fret assays with genetically targeted labels protein-protein interactions –heterotrimeric g...
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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