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

2. Outline Background Experimental Methods Results & Discussion

3. Background

4. Complexities of signal transduction pathways

5. 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.

6. 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

7. Overview of Chromatophores

8. Aggregation/Dispersion of Fish Chromatophores Before and after 100 nM Clonidine Before and after 10 µM Forskolin

9. G q mediated signaling

10. EXPERIMENTAL METHODS

11. 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

12. 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). AB CD

13. Cross-talk between G s and G q pathways R PLC Ca 2+  ACPLC cAMP PKA IP 3 DAG PKC   s R qq

14. Cross-talk between G i and G q pathways Ca 2+ R cAMP PKA IP 3 DAG PKC qq  ii ACPLC

15. 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

16. RESULTS AND DISCUSSION

17. ConcentrationPoint of actionResponse typeOptical density Forskolin10 µMAdenyl cyclase activator Hyper- Dispersion PMSF1 mMSerine protease inhibitor at / d/s of PKA Slight dispersion Clonodine100 nMG i activatorAggregation Cirazoline1 & 100 µMG q activatorAggregation H 891 & 100 µMPKA inhibitorAggregation MSH1 nMG s activatorDispersion Table 1: List of agents used with their concentrations and response patterns

18. Dilution curves for Clonidine, Cirazoline and L-15 control

19. Dose response curves for H-89 and DMSO controls

20. Dilution curves for Forskolin and MSH

21. Segmentation of the cAMP pathway by application of forskolin as the primary elicitor

22. Experiments with MSH as the primary elicitor

23. Elicitor experiments with PMSF applied after forskolin

24. DMSO and Ethanol controls

25. TARGETS FOR PRIMARY AND SECONDARY ELICITORS Gi ACForskolin cAMP PKA Aggregation Clonidine Gq PLC PIP2 Aggregation Cirazoline IP3 + DAG Ca ++ PKC H89

26. %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

27. 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.

28. 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.