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.