1. Cell Preparation and Experimental Lab Dallas

2. Cell Lab – Dallas

3. General Missions of Core C Production of cells: mouse splenic B cells WEHI cells mouse cardiac myocytes Ligand Screen: treatments sample preparation Assays for ligand screen: cAMP intracellular Ca ++ Protein traps: cellular expression of baits isolation of protein complexes

4. Zhen’s picture The Cells

5. 111 billion splenic B cells A milestone

6. April 15, 2002 Vol. 1, No. 1 BC AfCS Research Reports www.afcs.org Purification and Characterization of Mouse Splenic B Lymphocytes Scientific Staff Alliance for Cellular Signaling Laboratories University of Texas Southwestern Medical Center, Dallas, TX and Veteran’s Administration Medical Center, San Francisco, CA Abstract Initial efforts of Alliance for Cellular Signaling (AfCS) laboratories have focused on establishing standard and reproducible procedures for purifying B lymphocytes (B cells) from mouse spleen. Our goal was to obtain cultures that permit reliable measurement of short- term signaling events and changes in gene expression. This report describes a negative-selection procedure for B cell enrichment and characterization of the isolated cell population using multiparameter fluorescence- activated cell sorting (FACS) analysis. Results show that we obtain approximately 47 x 10 6 cells per spleen, of which 96% contain the B220 marker typically expressed on B cells. More than 80% of these cells can be further characterized as resting, mature B2 cells. Conditions for maintaining viable cells in serum-free medium for short-term culture were defined. Supplemented Iscove’s Modified Dulbecco’s Medium (SIMDM) was most consistent among several media in maintaining cell viability for 24 hours. B cells also showed consistent and reproducible signaling to manipulate gene expression in these cells based on reasonably complete knowledge of their genome. We also wish to study relatively normal cells from a mammalian organism. Based on these criteria one of the cells chosen was the resting B lymphocyte (B cell) of the mouse. By secreting antigen-specific immunoglobulin antibody, the B cell plays an essential role in host defense. Resting B cells reside in the circulation and migrate to the spleen and lymph nodes. Activation of the B-cell receptor by antigen in conjunction with appropriate co- stimulation causes the resting B cell to proliferate and differentiate into a plasma cell, which makes and secretes large amounts of immunoglobulin antibody. In addition to the antigen receptor, B cells exhibit a large number of additional cell surface receptors, which are activated by T-cell surface molecules, cytokines, bacterial endotoxin, and other ligands. Immunologists have begun to define key steps in signaling pathways downstream of these receptors that regulate steps in development of the mature B cell and modulate responses to antigen (1,2,3).

7. Ligand Screen – B cells Cell Lab in Dallas Produces Cells Treats Cells with Ligands Molecular Biology Lab Microarray analysis Protein Lab P-proteins Antibody Lab P-proteins Pilot project - Cornell Lipid analysis cAMP Calcium

8. Current Requirements 6 Outputs Calcium cAMP RNA (microarray) P-protein blots 2D gels Lipids B cells/assay (millions) ~1.5 1.5 18 6 Assays/year/6* 11,000 1000 3000 * Based on 100 billion B cells/year

9. Ligand Screen Where are we?

10. Survey of the ligands

12. Current Ligand List LigandListB.xls D:\LigandListB.xls

13. Current Ligand List 31 approved ligands for the initial B cell single-ligand screen yellow – tested and gave probable response (24) blue – not yet tested (4) tan – no response in original test but should be there (3) 4 additional ligands to be tried based on expression in GNF arrays. 4 ligands - no response in testing but still under re-evaluation 9 ligands - unresponsive in resting cells – probably due to absence of receptors, which are expressed in response to stimuli (use for ligand pairs?) 5 ligands have been rejected. 21 done in triplicate – up to 18 more to complete single ligand screen

14. Patterns of Response

16. Assays: cAMP

17. Challenges EIA – competition of Peroxidase-cAMP with acetylated cAMP from samples No PDE inhibitors Rapid response Assay interference Solution More sensitive assay – low fmol range switch to protocols using acetylation of cAMP

18. Effects of Propranolol on cAMP Responses Terbutaline: 10  M Propranolol: 1  M Forskolin: 100  M

19. 10  M terbutaline 10  M PGE2 0.34 nM IL4 1  M LPA

20. cAMP – Ligand Interactions

22. Calcium assay

23. Load cells with dye Incubate Measure basal fluorescence Add ligand – measure fluorescence over time Fmax – use detergent to lyse cells and expose dye to saturating Ca to measure Fmin - add EDTA to chelate Ca ++ to measure basal fluorescent of dye F – Fmin Free Ca++ = X Kd Fmax - F

25. 0.2  M BLC 1 mM LPA

26. Interactions ?

28. 3.8%55.5%86.0%40.4% cell number Fluo-3 (fluorescence intensity) Intracellular Calcium Response to Anti-IgM (0.3  M)

29. Response of Ca: (0-5 min) stimulation with anti-IgM

30. Assays with WEHI cells

31. Calcium Assay in WEHI-231 Cells Dose-dependent response to anti-IgM

32. cAMP assay in WEHI-231 cells

33. Protein-interaction traps 2-hybrid in yeast Pull-downs with exogenous tagged proteins Isolation of protein complexes formed with tagged proteins expressed in cells Co-immunoprecipitation of endogenous complexes

34. CO-precipitation/mass spectrometry approach Express FLAG-tagged bait Immunoaffinity pulldown Gel separation Excised band/trypsin digest MS/MS Database search Cell Lab

35. Expression of baits in WEHI cells Retroviral Infection

36. General Strategy Express tagged-protein driven by 5’-LTR of virus Bis-cistronic expression of a cell surface protein or antibiotic resistance gene for selection of cells expressing bait

37. Mscv-FTM-YFP Mscv-neo-EGFP Retroviral Expression of Flourescent protein in WEHI Mscv-neo-EGFP MSCV-FTM-YFP---CD4

38. How Long Can the Expression Last?

39. Current state of retroviral infection 50-70% infection efficiency (10 5 -10 6 cfu/ml) >90% viability Expression last over 8 wks Neomycin selection

40. Isolation of protein complexes Pilot experiments with CMV-3x-Flag-ERK2 (from Melanie Cobb) Transient expression in HEK293 cells

41. Prelimary Results: Erk2 Associated Proteins in 293 cells spectrin, alpha & beta ATP-dependent RNA helicase A chromatin-specific transcription elongatiopn factor splicing Factor 3b, subunit 1 & 2 ribosomal protein S6 kinase (Rsk2P90) methylosome protein 50 (MEP50) Human kinesin-related motor protein EG5 protein methyltransferase rErk2 kiaa0122 gene product Others… 3x-Flag- Erk2

42. Goals of the next year Ligand pair interaction in splenic B cells (reduce material required) Initiate screen in WEHI line Initiate screen in cardiac myocytes ?? Optimize protein trap protocols - streamline screens Continue cooperative development of output assays

43. Lead Scientist Zhen Yan Technical Staff Angela Alexander Julie Collins James Davis Richard Davis Jody Girouard Joella Grossoehme Read Pierce Jason Polasek Meghdad Rahdar Associate Directors Don Hilgemann Richard Scheuermann Scientific staff Robert Hsueh Keng-mean Lin Yan Ni The People