Transcript Trott Lecture 3 PPT

Signal Transduction
My favorite example… Vibrio fischeri
The light organ of the squid contains Vibrio fischeri, which under high cell densities
emits luminescence. The signal transduction pathway responsible for turning on
the genes responsible for luminescence is called Quorum Sensing.
Quorum Sensing in Vibrio fischeri
Signaling in prokaryotes:
http://wwwuser.gwdg.de/~genmibio/mascher/research1.html
Signal Transduction: The process by which a cell responds to an external signal
Ligand gated ion channels
Calmodulin and calcineurin are examples of calcium binding proteins involved in
multiple signaling pathways
http://www.answers.com/topic/ligand-gated-ion-channel?cat=technology
Transmembrane Receptors
>40% of the drugs on the market target specific GPCRs!
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
Enzyme-linked Receptors
Example: Insulin receptor
http://www.uic.edu/classes/bios/bios100/mike/spring2003/lect07.htm
MAPKKK
MAPKK
MAPK
http://www.uic.edu/classes/bios/bios100/mike/spring2003/lect07.htm
Unicellular eukaryotes are much more complex
Signaling pathways can overlap!
Klipp et al. BMC Neuroscience 2006 7(Suppl 1):S10 doi:10.1186/1471-2202-7-S1-S10
Multicellular eukaryotes
have very complex
signaling pathways!
Nuclear Factor of
Activated T cells
(NF-AT)
http://www.ambion.com/tools/pathway/pathway.php?pathway=
Activation%20of%20cAMP-Dependent%20PKA
B3Z
Helper T cells stimulate B cell antibody production as well as activate other T cells
http://www.unis.org/UNIScienceNet/Heart&CVS_knowledge.html
IL-2 production requires
activation of NF-AT
IL-2 binds to IL-2 receptor
on T cells and stimulates
T cell proliferation
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
Crabtree, 1999
Phospholipase C is activated
by T cell receptor activation
also
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
Crabtree, 1999
IL-2 Gene
mRNA
START
-286
-257 -256
-242
-208
-188
-158
-145
-93
-66
+1
TATA
NF-AT NFIL-2D
(OCT?)
NFIL-2C
(NF-KB)
NFIL-2B
ATG
+48
NFIL-2A
(OCT?)
DNA Binding Proteins
NFAT Z Construct
-70
+1
+47
TATA
TK
Promoter
Hygromycin
resistance gene
NF-AT NF-AT NF-AT
Trimer of the NF-AT
Binding Site
IL-2
Your B3Z cells have been transfected with the lacZ reporter
ATG
lacZ
The reporter we are using only has the lacZ gene from this operon
http://fig.cox.miami.edu/~cmallery/150/gene/operon.htm
http://www.answers.com/topic/beta-galactosidase?cat=technology
X-gal
Blue
Yellow
We will be using chlorophenol red galactoside (CPRG)
(Turns from yellow to purple in the presence of -galactosidase)
Activators and inhibitors you will be using….
ConA: T cell activator; crosslinks cell surface receptors
EGTA: T cell inhibitor; chelates Ca2+
Cyclosporin (CsA): T cell inhibitor; binds and inhibits the
cyclophilin receptor
Rapamycin: Neither a T cell activator or inhibitor; inhibits
phosphorylation and activation of p70 S6 kinase
Ionomycin: T cell inhibitor; a Ca2+ ionophore
PMA: T cell activator; specifically activates the PKC pathway
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
T-flask
Most cell types
B3Z cells
•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
Hemacytometer:
Use 10X
objective
1
2
5
3
4
Count the number of cells in squares 1-4; determine average # cells/square
Average # 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 data
so 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