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 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

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 factor

1 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