1. MCB 130L Lecture 7 Signal Transduction

2. This week’s lab T-cell line containing NFAT Z Activators and/or inhibitors CPRG Assay for  -galactosidase cleavage of CPRG Objective: To examine the effects of various inhibitors and activators on IL-2 expression

3. Cell Signaling Credit: Nicolle Rager, National Science Foundation 1.Synthesis of signaling molecule 2.Release/display of signaling molecule 3.Transport of signal to target cell 4.Detection of signal by receptor protein 5.Change in cell growth, differentiation, or metabolism

4. Intercellular signaling Fig.20-1

6. The Immune System

8. T cells Cytotoxic recognize antigens produced by the cell (e.g. viral proteins) interact with antigen-MHCI express glycoprotein CD8 kill infected cells Helper* recognize exogenous or ingested antigens interact with antigen-MHCII express glycoprotein CD4 stimulate T- and B-cell and macrophage proliferation and activity T cell response is mediated by activation of the T cell receptor after binding the antigen-MHC complex with the help of CD4 or CD8

9. T cell receptor signaling Macian 2005

10. T Cell Receptor (TCR) Signaling SIGMA-ALDRICH Ca 2+ CRAC

11. Reporter Genes Reporter genes can "report" many different properties and events: strength of promoters efficiency of gene delivery systems intracellular fate of a gene product, a result of protein traffic efficiency of translation initiation signals success of molecular cloning efforts. Transcriptional reporter Translational reporter http://www.wormbook.org/chapters/www_reportergenefusions/reportergenefusions.html

12. Commonly used reporter genes LacZ:  -galactosidase Indicator plates (X-gal) Colorimetric assays GFP Fluorescence of live cells Luciferase (firefly) chemiluminescence CAT: chloramphenicol acetyl-transferase ELISA Enzyme assay Selection for resistance

13. lac Z TK promoter Hygromycin Resistance gene NF-AT IL-2 TATA -70+1+47 NFAT Z Construct NF-ATNFIL-2DNFIL-2CNFIL-2BNFIL-2A IL-2 Gene -286-257-256-242-208-188-158-145-93-66 TATA +1+47

14. Detection of ß-Galactosidase: ß-galactosidase hydrolyzes chlorophenol red galactoside (CPRG), turning it from yellow to purple at alkaline pH. The color change provides a quantitative measure of gene transcription during T cell activation.

15. T-cell activators and inhibitors Activators Inhibitors Concavalin A (ConA)* - plant lectin that binds mannose residues - crosslinks T-cell receptors to activate PMA* - activates protein kinase C pathway Ionomycin* - Ca 2+ ionophore Cyclosporin (CsA)* - interacts with cyclophilin - blocks calcineurin FK506 - interacts with FKBP - blocks calcineurin Rapamycin* - inhibits p70 S6 kinase phosphorylation and activation EGTA* - Ca 2+ chelator

16. T Cell Receptor (TCR) Signaling SIGMA-ALDRICH ConA PMA ionomycin CsA FK506 EGTA Rapamycin = inhibits IL-2 response Ca 2+ CRAC

18. Use of NFAT Inhibitors in HIV Research Kinoshita et al. Cell 1998 CsA and FK506 Inhibit Completed HIV-1 Reverse Transcription. HIV-1 challenge of CD4+ T cells was performed at 3 days post treatment and assayed after 6 days.

19. Counting cells with a hemacytometer yellow box = 1 mm x 1 mm x 0.1 mm = 0.1 mm 3 =0.1  l # cells per box = # cells/0.1  l # cells/0.1 ml x 10 4 = # cells/ml *Calculate average of 4 boxes *Multiply by dilution factor 1 mm Depth = 0.1 mm Trypan blue stains dead cells

20. Aseptic Cell Culture Technique Laminar Flow Hood 70% Ethanol Surface of hood Bottles Gloves Autoclaved glass Flamed metal Opening of bottles, pipetting, etc. Placement of equipment Contaminated cells

21. Cell culture gone bad