Signal Transduction

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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 densities emits
luminescence. The signal transduction pathway
responsible for turning on the genes responsible
for luminescence is called Quorum Sensing.
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Quorum Sensing in Vibrio fischeri
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Signaling in prokaryotes
http//wwwuser.gwdg.de/genmibio/mascher/research1
.html
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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
Calmodulin and calcineurin are examples of
calcium binding proteins involved in multiple
signaling pathways
http//www.answers.com/topic/ligand-gated-ion-chan
nel?cattechnology
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Transmembrane Receptors
gt40 of the drugs on the market target specific
GPCRs!
http//www.sigmaaldrich.com/Area_of_Interest/Life_
Science/Cell_Signaling/Scientific_Resources/Pathwa
y_Slides__ _Charts/Diversity_of_G_Protein_Coupled_
Receptor_Signal_TDP.html
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Enzyme-linked Receptors
Example Insulin receptor
http//www.uic.edu/classes/bios/bios100/mike/sprin
g2003/lect07.htm
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MAPKKK
MAPKK
MAPK
http//www.uic.edu/classes/bios/bios100/mike/sprin
g2003/lect07.htm
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Unicellular eukaryotes are much more complex
Signaling pathways can overlap!
Klipp et al. BMC Neuroscience 2006 7(Suppl 1)S10
  doi10.1186/1471-2202-7-S1-S10
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Multicellular eukaryotes have very
complex signaling pathways!
Nuclear Factor of Activated T cells (NF-AT)
http//www.ambion.com/tools/pathway/pathway.php?pa
thway Activation20of20cAMP-Dependent20PKA
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B3Z
Helper T cells stimulate B cell antibody
production as well as activate other T cells
http//www.unis.org/UNIScienceNet/HeartCVS_knowle
dge.html
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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?article1
2
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Crabtree, 1999
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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/Articl
eLinking.asp?JournalCodeNPYear2007ManuscriptID
b407701fIss4
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Crabtree, 1999
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IL-2 Gene
mRNA START
ATG
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48
-257
-256
-242
-208
-188
-158
-145
-93
-66
-286
TATA
NFIL-2A (OCT?)
NF-AT
NFIL-2D (OCT?)
NFIL-2C (NF-KB)
NFIL-2B
DNA Binding Proteins
NFAT Z Construct
-70
ATG
1
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TATA
lacZ
Hygromycin resistance gene
TK Promoter
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
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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?ca
ttechnology
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X-gal
Blue
Yellow
We will be using chlorophenol red galactoside
(CPRG) (Turns from yellow to purple in the
presence of ?-galactosidase)
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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
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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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B3Z cells
Most cell types
T-flask
Mammalian cells normally grow as a single layer
and can be disrupted for subculturing by
treatment with proteolytic enzymes Growth
conditions are 37C 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
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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
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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 data so 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