USE OF ELICITOR SETS TO CHARACTERIZE CELLULAR SIGNAL TRANSDUCTION

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USE OF ELICITOR SETS TO CHARACTERIZE CELLULAR
SIGNAL TRANSDUCTION
Graduate Student Arthi Narayanan Major
Professor Dr. Frank Chaplen
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Outline

• Background
• Experimental Methods
• Results Discussion

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Background
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Complexities of signal transduction pathways
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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.

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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

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Overview of Chromatophores
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Aggregation/Dispersion of Fish Chromatophores
Before and after 100 nM Clonidine
Before and after 10 µM Forskolin
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Gq mediated signaling
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EXPERIMENTAL METHODS
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• 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

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A
B
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).
C
D
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Cross-talk between Gs and Gq pathways
aq
PLC
PLC
R
R
Ca2
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Cross-talk between Gi and Gq pathways
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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 (1100 µM H89, 1100
µM cirazoline, 100 nM clonidine) and monitored
the response for 42 minutes.
Plotted normalized change in OD Vs Time
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RESULTS AND DISCUSSION
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Table 1 List of agents used with their
concentrations and response patterns
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
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Dilution curves for Clonidine, Cirazoline and
L-15 control
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Dose response curves for H-89 and DMSO controls
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Dilution curves for Forskolin and MSH
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Segmentation of the cAMP pathway by application
of forskolin as the primary elicitor
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Experiments with MSH as the primary elicitor
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Elicitor experiments with PMSF applied after
forskolin
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DMSO and Ethanol controls
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TARGETS FOR PRIMARY AND SECONDARY ELICITORS
Gi
Clonidine
AC
Forskolin
cAMP
PKA
H89
Aggregation
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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.

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• 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.

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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.