FRET assays with genetically targeted labels Protein-protein interactions –heterotrimeric G proteins, transcription factors… GPCR conformational changes.

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Presentation transcript:

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 Protein kinase/phosphatase activities –EGFR, Src, Abl, PKA, PKB, PKC PIP 2 /IP 3, DAG, other lipid-related signals Protein knockouts in seconds by CALI

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

A generic design for indicators of kinase/phosphatase activity

FHA2 CFP ex YFP em FHA2 CFP YFP ex em -OH CKAR FRET Ratio 60min, 450x Frame Rate Low FRET High FRET +PKC substrate sequence designed with help from GGSGGRFRRFQ T LK I KAKAGGSGG cytosolic CKAR PM-anchored CKAR CKAR: C Kinase Activity Reporter Jon Violin, Alexandra Newton (UCSD)

MyrPalm-CKAR averaged peaks CKAR targeted to plasma membrane by acylation detects agonist-stimulated oscillations slightly lagging [Ca 2+ ] c Jon Violin, Alexandra Newton (UCSD)

PKC translocates in synchrony with Ca 2+ spikes CFP FRET YFP PKC  II YFP PKC  II Yellow/Cyan Emission Ratio Fura Red Intensity Time (Minutes) 10  M histamine Jon Violin

FRET-based PLC detector sees nonoscillatory response in HeLa cells YFPCFP PH  Yellow/Cyan Emission Ratio Fura Red Intensity Time (Minutes) 10  M histamine YFPCFP PH  PIP 2 PLC IP 3 DAG Jon Violin, Alexandra Newton (UCSD)

PLC, Ca 2+, DAG, PKC fluctuate together in MDCK cells Time (Minutes) Fura-2 Excitation Ratio Cyan/Yellow Emission Ratio 1  M ATP PLC Fura-2 Excitation Ratio Time (Minutes) Cyan/Yellow Emission Ratio 1  M ATP Time (Minutes) Fura-2 Excitation Ratio 1  M ATP YFPCFP C1 YFPCFP C1 DAG PKC Yellow/Cyan Emission Ratio Jon Violin, Alexandra Newton (UCSD)

17 W/cm 2 light ~ 1.7 W/cm 2 Electrical coupling ratio ReAsH fluorescence Transmitted light Strong illumination can inactivate ReAsH-stained connexins (= genetically targeted, chromophore-assisted light inactivation) Oded Tour

Raw date example 2 of CALI of a 1 C-(MPCCPGCC) m M ReAsH for 2 h in DMEM; 250 m M EDT wash for 30 minutes in DMEM 3 repeats of 10 second excitation Time (ms) Sw 3/19 (pA) ReAsH-mediated photoinactivation of L-type Ca 2+ channels Oded Tour; channel cDNA and cell line from R.W. Tsien Cl - channels in the membrane were simultaneously unaffected

Photoinactivation can be prevented by singlet oxygen quenchers and enhanced by D 2 O or 100% O 2 Oded Tour

Acute CALI reveals importance of synaptotagmin in endocytosis Endocytosis assayed with FM4-64

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/RNA i : 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