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

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

Cameleons: Ca2+ 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 http:/scansite.mit.eduGGSGGRFRRFQTLKIKAKAGGSGG

cytosolic CKAR

PM-anchored CKAR

CKAR: C Kinase Activity Reporter

Jon Violin, Alexandra Newton (UCSD)

0 2 4 6 8 10 12 14 161.02

1.00

0.98

0.96

0.94

0.92

0.90

MyrPalmCKAR in HeLa

10M Histamine

Fur

a R

ed In

tens

ity

FR

ET

Rat

io

Time (Minutes)

88

84

80

76

72

68

0 20 40 60 80 100 120

0.96

0.95

0.94

0.93

0.92F

ura

Red

Inte

nsity

FR

ET

Rat

io

Time (Seconds)

78

76

74

72

70

MyrPalm-CKAR

averaged peaks

CKAR targeted to plasma membrane by acylation detects agonist-stimulated oscillations slightly lagging [Ca2+]c

Jon Violin, Alexandra Newton (UCSD)

0 5 10 15 20 25

1.9

1.8

1.7

1.6

1.5

1.4

FRET Intensity = 140 FRET Intensity = 75 FRET Intensity = 45

FR

ET

Rat

io

Time (Minutes)

PKC translocates in synchrony with Ca2+ spikes

CFP

FRET

YFPYFP

PKCII

YFPYFPPKCII

0.61

0.63

0.65

0.67

0.69

0 4 8 12

0.7

0.8

0.9

1

Yel

low

/Cya

n E

mis

sio

n R

atio

Fu

ra R

ed In

ten

sity

Time (Minutes)

10 M histamine

Jon Violin

1.5

1.55

1.6

1.65

0 4 8 12

0.2

0.3

0.4

0.5

0.6

0.7

FRET-based PLC detector sees nonoscillatory response in HeLa cells

YFPCFP

PH

Yel

low

/Cya

n E

mis

sio

n R

atio

Fu

ra R

ed I

nte

nsi

ty

Time (Minutes)

10 M histamine

YFPCFP

PHPIP2 PLC

IP3

DAG

Jon Violin, Alexandra Newton (UCSD)

PLC, Ca2+, DAG, PKC fluctuate together in MDCK cells

0.45

0.5

0.55

0.6

0 5 10 15 20

0.2

0.3

0.4

0.5

Time (Minutes)

Fur

a-2

Exc

itatio

n R

atio

Cya

n/Y

ello

w E

mis

sion

Rat

io

1 M ATP PLC

0.58

0.6

0.62

0.64

0.66

0 4 8 12

0.15

0.25

0.35

0.45

Fur

a-2

Exc

itatio

n R

atio

Time (Minutes) Cya

n/Y

ello

w E

mis

sion

Rat

io 1 M ATP

1.22

1.24

1.26

1.28

0 4 8 12

0.2

0.4

0.6

0.8

1

Time (Minutes)

Fur

a-2

Exc

itatio

n R

atio

1 M ATP

YFPCFP

C1

YFPCFP

C1DAG

DAG

PKC

Yel

low

/Cya

n E

mis

sion

Rat

io

Jon Violin, Alexandra Newton (UCSD)

17 W/cm2light ~ 1.7 W/cm2

Ele

ctri

cal c

oupl

ing

rati

o

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 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

•600•400•200•0

Time (ms)•Sw 3/19

(pA

)

•-2400

•-2200

•-2000

•-1800

•-1600

•-1400

•-1200

•-1000

•-800

•-600

•-400

•-200

•0

•200

25 50 75 100 125 150 175

-2000

-1400

-800

-200

time (sec)

curr

ent

(pA

)

ReAsH-mediated photoinactivation of L-type Ca2+ channels

Oded Tour; channel cDNA and cell line from R.W. Tsien

Cl- channels in the membrane were simultaneously unaffected

Acute CALI reveals importance of synaptotagmin in endocytosis

559-563

Endocytosisassayed withFM4-64

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/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