FRET assays with genetically targeted labels Proteinprotein interactions

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 c. AMP, c. GMP, 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 Ca. M + GFP mutants Atsushi Miyawaki

A generic design for indicators of kinase/phosphatase activity

CKAR: C Kinase Activity Reporter cytosolic CKAR FRET Ratio High FRET YFP +PKC FHA 2 -OH CFP YFP lex PM-anchored CKAR lem lex lem 60 min, 450 x Frame Rate Low FRET GGSGGRFRRFQTLKIKAKAGGSGG substrate sequence designed with help from http: /scansite. mit. edu Jon Violin, Alexandra Newton (UCSD)

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

PKC translocates in synchrony with Ca 2+ spikes CFP FRET

FRET-based PLC detector sees nonoscillatory response in He. La cells CFP YFP PHd CFP YFP Fura Red Intensity PHd Yellow/Cyan Emission Ratio 10 m. M histamine Time (Minutes)

Time (Minutes) 1 m. M ATP Fura-2 Excitation Ratio PLC Cyan/Yellow Emission Ratio 1 m. M ATP Fura-2 Excitation Ratio Cyan/Yellow Emission Ratio PLC, Ca 2+, DAG, PKC fluctuate together in MDCK cells Time (Minutes) Fura-2 Excitation Ratio 1 m. M ATP C 1 CFP YFP Time (Minutes) PKC

Strong illumination can inactivate Re. As. H-stained connexins (= genetically targeted, chromophore-assisted light inactivation) Re. As. H fluorescence Transmitted light Electrical coupling ratio light ~ 1. 7 W/cm 2 17 W/cm 2 Oded Tour

Raw date example 2 of CALI of aof 1 C-(MPCCPGCC) 2 Re. As. H-mediated photoinactivation L-type Ca 2+ channels 2. 5 m. M Re. As. H for 2 h in DMEM; 250 m. M EDT wash for 30 minutes in DMEM 3 repeats of 10 second excitation • 200 • -200 • -400 • -600 (p. A) • -800 • -1000 • -1200 • -1400 • -1600 • -1800 • -2000 • -2200 • -2400 • 0 • 200 • 400 Time (ms) • 600 • Sw 3/19 Cl- channels in the membrane were simultaneously unaffected Oded Tour; channel c. DNA and cell line from R. W. Tsien

Acute CALI reveals importance of synaptotagmin in endocytosis Endocytosis assayed with FM 4 -64 559 -563

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