USE OF ELICITOR SETS TO
CHARACTERIZE CELLULAR SIGNAL
TRANSDUCTION
Graduate Student: Arthi Narayanan
Major Professor: Dr. Frank Chaplen
Outline
Background
Experimental Methods
Results & 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
A
C
B
D
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).
Cross-talk between Gs and Gq pathways
aq
bg
as
bg
AC
PLC
PLC
IP3
cAMP
RR
Ca2+
PKA
PKC
DAG
Cross-talk between Gi and Gq pathways
bg
aq
ai
bg
AC
PLC
IP3
cAMP
R
Ca2+
PKA
PKC
DAG
EXPERIMENTAL SET-UP
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
RESULTS AND DISCUSSION
Table 1: List of agents used with their
concentrations and response patterns
Concentration
Point of action
Response type
Forskolin
10 µM
Adenyl cyclase
activator
HyperDispersion
PMSF
1 mM
Serine protease
Slight dispersion
inhibitor at / d/s of
PKA
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
Optical
density
Dilution curves for Clonidine, Cirazoline and
L-15 control
120
NORMALIZED % OD CHANGE
100
80
60
AVG:500 nm CLO
AVG:100 nm CLO
AVG:10 nm CLO
AVG:1 um CRZ
AVG:100 um CRZ
AVG:10 um CRZ
AVG:L-15
40
20
0
0
500
1000
1500
2000
TIME, SECONDS
2500
3000
3500
Dose response curves for H-89 and DMSO controls
120
NORMALIZED % OD CHANGE
100
80
AVG:10 nm H89
AVG:100 nm H89
60
AVG:1 um H89
AVG:10 um H89
AVG:100 um H89
40
AVG:DMSO control 100 nm
AVG:DMSO control 10 nm
AVG:DMSO control 1 um
20
AVG:DMSO control 10 um
AVG:DMSO control 100 um
0
0
200
400
600
TIME, SECONDS
800
1000
1200
Dilution curves for Forskolin and MSH
160
NORMALIZED % OD CHANGE
140
120
AVG:100 um Fors
100
AVG:1 um Fors
AVG:10 um Fors
80
AVG:10 nm MSH
AVG:1 nm MSH
60
AVG:0.1 nm MSH
40
20
0
0
500
1000
1500
2000
TIME, SECONDS
2500
3000
3500
Segmentation of the cAMP pathway by
application of forskolin as the primary elicitor
140
NORMALIZED % OD CHANGE
120
AVG: Fors_H89 1 uM
100
AVG: Fors_H89 100 uM
AVG: Fors_CRZ 1 uM
AVG: Fors_CRZ 100 uM
80
AVG: Fors_CLO 100 nM
AVG: DMSO_H89 1 um
60
AVG: DMSO_H89 100 um
AVG: DMSO_CRZ 1 um
AVG: DMSO_CRZ 100 um
40
AVG: DMSO_CLO 100 nm
20
0
0
500
1000
1500
TIME, SECONDS
2000
2500
Experiments with MSH as the primary elicitor
140
NORMALIZED % OD CHANGE
120
100
AVG: MSH_H89 1uM
AVG: MSH_H89 100 uM
AVG: MSH_CRZ 1 uM
80
AVG: MSH_CRZ 100 uM
AVG: MSH_CLO 100 nM
AVG: L15_H89 1 uM
60
AVG: L15_H89 100 uM
AVG: L15_CRZ 1 uM
AVG: L15_CLO 100 nM
40
AVG: L15_CRZ 100 uM
20
0
0
500
1000
1500
TIME, SECONDS
2000
2500
Elicitor experiments with PMSF applied
after forskolin
180
160
NORMALIZED % OD CHANGE
140
120
avg FOR_PMSF_1 um H89
100
avg FOR_PMSF_100 um H89
avg FOR_PMSF_1 um CRZ
80
avg FOR_PMSF_100 um CRZ
avg FOR_PMSF_100 nm CLO
60
40
20
0
0
2000
4000
6000
8000
10000
TIME, SECONDS
12000
14000
16000
DMSO and Ethanol controls
220
200
NORMALIZED % OD CHANGE
180
160
avg FOR_EtOH_1 um CRZ
avg FOR_EtOH_100 um CRZ
140
avg FOR_EtOH_100 nm CLO
avg DMSO_EtOH_100 nm CLO
120
avg DMSO_EtOH_100 um CRZ
avg DMSO_EtOH_1 um CRZ
100
avg DMSO_EtOH_1 um H89
80
avg DMSO_EtOH_100 um H89
avg FOR_EtOH_1 um H89
60
avg FOR_EtOH_100 um H89
40
20
0
0
2000
4000
6000
8000
10000 12000 14000 16000
TIME, SECONDS
TARGETS FOR PRIMARY AND SECONDARY
ELICITORS
Clonidine
Gq
Gi
Cirazoline
PLC
AC
Forskolin
PIP2
IP3 + DAG
cAMP
Ca++
H89
PKA
PKC
Aggregation
Aggregation
Mechanistic interpretation from PMSF action
%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.
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.