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Microscopy Based Biosensors and Functional Assays
Receptor Inputs
Signaling Proteins Second Messengers
Secretion Endocytosis Pinocytosis Phagocytosis
Cell Polarity Cell-Cell Contact Adhesion Migration Chemotaxis
Cell Cycle Apoptosis Cell Size (DNA Damage, Nutrition State)
Functional Outputs
Functional Outputs
Dissecting cellular signaling systems: Perturbations and
Biosensors
Perturbations
Biosensors, Functional assays
Receptor Inputs
1. Microscopy Strategies to Explore Signaling Systems
1. Epifluorescence Imaging
2. Confocal Imaging
3. Total Internal Reflection Microscopy
Anti FLAGM1 monoclonal Ab
YFP YFPYFP
YFP
Automated assays for receptor endocytosis
Epinephrin
Internalization (~30%)
YFP
+ EDTA 2mM
YFP
YFP
YFPFix, perm, 2ndary Ab
A568
0
5
10
15
20
25
30
35
0.1 0.7 1.3 1.9 2.5 3.1 3.7 4.3 4.9 More
Control
ARF1
Automated immunofluorescence analysis (epifluorescence)
YF
PT
exas
Red
CF
P
CFP-Arf1 (DN)
YFP-2-adrenergic receptor
Internalized receptor
Reduced endocytosis rates in the presence of some constitutively active
small GTPases
0
20
40
60
80
100
120
Rat
io I 5
94/I Y
FP
(% C
TR
)
CTR
Potentially involved in regulating endocytosis
ARL4ARF1 ARF3
2-adrenergic receptor internalization kinetics (confocal imaging)
45 minute movie, YFP-tagged beta2-adrenergic receptor; epinephrine stimulation after 15 minutes
GFP-PKC
Calcium-Crimson
Calcium signals versus PKC translocation (confocal imaging)
Antigen stimulation of tumor mast cells
Membrane translocation measured by total internal reflection fluorescence microscopy
+Receptor stimulus
PKC-GFP
The evanescent wave field in TIRF imaging
Monitoring plasma membrane translocation in large numbers of cells
Evanescent wave Single Cell Array Technology (E-SCAT)
Adherent cells
Laser
Teflon ring
Low magnificationprojection(9mm x 7mmimaged area)
Camera
Recording of GFP-C2 domain plasma membrane translocation in many cells
PAF ionomycin
0
1Plasma membrane
Cytosol50 s
Rel
. flu
ores
cenc
e
Teruel and Meyer, Science, 2002
2. Perturbation strategies
suitable for microscopy
1. RNAi
2. Expression constructs (wt, DN, CA)
3. Small molecule perturbations (for example induced translocation)
Generating d-siRNA sets based on in vitro dicing
*
*
Transfection
Pool ofdsiRNAs
r-DicerLarge dsRNA
Myers, Jones, Meyer and Ferrell Nature Biotech, 2003
In Vitro Dicing and
Purification
PCR
In Vitro Txn
X 24 = 2304 siRNA pools targeted to signaling domain selected proteins
In vitro Dicer method developed by Jason Myers in Jim Ferrell’s lab
R-R
AS
2R
-RA
SR
-RA
S3
H-R
AS
K-R
AS
N-R
AS
RIT
RIN
RA
P1A
RA
P1B
RA
P2B
RA
P2A
RA
LA
RA
LB
RA
DG
EM
RE
MA
RH
IA
GS
1R
RP
22kB
-RA
S1
kB-R
AS
2R
HE
B2
RA
C3
RA
C2
RA
C1
RH
OG
CD
C42
hC
DC
42T
C10
TC
LR
HO
EA
RH
ER
HO
7R
HO
6R
HO
CR
HO
AR
HO
BR
HO
HR
HO
DA
RF
1A
RF
3A
RF
5A
RF
4A
RF
6A
RL
1A
RL
5A
RL
3A
RL
2A
RL
7AA
RL
7BA
RL
4A
RF
4LA
RF
RP
1S
AR
1S
AR
AR
AG
AR
AG
B
RA
B8B
RA
B8
RA
B10
RA
B13
SE
C4L
RA
B1A
RA
B1B
RA
B35
RA
B27
AR
AB
27B
RA
B26
RA
B3B
RA
B3A
RA
B30
RA
B33
AR
AB
RP
RA
B4
RA
B4B
RA
B14
RA
B2
RA
B11
AR
AB
11B
RA
B25
RA
B18
RA
B5A
RA
B5C
RA
B5B
RA
B22
AR
AB
22B
RA
B21
RA
B9
RA
B9L
RA
B7
RA
NR
AB
L2B
RA
B32
RA
B38
RA
B7L
1R
AB
23R
AB
6R
AB
6CR
AB
28
Polar
EyelashesLamellipodia
FilopodiaRounding
Stress fibersMultiple Shrunk
Multiple Local spread
CA small GTPases and cell morphology
Heo and Meyer, Cell, 2003
Rac PM translocation induced by a rapamycin analog
+ FKBP-YFP-Rac1(CA)
Rapamycin analog synthesized by Tom Wandless
NIH3T3 cells
w. PM-FRB
3. Automated Microscopy Based Biosensors and Functional
Assays
1. FRET Biosensors
2. Phosphospecific Antibody and Related Fixed Cell Assays
3. Translocation Biosensors
4. Live and Fixed Cell Functional Assays (Outputs)
5. Many critical assays are lacking
Activation of c-jun by CA small GTPases
(example of rapid survey assay)
Constitutively active small GTPases (HS68-cells)
Activation of c-jun phosphorylation by small GTPases
0
200
400
600
800
1000
1200
1400
1600
1800
2000
NO
NO
TN
F-a
CD
C42
TC
10C
DC
42h
RA
C1
AR
L7
RA
C2
RA
B40
BR
AB
7L1
RA
LB
RA
B26
RA
B2
RA
C3
RA
B5C
RH
OG
GE
MN
-RA
SR
HO
BA
RH
ER
-RA
S2
RA
DK
-RA
ST
CL
AG
S1
AR
L5
RH
OD
RA
B23
RA
B8B
AR
L4
SA
R1B
RH
O7
R-R
AS
1R
-RA
S3
RA
B21
RH
O6
RA
GB
RH
OA
RA
B5A
SA
R1A
RA
P1A
RR
P22
RH
OC
AR
HI
H-R
AS
RH
O8
RA
B3A
RA
B33
AR
AB
1AR
AB
39L
AR
L7
RA
B22
BR
AB
4BR
HE
B2
RA
B6A
RA
B9B
RA
NR
AB
25R
AG
AR
AB
18R
AB
11A
RA
B10
RA
B8
RA
B4A
RA
B27
AR
AB
22B
RA
B38
RIN
RH
OH
RA
B1B
RA
BL
2BA
RF
4R
AB
22A
AR
F3
RIT
AR
L3
AR
F1
RA
LA
RA
B27
BR
AB
7R
AB
5BR
AB
35R
AB
30R
AB
28R
AP
2BR
AB
6CR
EM
RA
B11
BR
AB
9R
AB
3B
Inte
ns
ity
of
ph
os
ph
o-c
-ju
n i
n n
uc
leu
s
No/low expression
High expression
Apoptosis induced by d-siRNA against human signaling proteins (important
control)Plate #2 siRNA-1 (MCF-7)
0
10
20
30
40
50
60
70
A01
A03
A05
A07
A09
A11
A13
A15
A17
A19
A21
A23
B01
B03
B05
B07
B09
B11
B13
B15
B17
B19
B21
B23
C0
1C
03
C0
5C
07
C0
9C
11
C1
3C
15
C1
7C
19
C2
1C
23
D0
1D
03
D0
5D
07
D0
9D
11
D1
3D
15
D1
7D
19
D2
1D
23
E01
E03
E05
E07
E09
E11
E13
E15
E17
E19
E21
E23
F0
1F
03
F0
5F
07
F0
9F
11
F1
3F
15
F1
7F
19
F2
1F
23
G0
1G
03
G0
5G
07
G0
9G
11
G1
3G
15
G1
7G
19
G2
1G
23
H0
1H
03
H0
5H
07
H0
9H
11
H1
3H
15
H1
7H
19
H2
1H
23
well
Ap
op
toti
c ce
lls/t
ota
l cel
ls (
%)
Anti-Lamin B antibody apoptosis assay
Fluorescent Translocation Biosensors: Non-perturbing versus endpoint
indicatorsSH2-domains to monitor local tyrosine phosphorylation
(Stauffer et al., JCB 1997)
C1-domains to monitor localized diacylglycerol signals (Oancea et al., JCB 1998)
C2-domains to monitor local Ca2+/PS-signals (Oancea et al., Cell 1998)
PH-domains to monitor local changes of phosphoinositides (Stauffer et al., Current Biology 1998, PLC-delta;
Kontos et al., Mol. Pharm. 1998, Akt)
Potentially many other useful domains (FYVE, PTB, …)
PH Domain
Binding selectivity
CTH3(PH1A) Akt3(PH1A) PLCd1(PH1A) RalGPS2(PH1A) Hapip1(PH1A)
PI(3,4,5)P3 PI(3,4,5)P3
PI(3,4,)P2 PI(3,4,5)P3
PI(4,5)P2PI(3,4)P2
PI(4,5)P2
PI(3,4,5)P3
PI(3,4)P2
127 PH Domain constructs tested:PI(3,4,5)P3 CTH3(PH1A), Myo10(PH1A), ITK(PH1A), H056(PH2A), EtOHD4(PH1A), APS(PH1A), Afap(PH1A), TEC(PH1A)
PI(3,4)P2 and PI(3,4,5)P3
Gab1(PH1A), Gab2(PH1A), Bam32(PH1A), CTH2(PH1A), IRS-1(PH1A), Osbp13(PH1A), Plek(PH1A), TNFidp(PH1A), Akt2(PH1A), Akt3(PH1A), Akt1(PH1A), LL5(PH1A), Arl61(PH1A), BCRa(PH1a)
PI(4,5)P2 and PI(3,4,5)P3 PLCd1(PH1A), Spnb2(PH1A), RalGPS2*(PH1A), Centb5(PH1A), Cnk2(PH1A)
PI(3,4)P2 Plek2(PH2A), Hapip1(PH1A)
Biosensors & PerturbationsPH-domain selectivity
Wei Sun Park, James Whalen, Takako Mukai & Nancy O’Rourke
PIP3 production by constitutively active small GTPases
ECFP H-RAS K-RAS RALA RAP2B
RAP2ARAC1 RHOG CDC42RHOH TC10
RAB1A RAB2 RAB23 RAB30 ARF1
Ras subfamily
Rho subfamily
Rab & Arf subfamily
Morphology changes make automated analysis more difficult
Functional assays (Outputs)
YFP
NLS PM targeting
Mitotic biosensor
Jones, Myers, Ferrell & Meyer, Nat. Biotech., 2004
Watching 3 hours in the life of cycling cells
Mitosis biosensor
10
100
1000
10000
Mitosis import G1 G1/S S/G2
Phase
Tim
e (m
inut
es)
HeLa
MCF7
Measuring cell cycle timing
Time (min)0 40 80 120 160 200 240 280
Re
l. F
luo
res
c. I
nt.
4
32
1
Mitosis in RBL’s
Mitosis is accelerated by a loss of the spindle checkpoint
25 nM15 min
5 nM25 min
3 nM35 min
Untreated45 min
0
10
20
30
40
50
60
0 GL3 3 5 10 15 25 50 75
[d-siRNA] nM
Tim
e (m
in)
NEB-AnaphasePrometaphaseMetaphase
Mad2 targeted by d-siRNA
Examples of mitosis defects observed with the mitosis
biosensor
Cytokinesis Defects (Rab 21)
Abnormal Spindles (Rab 3B)
Normal
Automated measurements of dynamic parameters in cell migration
Dendritic cell migration in presence of C5a
TIRF measurements of secretion, endocytosis and signaling
processes
CFP-PH(Akt) GLUT4-YFP
TIRF Assay for PIP3 and the PM insertion and endocytosis of GLUT4 transporter
AcknowledgmentsJosh JonesAngie Hahn Onn Brandman Annette Salmeen Cecile ArrieumerlouTakanari InoueMarc FivazMadeleine CraskeThierry GalvezMichael BradshawChuck FinkMary TeruelMan Lyiang KimWon Do HeoJen Liou
Calif. Ave. AfCS Microscopy lab:Grischa Chandy Nancy O’Rourke Wei Sun Park Jim WhalenTakako Mukai Mary VergheseLiz Gehrig Sarah Lim
James Ferrell, Jason Myers,Michal RonenTom Wandless
Automated microscopy based signaling and functional assays
1. Phosphospecific antibodies and other fixed cell assays (analysis procedures can readily be developed; more suitable phosphospecific antibodies needed)
2. FRET biosensors (implementation of automated assays of existing biosensors is first needed)
3. Translocation biosensors (PM, nucleus, Golgi and vesicular structures could be automatically analyzed; development of new assays and implementation of automated assays needed)
4. Functional output assays (apoptosis, cell cycle, secretion endocytosis, phagocytosis, pinocytosis, cell migration, cell adhesion; Automated assays still need development)
5. Still fairly low biosensors coverage. More assays needed (how many?). Microscopy can provide suitable assays for many of them.