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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?.

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slide1

USE OF ELICITOR SETS TO CHARACTERIZE CELLULAR SIGNAL TRANSDUCTION

Graduate Student: Arthi Narayanan

Major Professor: Dr. Frank Chaplen

outline
Outline
  • Background
  • Experimental Methods
  • Results & Discussion
slide5

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.
slide6

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
slide8

Aggregation/Dispersion of Fish Chromatophores

Before and after

100 nM Clonidine

Before and after

10 µM Forskolin

slide11

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
slide12

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

cross talk between g s and g q pathways

as

IP3

DAG

bg

bg

cAMP

PKC

PKA

AC

PLC

Cross-talk between Gs and Gq pathways

aq

PLC

PLC

R

R

Ca2+

cross talk between g i and g q pathways

bg

bg

ai

aq

AC

PLC

IP3

DAG

cAMP

R

Ca2+

PKC

PKA

Cross-talk between Gi and Gq pathways
slide15

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

targets for primary and secondary elicitors

Gq

Cirazoline

PLC

PIP2

IP3 + DAG

Ca++

PKC

Aggregation

TARGETS FOR PRIMARY AND SECONDARY ELICITORS

Clonidine

Gi

AC

Forskolin

cAMP

PKA

H89

Aggregation

slide26

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.
slide27

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
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.