A Single Cell PIP3 Assay Based on Akt PH domain Translocation

Nancy O’Rourke

Microscopy Lab, Stanford University

Jim Whalen, Takako Mukai, Wei Sun Park, Liz Gehrig

Grischa Chandy, Tobias Meyer

Macrophage Lab, UCSFTamara RoachRobert Rebres

Bioinformatics Lab, UCSDIlango VadiveluBob Sinkovits

Molecular Biology Lab, CaltechIain Fraser

Joelle Zavzavadjian Leah Santat Jamie Liu

Estelle Wall Kavitha Dhandapani

A Single Cell PIP3 Assay Based on Akt PH domain Translocation

Assay Protocol and Analysis

Testing the FXM Ligands

Perturbation of the System with RNAi and Chemical Reagents

Assay Protocol

Transfect YFP Akt PH domainand CFP KRAS CAAX

Cells in 8-well chamber slide

Serum Deprive 1 hour

Run at 37o

Image on confocal microscope

40 scans w/ 10 second interval

Ligand added at 50 seconds

-0.3238 0.4148CorrelationCoefficient

Image Analysis

Time 0 secs. 140 secs.

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0 50 100 150 200 250 300 350 400

Seconds

Cor

rela

tion

Coe

ffic

ien

t

C5a

100 nM C5a

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0 100 200 300 400

Seconds

Co

rrela

tio

n C

oeffi

cie

nt

ts14-2

ts14-3

ts14-4

ts14-5

average

Individual and Average Responses

Akt PH Domain Translocation after 100 nM C5a Stimulation

Diverse Patterns of Akt PH Domain Translocation

Responses to C5a, UTP and FcR1

C5a responses are consistent and robust.C5a stimulation used for current perturbation studies.

Translocation does not occur followingUDP/UTP stimulation.

Responses to FcR1stimulation vary with cell batch.

100 M UTP0.44 M F(ab’)2

100 nM C5a

Akt PH Translocation with UTP, FcR1, and C5a Stimulation

0

10

20

30

40

50

60

70

80

90

100

UTP UTP F(ab')2 F(ab')2-UCSF

C5a C5a

Ligand

% C

ells

w/

Tra

nsl

ocat

ion

Akt PH Domain Translocation with UTP, FcgR1 and C5aStimulation

Percent Cells with FcR1 Response

0

10

20

30

40

50

60

70

80

90

100

S1 S1 S1 U1 U1 U2 U2 U3

Cell Batch #

% C

ells

w/

Tra

nsl

ocat

ion

Variability in the FcR1 Response

Perturbation Experiments

C5a stimulation utilized for perturbation studies.

Lentiviral cell lines expressing GFP interfere with the YFP-Akt PH fluorescence signal.

Three CD4 lentiviral cell lines: SHIP1, G4 and Gi3.

Chemical perturbation studies initiated with LY

Knockdown of SHIP1 Increases Translocation

100 nM C5a Stimulation in SHIP1-shRnai and Vector Control

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0 100 200 300 400

Seconds

Co

rrela

tion C

oeffi

cie

nt

pL_Gene-UCI2P

pLX_mH1-SHIP-A-UC

0102030405060708090

100

co

ntr

ol

SH

IP1

KD

% C

ell

s w

ith

Tra

nslo

cati

on

Testing of lentiviral lines for knockdown efficacy

IB: G alpha i3

UCIPSHIP

1G

i3

49

38

SHIP1

Gi3

IB: SHIP1

UCIP

188

98

99% Knockdown in UC lines.

Caltech Lab

Knockdown of G4 does not alter Translocation

100 nM C5a Stimulation in G4-shRnai and Vector Control

-0.4

-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0 100 200 300 400

Seconds

Co

rre

lati

on

Co

effi

cie

nt

pL_ Gene-UCI2P

Gb4-UCI2P

G-proteins 4 Gi2 1 0.23 2.4 1.0‡ 1.3

and related 2 0.01 2.1 1.3 1.05 Gi3 1 0.28 0.9 1.1 1.56 Gq 1 0.01 1.2 0.6 2.07 G1 1 0.07 0.9 1.0‡ 0.58 G2 1 0.07 0.5 0.9‡ 0.2

2 0.01 0.0 0.8‡ 0.93 0.15 0.5 1.2 0.6

9 G4 1 pending 1.1 1.3 1.710 G5 1 pending 1.5 1.0 1.611 GRK2 1 0.53 2.1 0.8 1.512 Arr3 1 pending 1.3 1.0 1.1

PI Metabolism 13 PTEN 1 0.01 3.3 0.7 0.92 0.01 1.5 1.0 1.0

14 SHIP1 1 0.12 1.3 1.0 1.815 PI3K p85 1 pending 1.0 1.1 0.516 PI3K p85 1 pending 0.4 1.0 0.417 PI3K p101 1 0.04 1.4 1.4 2.118 PI3K p110 1 0.01 0.5 0.8 0.719 PI3K p110 (A**) 1 0.01 0.9 1.0 1.2

PI3K p110 (B**) 2 0.01 1.9 1.4 1.020 PDK1 1 0.8 1.0 0.9 1.021 PLC2 1 0.16 0.8 0.9 1.922 PLC3 1 pending 0.6 1.0 0.923 IP3R1 1 0.01 0.8 0.9 1.224 IP3R2 1 pending 1.2 1.0 1.925 IP3R3 1 0.01 0.9 1.1 1.226 SERCA2 1 pending 1.2 0.9 0.8

UCSF lab

Results are Consistent with Population Ca2+ Results

LY Pretreatment Decreases the Translocation

100 nM C5a Stimulation with 100 M LY Pretreatment

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0 100 200 300 400

Seconds

Co

rrel

atio

n C

oef

fici

ent

C5a only

LY, C5a

10 minute pretreatment with100 M LY

0

10

20

30

40

50

60

70

80

90

C5a LY, C5a

% C

ells w

/ T

ran

slocati

on

GRK2 (4,5,6)

Arrestin 2,3

Gq, G11

P2Y2,6

G1,2 (4,5)

Gi2,3

CD64

Lyn, HckSyk

PTEN

SHIP-2 (1)

PLC2,3 (4) PLC1,2

Akt1,2,3

PI3K p85, p110

p101, p110

IP3R1,2,3IP3KTB(C)

SERCA2,3 PMCA1,3 (4)

Btk, Tec

C5aR

LentiviralLines withCD4 marker

LY

PI(3,4,5)P3

Gil SambranoLily Jiang

FXM Translocation Data Display

N - 8 cells

% cells with Translocation – 88%

100 nM C5a

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0 100 200 300 400

Seconds

Co

rrela

tio

n C

oeffi

cie

nt

ts14-2

ts14-3

ts14-4

ts14-5

average

Treatment Summary

Conclusions

The Akt PH domain translocation assay can be employed to probe PIP3 signaling pathway

C5a provides a consistent and robust response in the translocation assay

Results from preliminary studies confirm the roles of PI3K and SHIP1 in PIP3 signaling.

FXM Translocation Display Available

Future Directions

Increase throughput of assay.

Screen Additional Non-fluorescent Lentiviral Cell Lines

Develop Methods for Using Duplex RNAi with Translocation Assay

Refine FXM Display