Alliance for Cellular Signaling

www.signaling-gateway.org

4th Annual Meeting; Dallas, May 23-26, 2004

Announcements

• You want to see me suffer?

• Others?

This Talk

• Brief review of goals and organization• Transition from primary cells (B cells, myocytes)

to the RAW 264.7 macrophage• Current status of major projects/goals• Issues: capabilities to be strengthened or

developed• Everything I say will be (or should be) discussed

in greater depth as the meeting proceeds• I have not stolen anyone’s slides

Central Questions of the AfCS: I

Question 1: How complex is signal processing in cells? The set of ligands for cellular receptors is the potential combinatorial code of inputs. How much of this input complexity can a cell uniquely decode as outputs?

Experiments: Systematic single- and double- (multi?) ligand screens. Classify output responses; determine degree of crosstalk; identify “hotspots” for later quantitative analysis.

Central Questions of the AfCS: II

Question 2: What is the structure of the whole signaling network? Is the connectivity sparse or dense?

Experiments: Wholesale mapping of relevant protein-protein and small molecule-protein interactions.

Central Questions of the AfCS: III

Question 3: How much does network topology constrain signal processing capability? How much function is specified by the nature of the connections, rather than by the specific biochemical constants of individual activities.

Experiments: Perturbation methods; gain and loss of function, coupled with functional assays.

Central Questions of the AfCS: IV

Question 4: What are the dynamics of the signaling network? Can we visualize how information propagates through the network and emerges as functional activities?

Question 5: Can functional modules be abstracted mathematically? Can we make physical models and predict input-output relationships

Question 6: Why is the network the way it is? Why have the observed solutions been chosen? What is being optimized?

Management• Steering Committee

– Gilman, chair; Taussig (COO): monthly meetings

• Macrophage Committee– Bourne, chair; monthly meetings

• Analysis Group– Simon, chair; monthly meetings

• Editorial Board– Casey, chair

• Lab Meetings (AfCS wide); twice/mo., two labs each• Ad hoc Groups

– e.g., FXM issues

External Advisory Committee

• Joan Brugge, Chair (Harvard) • David Botstein (Princeton)• Bill Catterall (U. of Washington)• Jack Dixon (UC San Diego)• Tony Hunter (Salk Institute)• Bob Lefkowitz (Duke)• Tony Pawson (Samuel Lunenfeld Research Institute)• Paul Sternberg (Caltech)

Talent: AfCS Laboratories

• Bioinformatics: Subramaniam (UCSD)– Sinkovits, Ning, Johnson, Saunders

• Cell Preparation and Analysis: Sternweis (UTSW)– Hseuh, Lin, DeCamp, Ni

• Macrophage Physiology: Seaman (UCSF)– Roach, Rebres

• Molecular Biology: Simon (Caltech)– Fraser, Choi, Eversole-Cire

• Protein Chemistry: M. Mumby (UTSW)– Shu, Brekken

• Microscopy: Meyer (Stanford)– Chandy, O’Rourke, Verghese, Whalen, Heo, Liou

• Antibody: S. Mumby (UTSW)– Han, Levitz

• Lipidomics: Brown (Vanderbilt)– Forrester, Milne, Ivanova

Other VIPs

• Lily Jiang: COO FXM

• Madhu Natarajan, Michal Ronen, Xiaocui Zhu, Dennis Mock, Jeff Forrestor, Stuart Johnson: Analysis and modeling

• Gil Sambrano: MOATLily

Bridging Projects

• Data Modeling and Network Analysis; Arkin• Monitoring Plasma Membrane Signaling Events

by Evanescent Wave (TIRF) Microscopy (Meyer)• Genetically Encoded Indicators of

Phosphorylation and Protein Interaction (Tsien)• Mapping Signal Transduction Pathways in Living

Cells Using PCAs (Michnick)

• Former BP: Lipidomics (Brown)

Collaborations

• Nature Publishing Group (Signaling Gateway)

• Myriad Genetics (Y2H)• Cell Signaling Technology (Antibodies)• Agilent Technologies (DNA microarrays)• Biorad (Bio-Plex; phosphoprotein analysis)• Isis Pharmaceuticals (Antisense)• ATCC (Plasmid distribution)

Census

• Participating investigators: 41

• Ph.D. staff scientists: 30

• Programmers, bioinformatics staff: 15

• Technicians: 40

• Administrative: 6

• Total ~ 130

Current Sponsors

• NIGMS (Rochelle Long, Mike Rogers)• NIAID (Conrad Mallia)• NCI• Eli Lilly (AfCS & Signaling Gateway; Alph

Bingham)• Aventis (Bruce Harris)• Merck• Johnson and Johnson• Anonymous Foundation• Genentech (Signaling Gateway)

The AfCS Cell Systems

• Initial decision to work with primary cells– mouse B lymphocytes– mouse cardiac myocytes– retrospectively, choice of two was unwise

• Risky but worth it; use a surrogate B cell line and attempt to develop a cardiac myocyte line

• Several issues, but the ultimate ironic shaft from the WEHI-231 cell

May/June 2003

• Terminate cardiac myocytes

• Phase out B lymphocytes– Complete single ligand screen (Ca2+, cyclic

AMP, phosphoproteins [11], transcripts)– Complete double ligand screen with Ca2+ &

cyclic AMP

• Initiate work with RAW 264.7 macrophage

What has happened since?In a few Words:

• Last year I came with major problems

• This year I come with major progress and praise

The RAW264.7 Macrophage

• Diploid; mouse• Adherent; easily grown• Transfectable; RNAi• Receptors: GPCRs, TLRs,

TKs, ILs, others• Downstream responses

– Cytokine secretion– Macropinocytosis– Phagocytosis– Chemotaxis

Current Status

• Major Efforts: three– Single/double ligand screens (Monday)

• Ca2+, cAMP, phosphoproteins (21), cytokines (18)• Glycerophospholipids, transcripts (arrays & PCR)

– FXM project: Focus on X Modules (x = Ca2+ & PIP3) (Tuesday)

– Dissemination of information; enhancements for the signaling research community (Tuesday)

July-October 2003

• Culture conditions; frozen stocks• Chromosome number and stability plus stability

of responses• Define ligand list (25); dose-response & time

relationships– Note: ~25 ligands means 252/2 = >300 combinations

for double ligand screen• Adapt and expand old assays

– Ca2+, phosphoproteins, lipids• Add new assays

– Cytokines– Macropinocytosis

Ligand ScreenNovember 2003-May 2004

• Single/double ligand screen with Ca2+ and cyclic AMP essentially completed

• Single/double ligand screen with phosphoproteins (21) 50% complete

• Single/double ligand screen with cytokines one month from completion

• Extensive data on glycerophospholipid composition and ligand-induced changes

• Transcript changes with some ligands and combinations

FXM ProjectOctober 2003-May 2004

• Focus on a module (or two): Ca2+ & PIP3– Dissect a portion of the network; crawl before we walk

and run; integrate all technologies and analysis• Maps: cartoons and Pathway Builder

(computational)• Parts List: Legacy data; arrays and RT-PCR• Assays: populations and single-cells: Ca2+, PIP3,

phosphoproteins (but), lipids• Perturbations (the key)

– Drugs– RNAi: 41 targeted cell lines (34 targets); more at

four/week; I gratefully pay my debt

Contributions to the Research Community

• Website (www/signaling-gateway.org) (NPG)• Data

– Ligand screen and FXM– Phosphoproteins; phosphorylation sites– Images; locations/translocations of protein & domain families

• Plasmid database/Reagents/ATCC• Yeast Two-Hybrid (Myriad)• Antibody Database• Protocols• Research Reports• Molecule Pages (NPG)• Newsletter

Issues: Ligand Screen

• Finish it and realign internal resources• Primary data: Ca2+, cAMP, phosphoproteins,

cytokines: will be complete “now” and “in December”• Other data (transcripts and lipids): experiments will

be guided by primary data; full coverage not possible; we have to snoop around

• Curation and analysis of data: ongoing– Internal debate/inconsistency on extent of analysis– Profiling of ligands; extent of interactions; selection of

interacting ligands; placement of biosensors – Pathways and mechanisms; magical inductionism

Issues: FXM and Beyond

• Phenotypes: are they “real”?– Variability, adaptation, selection, off-target hits– If real, ……– Tension: explore phenomena of interest now or later?

• Multiple knock-downs• Scope; when and how to expand; ooze or jump?• Additional assays: biosensors and protein

phosphorylation• Data analysis and modeling: integration of

disparate types of data, perturbations, pathway data (Y2H etc.), modeling

Issues: Contributions to the Research Community

• We provide a great deal, but awareness and use can always be improved.– Listen to Timo Hannay (Tuesday)

• Data is our primary product– Increasing indications of analysis by others

• What will help?– Publications– “Advertisement”: can we have a story in Nature at the proper

moment?– Specific outreach to the Immunology community

• What else can we monitor?– ATCC; specific information on page hits; data downloads?

Issues: Molecule Pages• A great deal of talent and effort has gone into this

– Has taken significantly longer than expected– Further enhancements are needed, especially a facile

search function

• Idealistic pipedream or major contribution?– The next several months will tell; positive feedback– Truly valuable only if reasonably comprehensive

• Authorship of a MP is a serious effort; so is authorship of a scholarly review article

• The carrot: Nature; link to lab web site; others?• The stick?

Issues: Renewal Application

• Deadline: October 2004

• Cooperation is essential; everyone has to do their job magnificently!

• Recall the thorny funding problem: we operate on a $10M budget with a $5M cap from NIGMS on awards for glue grants