| 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 |
FRET - dual chain |
Completed - Tzima et al., 2003 |
Schwartz |
| 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 |
| 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 |
| FAK |
FRET - single chain |
Completed - Cai et al., 2008 (CMC Update Article) |
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 |
| 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 Article) |
Imperiali |
| Prkc - (PKC) |
Chelation-enhanced fluorophore |
Completed - Shults and Imperiali, 2003 (CMC Update Article); Shults et al., 2005 (CMC Update Article) |
Imperiali |
| Protein - (any) |
Environment sensitive fluorophore |
Completed - A combinatorial approach to merocyanines for live cell imaging: a toolchest of dyes for different biosensor designs.
Publication in Progress |
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 - (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) |
| 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 - (environmentally sensitive dye) |
Completed - Gulyani et al., 2011 (CMC Update Article) |
Hahn |
| Src |
Chelation-enhanced fluorophore |
Completed - Sox based chelation-enhanced fluorophore |
Imperiali |
| STK11 - (LKB-1) |
FRET - dual chain |
Completed - Sebbagh et al., 2009 |
Schwartz |
| paxillin - (Tyr118) |
Environment sensitive fluorophore |
On hold - Intact paxillin via unnatural amino acid mutagenesis DANA & 6-DMN based environment sensitive fluorophore |
Imperiali |
| Cdc42 |
Affinity reagent for endogenous Cdc42 - (genetically encode FRET) |
In progress |
Hahn |
| 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 |
| 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 |
| 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 - (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 promising. 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 |
| rac - (Rac1) |
FRET - single chain |
In progress |
Hahn |
| Src |
Affinity reagent for endogenous Src - (genetically encode FRET) |
In progress |
Hahn |
| Tension |
Mechanical tension sensor - (titin-based FRET sensor) |
In progress - 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) |
| 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 |
| 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 |
