signal transduction. my favorite example… vibrio fischeri the light organ of the squid contains...
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Signal Transduction
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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.
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Quorum Sensing in Vibrio fischeri
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http://wwwuser.gwdg.de/~genmibio/mascher/research1.html
Signaling in prokaryotes:
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Signal Transduction: The process by which a cell responds to an external signal
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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
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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!
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http://www.uic.edu/classes/bios/bios100/mike/spring2003/lect07.htm
Enzyme-linked Receptors
Example: Insulin receptor
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http://www.uic.edu/classes/bios/bios100/mike/spring2003/lect07.htm
MAPKKK
MAPKK
MAPK
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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!
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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)
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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
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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
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Crabtree, 1999
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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
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Crabtree, 1999
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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
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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
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X-gal
We will be using chlorophenol red galactoside (CPRG)(Turns from yellow to purple in the presence of -galactosidase)
Blue Yellow
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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….
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Day 1:•Data presentations and journal club
•Watch video
•Count cells using a hemacytometer
•Subculture B3Z cells for next lab class
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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
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•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
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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
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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
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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