1. 5 min for questions Alliance for Cellular Signaling Pasadena, CA May 19, 2003Monday Morning Single and Double Ligand Screens 9:00 Rama Ranganathan Single ligand screen: What did we do; what did we learn? Signaling experiments 9:25 Mel Simon Single ligand screen: What did we do; what did we learn? Microarray experiments 9:55 Break 10:25Paul Sternweis Double ligand screen: experimental design and initial patterns of interaction 10:55 Rama Ranganathan Analysis and results of double ligand screen data 11:20 Discussion 11:50 Wrap-up

2. Ligand Screens AfCS Goal : Quantitative understanding of the signaling network in a mammalian cell 1. Independent of cell type 2. Focused on pathway structure and interactions 3. Complex system: numerically, spatially and organizationally 4. Includes processes in multiple time domains 5. Includes both mass-action and stochastic processes why is that so? The ligand screen is our first real experiment

3. Ligand Screens AfCS Goal : Quantitative understanding of the signaling network in a mammalian cell 1. Independent of cell type 2. Focused on pathway interactions 3. Complex system: numerically, spatially and organizationally 4. Includes processes in multiple time domains 5. Includes both mass-action and stochastic processes 6. Not amenable to simply exhaustive data gathering 7. Requires black box (top down) models to direct choice of cellular signaling probes 8. Requires black box (behavioral) data for experimental design and, later, to evaluate bottom-up models why is that so? Experiments will depend on general understanding of the nature and exent of the complexity of our cells The ligand screen is our first real experiment

4. Ligand Screens What behaviors lie within the black box? What are its rules? Challenge: acquire, organize and interpret a data set that will allow us to  choose the cellular signaling probes that will be most informative  assemble and evaluate input/output data  constrain models of how the signaling pathways work in cells according to overall cellular behaviors The ligand screen is our first real experiment single- ligand screen is...

5. Single Ligand Screen How many inputs are there? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized?

6. Single Ligand Screen How many inputs are there ? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized? Are there rules for network organization? What are the rules? How many regulatory ligands does a cell respond to ? How many do we have to look at ? What is the most efficient way to look ? Depends on # of signaling patterns

7. Single Ligand Screen How many inputs are there? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized? Are there rules for network organization? What are the rules? What ligands are redundant? What ligands give overlapping outputs? How many ligands with “identical” outputs should we consider? How do we identify “identical” experimentally? Can response patterns be productively clusterd ?

8. Single Ligand Screen How many inputs are there? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized? Are there rules for network organization? What are the rules? Can we identify conserved patterns of output patterns even if individual outputs differ? How do we look for them experimentally? Do different response patterns draw from a listable toolbox of signal outputs? Do different response patterns reflect branched pathways, mulitple receptors,...?

9. Single Ligand Screen How many inputs are there? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized? Are there rules for network organization? What are the rules? Distinctive groupings of individual responses? Relative dynamics and time courses?

10. Single Ligand Screen How many inputs are there? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized? Are there rules for network organization? What are the rules? How does a cell assemble its signaling network? Buzzwords: module, heirarchy, parallel How do you rigorously identify such patterns?

11. Double Ligand Screen R A+B = R A + R B ? Conceptual violations of signal additivity 0+0=1 1+1=0 1+0=0 1+1=17 1+0=17 How often do two signaling pathways interact to produce a response other than the sum of the two individual responses This screen is designed to tell us

12. Double Ligand Screen How numerous (dense) are ligand - ligand interactions? Which ligands interact? Are interactions determined primarily by individual outputs? How do you identify such interactions conceptually? How do you identify them experimentally? What are the mechanisms of these interactions? Are the interactions regulated? (Triple-ligand screen...) Are there rules for network organization? What are they? R A+B = R A + R B ?

13. Double Ligand Screen How numerous (dense) are ligand - ligand interactions? R A+B = R A + R B ? How many ligand pairs must we consider? How complex a network should we plan on studying? How do we prepare our models to direct our experimental design? to map such a system? Does a cell process information more through complex interactions than with multiple signals ?

14. Double Ligand Screen How numerous (dense) are ligand - ligand interactions? Which ligands interact? Are interactions determined primarily by individual outputs? R A+B = R A + R B ? Will receptors with similar individual outputs demonstrate similar spectra of double-ligand interactions? How does this result impact on our experiments?

15. Double Ligand Screen How numerous (dense) are ligand - ligand interactions? Which ligands interact? Are interactions determined primarily by individual outputs? How do you identify such interactions conceptually? R A+B = R A + R B ? What do “additive” and “non-additive” mean in systems that saturate? Cooperativity, topping out, competition, inhibition, cross-talk, weirdness...

16. Double Ligand Screen How numerous (dense) are ligand - ligand interactions? Which ligands interact? Are interactions determined primarily by individual outputs? How do you identify such interactions conceptually? How do you identify them experimentally? What are the mechanisms of these interactions? Are the interactions regulated? (Triple-ligand screen...) Are there rules for network organization? What are they? R A+B = R A + R B ?

17. established strategies for the single ligand screen. set up some of the wide array of necessary assays. executed a single-ligand screen in B cells and WEHI. begun to analyze single-ligand data. provocative enough that it won’t be “done” soon established strategies for the double-ligand screen. non-trivial and still under development time and concentration domains different kinds of interactions choice of measured outputs choices influenced by types of assays run partial double-ligand screen on B cells. How do we best and most efficiently execute and analyze the single- and double-ligand screen on a new cell type? So far, we have...

18. Title Single and Double Ligand Screens What’s next? Evaluate applicability of microarrays to double-ligand screen Lots of data; not quite quantitative; expensive Replace with a few well-chosen RT-PCR’s? Bring multiplex lipid analysis on-line Extend to inositol lipids Adopt standards for unified data normalization Develop and adopt formulas to choose ligand concentrations Automate data normalization and integration Add new assays as feasible

19. Criteria for New AfCS Assays Highly multiplexed or complex (integrative) readouts Quantitative data Large dynamic range Reproducible High throughput Responsive to many inputs (  or  ) Continuous time-base readout    15 min Assorted depths into pathways Single cell measurements, but lots of them Data obtainable in < 12 months; established assays

20. Title Single and Double Ligand Screens What’s next? Evaluate applicability of microarrays to double-ligand screen Lots of data; not quite quantitative; expensive Replace with a few well-chosen RT-PCR’s? Bring multiplex lipid analysis on-line Extend to inositol lipids Adopt standards for unified data normalization Develop and adopt formulas to choose ligand concentrations Automate data normalization and integration Add new assays as feasible Initiate the screens on our new cell type

21. Title Alliance for Cellular Signaling Pasadena, CA May 19, 2003 Single and Double Ligand Screens 9:00 Rama Ranganathan Single ligand screen: What did we do; what did we learn? Signaling experiments 9:25 Mel Simon Single ligand screen: What did we do; what did we learn? Microarray experiments 9:55 Break 10:25Paul Sternweis Double ligand screen: experimental design and initial patterns of interaction 10:55 Rama Ranganathan Analysis and results of double ligand screen data 11:20 Discussion 11:50 Wrap-up