrett, S. C. et al, 2008
Others Voltage Intramolecular FRET between FPs CFP-YFP-Ci-VSP Dimitrov, D. et al, 2007
Proteases Calpain Intramolecular FRET between FPs YFP-Calpain cleavage site (alpha-spectrin)-CFP Vanderklish, P. W. et al, 2000
Proteases Caspase-3 Intramolecular FRET between FPs GFP-Caspase-3 substrate-BFP Xu, X. et al, 1998
Proteases Caspase-3 Intramolecular FRET between FPs CFP-Caspase-3 substrate-Venus (improvement) Takemoto, K. et al, 2003
Small molecules cAMP Intermolecular FRET between FPs PKA-GFP; PKA RII-EBFP Zaccolo, M. et al, 2000
Small molecules cAMP Intramolecular FRET between FPs CFP-Epac1-YFP DiPilato, L. M. et al, 2004
Small molecules cGMP Intramolecular FRET between FPs CFP-cGPK-YFP Honda, A. et al, 2001
Small molecules glutamate Intramolecular FRET between FPs CFP-ybeJ-YFP Okumoto, S. et al, 2005
Small molecules maltose Intramolecular FRET between FPs CFP-MBP-YFP Fehr, M. et al, 2002
Small molecules phosphate Intramolecular FRET between FPs CFP-PiBP-YFP Gu, H. et al, 2006
Small molecules Tryptophan Intramolecular FRET between FPs CFP-TrpR-YFP Kaper, T. et al, 2007
Transcription factors bZIP and Rel BiFC bZIP and Rel-fragments of YFP Hu, C. D. et al, 2002
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