use of elicitor sets to characterize cellular signal transduction graduate student: arthi narayanan...
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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.