USE OF ELICITOR SETS TO CHARACTERIZE CELLULAR SIGNAL

TRANSDUCTION

Graduate Student: Arthi NarayananMajor Professor: Dr. Frank Chaplen

Outline

BackgroundExperimental MethodsResults & Discussion

Background

Complexities of signal transduction pathways

What is systems biology?

Does not investigate individual genes or proteins, but investigates the behavior and relationships of all of the elements in a particular biological system while it is functioning.

Study of a biological system by a systematic and quantitative analysis of all of the components that constitute the system.Biological information has several important features:

Operates on multiple hierarchical levels of organization.

Processed in complex networks.

Key nodes in the network where perturbations may have profound effects; these offer powerful targets for the understanding and manipulation of the system.

Problem Statement

Use the elicitor method - an experimental framework designed to monitor information flows through the G-protein signal transduction network.

To derive mechanistic interpretations from the action of Phenylmethylsulfonyl Fluoride (PMSF), a serine protease inhibitor and nerve agent analog.

Model System: Fish Chromatophores

Overview of Chromatophores

Aggregation/Dispersion of Fish Chromatophores

Before and after 100 nM Clonidine

Before and after 10 µM Forskolin

Gq mediated signaling

EXPERIMENTAL METHODS

Elicitor sets method

What is an elicitor panel?

Known effectors of checkpoints in the signaling cascade.

Elicitor = effector + application method

Why elicitor sets?

Enable identification of the key nodes in the signaling pathway

Segregation of the pathway into different modules

Perturbation of the signaling cascade by adding different effectors will help investigate the cross-talk mechanisms

Enable signature identification of biologically active compounds

20-D mechanism space defined by elicitor panel described below and represented as 3-D projection(A) Cluster map for PMSF; (B) Cluster map for BC 1; (C) Cluster map for BC 5; (D) Cluster map for BC 6. The cluster map for each agent represents a unique complex signature defined by its biological mechanism of action. Elicitors are clonidine (100 and 50 nM), melanin stimulating hormone (10 nM) and forskolin (100 µM).

A B

C D

Cross-talk between Gs and Gq pathways

R

PLCPLC

Ca2+

AC PLC

cAMP

PKA

IP3 DAG

PKC

s

R

q

Cross-talk between Gi and Gq pathways

Ca2+

R

cAMP

PKA

IP3 DAG

PKC

q

i

AC PLC

Day 0: Plated cultured fish chromatophores in 24 well plates Day 1: Media change Day 2: Experiments

Measured OD of cells at ground state

Exposed cells to 10 µM forskolin for 24 minutes with OD being measured at regular intervals

Added 1 mM PMSF to cells and measured OD values for 2.77 hours

Added secondary elicitors (1&100 µM H89, 1&100 µM cirazoline,100 nM clonidine) and monitored the response for 42 minutes.

Plotted normalized % change in OD Vs Time

EXPERIMENTAL SET-UP

RESULTS AND DISCUSSION

Concentration Point of action Response type Optical density

Forskolin

10 µM Adenyl cyclase activator

Hyper-Dispersion

PMSF 1 mM Serine protease inhibitor at / d/s of PKA

Slight dispersion

Clonodine

100 nM Gi activator Aggregation

Cirazoline

1 & 100 µM Gq activator Aggregation

H 89 1 & 100 µM PKA inhibitor Aggregation

MSH 1 nM Gs activator Dispersion

Table 1: List of agents used with their concentrations and response patterns

0

20

40

60

80

100

120

0 500 1000 1500 2000 2500 3000 3500

TIME, SECONDS

NO

RM

ALIZ

ED

% O

D C

HA

NG

E

AVG:500 nm CLOAVG:100 nm CLOAVG:10 nm CLOAVG:1 um CRZAVG:100 um CRZAVG:10 um CRZAVG:L-15

Dilution curves for Clonidine, Cirazoline and L-15 control

0

20

40

60

80

100

120

0 200 400 600 800 1000 1200

TIME, SECONDS

NO

RM

ALIZ

ED

% O

D C

HA

NG

E

AVG:10 nm H89

AVG:100 nm H89

AVG:1 um H89

AVG:10 um H89

AVG:100 um H89

AVG:DMSO control 100 nm

AVG:DMSO control 10 nm

AVG:DMSO control 1 um

AVG:DMSO control 10 um

AVG:DMSO control 100 um

Dose response curves for H-89 and DMSO controls

0

20

40

60

80

100

120

140

160

0 500 1000 1500 2000 2500 3000 3500

TIME, SECONDS

NO

RM

ALIZ

ED

% O

D C

HA

NG

E

AVG:100 um Fors

AVG:1 um Fors

AVG:10 um Fors

AVG:10 nm MSH

AVG:1 nm MSH

AVG:0.1 nm MSH

Dilution curves for Forskolin and MSH

0

20

40

60

80

100

120

140

0 500 1000 1500 2000 2500

TIME, SECONDS

NO

RM

ALIZ

ED

% O

D C

HA

NG

E

AVG: Fors_H89 1 uM

AVG: Fors_H89 100 uM

AVG: Fors_CRZ 1 uM

AVG: Fors_CRZ 100 uM

AVG: Fors_CLO 100 nM

AVG: DMSO_H89 1 um

AVG: DMSO_H89 100 um

AVG: DMSO_CRZ 1 um

AVG: DMSO_CRZ 100 um

AVG: DMSO_CLO 100 nm

Segmentation of the cAMP pathway by application of forskolin as the primary

elicitor

0

20

40

60

80

100

120

140

0 500 1000 1500 2000 2500

TIME, SECONDS

NO

RM

ALIZ

ED

% O

D C

HA

NG

E

AVG: MSH_H89 1uM

AVG: MSH_H89 100 uM

AVG: MSH_CRZ 1 uM

AVG: MSH_CRZ 100 uM

AVG: MSH_CLO 100 nM

AVG: L15_H89 1 uM

AVG: L15_H89 100 uM

AVG: L15_CRZ 1 uM

AVG: L15_CLO 100 nM

AVG: L15_CRZ 100 uM

Experiments with MSH as the primary elicitor

0

20

40

60

80

100

120

140

160

180

0 2000 4000 6000 8000 10000 12000 14000 16000

TIME, SECONDS

NO

RM

ALIZ

ED

% O

D C

HA

NG

E

avg FOR_PMSF_1 um H89

avg FOR_PMSF_100 um H89

avg FOR_PMSF_1 um CRZ

avg FOR_PMSF_100 um CRZ

avg FOR_PMSF_100 nm CLO

Elicitor experiments with PMSF applied after forskolin

0

20

40

60

80

100

120

140

160

180

200

220

0 2000 4000 6000 8000 10000 12000 14000 16000

TIME, SECONDS

NO

RM

ALIZ

ED

% O

D C

HA

NG

E avg FOR_EtOH_1 um CRZ

avg FOR_EtOH_100 um CRZ

avg FOR_EtOH_100 nm CLO

avg DMSO_EtOH_100 nm CLO

avg DMSO_EtOH_100 um CRZ

avg DMSO_EtOH_1 um CRZ

avg DMSO_EtOH_1 um H89

avg DMSO_EtOH_100 um H89

avg FOR_EtOH_1 um H89

avg FOR_EtOH_100 um H89

DMSO and Ethanol controls

TARGETS FOR PRIMARY AND SECONDARY ELICITORS

Gi

AC Forskolin

cAMP

PKA

Aggregation

ClonidineGq

PLC

PIP2

Aggregation

Cirazoline

IP3 + DAG

Ca++

PKC

H89

%OD change due to H-89 in: wells treated with PMSF - 26% control wells - 44%

Our experimental results predict that PMSF acts at or downstream of PKA.

An interpretation of the results suggests an interaction between a serine protease and PKA, that makes the latter less susceptible to H89.

When PMSF, a serine protease inhibitor is added to the cells, this interaction is hampered thereby allowing H-89 to totally exert its inhibitory effect on PKA.

Mechanistic interpretation from PMSF action

Discussion and Conclusion

Choice of AC as reference node and forskolin as primary elicitor simplifies the determination of the mechanism of action of PMSF.

Application of PMSF after forskolin localized the measurable effect of PMSF to regions of the signaling cascade, below AC

Perturbation by addition of secondary elicitors provided more information within the simplex scenario created by forskolin.

Increased information resolution is evident in the heightened sensitivity of PKA to H-89 in the presence of PMSF, while the upper segment of the pathway is decoupled through application of forskolin

help identify cross-talks. Failure of cirazoline to elicit a response when applied after forskolin shows an evidence of cross-talk.

Thanks To:• Dr.Frank Chaplen for his indispensable support and guidance at every

step during my research.

• Dr. Rosalyn Upson for her guidance and encouragement.

• Elena, Linda, June, Ruth, Christy, Bob and Indi for all your help along the way.

• Dr.Michael Schimerlik and Dr. Skip Rochefort for serving on my committee.

• Jeanine Lawrence, Ljiljana Mojovic and Ned Imming for your help in the lab.

• Ganesh and my family back in India for everything.

• NSF and AES for funding this work.