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FRET assays with genetically targeted labels

FRET assays with genetically targeted labels. Protein-protein interactions heterotrimeric G proteins, transcription factors… GPCR conformational changes at ms time resoln. GTP/GDP-bound status of small G proteins cAMP, cGMP, NO, Zn 2+ Proteases such as caspases Ca 2+ : cytosol vs. ER

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FRET assays with genetically targeted labels

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  1. FRET assays with genetically targeted labels • Protein-protein interactions • heterotrimeric G proteins, transcription factors… • GPCR conformational changes at ms time resoln. • GTP/GDP-bound status of small G proteins • cAMP, cGMP, NO, Zn2+ • Proteases such as caspases • Ca2+: cytosol vs. ER • Protein kinase/phosphatase activities • EGFR, Src, Abl, PKA, PKB, PKC • PIP2/IP3, DAG, other lipid-related signals Protein knockouts in seconds by CALI

  2. Cameleons: Ca2+ indicators based on CaM + GFP mutants Atsushi Miyawaki

  3. A generic design for indicators of kinase/phosphatase activity

  4. CKAR: C Kinase Activity Reporter High FRET cytosolic CKAR CKAR FRET Ratio YFP +PKC FHA2 FHA2 CFP -OH CFP YFP lex lem lex 60min, 450x Frame Rate Low FRET lem GGSGGRFRRFQTLKIKAKAGGSGG PM-anchored CKAR substrate sequence designed with help from http:/scansite.mit.edu Jon Violin, Alexandra Newton (UCSD)

  5. CKAR targeted to plasma membrane by acylation detects agonist-stimulated oscillations slightly lagging [Ca2+]c MyrPalm-CKAR averaged peaks Jon Violin, Alexandra Newton (UCSD)

  6. PKC translocates in synchrony with Ca2+ spikes 10 mM histamine PKCbII YFP YFP CFP FRET Yellow/Cyan Emission Ratio Fura Red Intensity PKCbII YFP YFP Time (Minutes) Jon Violin

  7. PHd CFP YFP FRET-based PLC detector sees nonoscillatory response in HeLa cells 10 mM histamine DAG PHd PLC PIP2 CFP YFP IP3 Yellow/Cyan Emission Ratio Fura Red Intensity Time (Minutes) Jon Violin, Alexandra Newton (UCSD)

  8. C1 CFP YFP PLC, Ca2+, DAG, PKC fluctuate together in MDCK cells PKC 1 mM ATP PLC 1 mM ATP Fura-2 Excitation Ratio Fura-2 Excitation Ratio Cyan/Yellow Emission Ratio Cyan/Yellow Emission Ratio Time (Minutes) Time (Minutes) DAG 1 mM ATP C1 DAG CFP YFP Fura-2 Excitation Ratio Yellow/Cyan Emission Ratio Time (Minutes) Jon Violin, Alexandra Newton (UCSD)

  9. Strong illumination can inactivate ReAsH-stained connexins(= genetically targeted, chromophore-assisted light inactivation) 17 W/cm2 light ~1.7 W/cm2 Electrical coupling ratio ReAsH fluorescence Transmitted light Oded Tour

  10. ReAsH-mediated photoinactivation of L-type Ca2+ channels Raw date example 2 of CALI of a1C-(MPCCPGCC)2 2.5 mM ReAsH for 2 h in DMEM; 250 mM EDT wash for 30 minutes in DMEM 3 repeats of 10 second excitation • 200 • 0 • -200 • -400 • -600 • -800 • -1000 (pA) • -1200 • -1400 • -1600 • -1800 • -2000 • -2200 • -2400 • 0 • 200 • 400 • 600 Time (ms) • Sw 3/19 Cl- channels in the membrane were simultaneously unaffected Oded Tour; channel cDNA and cell line from R.W. Tsien

  11. Acute CALI reveals importance of synaptotagmin in endocytosis Endocytosis assayed with FM4-64 559-563

  12. Tetracysteine-biarsenical CALI • Compared to traditional CALI, eliminates need to raise innocuous Abs, label with dye, microinject just the right amount • Compared with noncovalent small molecule inhibitors, avoids need for custom drug development/med chem, allows isoform specificity • Compared with gene knockout/RNAi: much higher temporal/spatial resolution, less chance for compensation or avalanche of effects • But only eliminates exogenous tagged copies. Ultimately, one would knock out endogenous copies, replace by tagged copies, show function is normal until CALI suddenly initiated

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