MCB 130L Lecture 7
Signal Transduction
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
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
Intercellular signaling
Fig.20-1
The Immune System
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
T Cell Receptor (TCR) Signaling SIGMA-ALDRICH
Ca2+
CRAC
Reporter GenesReporter 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
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
lac ZTKpromoter
HygromycinResistance gene
NF-AT NF-AT NF-AT IL-2
TATA-70 +1 +47
NFAT Z Construct
NF-AT NFIL-2D NFIL-2C NFIL-2B NFIL-2A
IL-2 Gene
-286 -257 -256 -242 -208 -188 -158 -145 -93 -66TATA
+1 +47
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.
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 pathwayIonomycin* - Ca2+ ionophore
Cyclosporin (CsA)* - interacts with cyclophilin - blocks calcineurin
FK506 - interacts with FKBP- blocks calcineurin
Rapamycin* - inhibits p70 S6 kinase phosphorylation and activation
EGTA* - Ca2+ chelator
T Cell Receptor (TCR) Signaling SIGMA-ALDRICH
ConA
PMA
ionomycinCsAFK506
EGTA
Rapamycin = inhibits IL-2 response
Ca2+
CRAC
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.
Counting cells with a hemacytometer
yellow box = 1 mm x 1 mm x 0.1 mm = 0.1 mm3
=0.1 l
# cells per box = # cells/0.1 l
# cells/0.1 ml x 104 = # cells/ml
*Calculate average of 4 boxes
*Multiply by dilution factor1 mm
1 mm Depth = 0.1 mm Trypan blue stains dead cells
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
Cell culture gone bad