Signal Transduction

My favorite example… Vibrio fischeri

The light organ of the squid contains Vibrio fischeri, which under high cell densitiesemits luminescence. The signal transduction pathway responsible for turning onthe genes responsible for luminescence is called Quorum Sensing.

Quorum Sensing in Vibrio fischeri

http://wwwuser.gwdg.de/~genmibio/mascher/research1.html

Signaling in prokaryotes:

Signal Transduction: The process by which a cell responds to an external signal

Ligand gated ion channels

http://www.answers.com/topic/ligand-gated-ion-channel?cat=technology

Calmodulin and calcineurin are examples of calcium binding proteins involved inmultiple signaling pathways

Transmembrane Receptors

http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Cell_Signaling/Scientific_Resources/Pathway_Slides___Charts/Diversity_of_G_Protein_Coupled_Receptor_Signal_TDP.html

>40% of the drugs on the market target specific GPCRs!

http://www.uic.edu/classes/bios/bios100/mike/spring2003/lect07.htm

Enzyme-linked Receptors

Example: Insulin receptor

http://www.uic.edu/classes/bios/bios100/mike/spring2003/lect07.htm

MAPKKK

MAPKK

MAPK

Klipp et al. BMC Neuroscience 2006 7(Suppl 1):S10   doi:10.1186/1471-2202-7-S1-S10

Unicellular eukaryotes are much more complex

Signaling pathways can overlap!

http://www.ambion.com/tools/pathway/pathway.php?pathway=Activation%20of%20cAMP-Dependent%20PKA

Multicellular eukaryoteshave very complexsignaling pathways!

Nuclear Factor ofActivated T cells(NF-AT)

http://www.unis.org/UNIScienceNet/Heart&CVS_knowledge.html

Helper T cells stimulate B cell antibody production as well as activate other T cells

B3Z

Helper T cells recognize antigens presented by an antigen-presenting cell in combination with Class II major histocompatibility complex (MHC)

http://www.blobs.org/science/article.php?article=12

IL-2 production requires activation of NF-AT

IL-2 binds to IL-2 receptoron T cells and stimulatesT cell proliferation

Crabtree, 1999

The drop in ER Ca2+ stimulated Ca2+ entry into the cell via CRAC

http://www.rsc.org/delivery/_ArticleLinking/ArticleLinking.asp?JournalCode=NP&Year=2007&ManuscriptID=b407701f&Iss=4

Phospholipase C is activatedby T cell receptor activationalso

Crabtree, 1999

TATA

IL-2 Gene

NF-AT NFIL-2D (OCT?)

NFIL-2C(NF-KB)

NFIL-2B NFIL-2A(OCT?)

+1 +48

mRNASTART ATG

DNA Binding Proteins

-286 -257 -256 -242 -208 -188 -158 -145 -93 -66

NFAT Z Construct

lacZTK

PromoterHygromycin

resistance geneTrimer of the NF-AT

Binding Site

-70

NF-AT NF-AT NF-AT

IL-2

TATA+1 +47 ATG

Your B3Z cells have been transfected with the lacZ reporter

http://www.answers.com/topic/beta-galactosidase?cat=technology

http://fig.cox.miami.edu/~cmallery/150/gene/operon.htm

The reporter we are using only has the lacZ gene from this operon

X-gal

We will be using chlorophenol red galactoside (CPRG)(Turns from yellow to purple in the presence of -galactosidase)

Blue Yellow

ConA: T cell activator; crosslinks cell surface receptors

EGTA: T cell inhibitor; chelates Ca2+

Cyclosporin (CsA): T cell inhibitor; binds and inhibits thecyclophilin receptor

Rapamycin: Neither a T cell activator or inhibitor; inhibitsphosphorylation and activation of p70 S6 kinase

Ionomycin: T cell inhibitor; a Ca2+ ionophore

PMA: T cell activator; specifically activates the PKC pathway

Activators and inhibitors you will be using….

Day 1:•Data presentations and journal club

•Watch video

•Count cells using a hemacytometer

•Subculture B3Z cells for next lab class

Goal: To avoid contamination…

70%Ethanol

Laminar Flow Hood

Must practice exceptional aseptic technique!!!!

•Wash hands/gloves before beginning

•Wipe area before/after work and if spills occurring during work with 70% ethanol

•Work quickly to minimize exposure

•Mammalian cells normally grow as a single layer and can be disrupted for subculturing by treatment with proteolytic enzymes

•Growth conditions are 37°C with 5% CO2 and require a “CO2 incubator”

•Cells are usually grown until confluency is reached

•You will be using T cells called “B3Z” which have little to no attachment to flask

surface and thus do not need to be treated with proteolytic enzymes

T-flaskB3Z cellsMost cell types

Hemacytometer:

Use 10Xobjective

1 2

3 4

5

Count the number of cells in squares 1-4; determine average # cells/squareAverage # cells/square X 104 = # cells/ml

Day 2:•Harvest cells

•Treat with activators/inhibitors

•Harvest cells

•Perform -galactosidase assay

•The plates will be read for you but make sure to get the dataso that you can do the calculations

Notebooks are due at the BEGINNING of the last class-This is Wed Dec 5th for the M/W section and Thurs Dec 6th for the T/Th section