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:

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

Transmembrane Receptors _Charts/Diversity_of_G_Protein_Coupled_Receptor_Signal_TDP.html >40% of the drugs on the market target specific GPCRs!

Enzyme-linked Receptors Example: Insulin receptor

MAPKKK MAPKK MAPK

Klipp et al. BMC Neuroscience (Suppl 1):S10 doi: / S1-S10 Unicellular eukaryotes are much more complex Signaling pathways can overlap!

Activation%20of%20cAMP-Dependent%20PKA Multicellular eukaryotes have very complex signaling pathways! Nuclear Factor of Activated T cells (NF-AT)

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) IL-2 production requires activation of NF-AT IL-2 binds to IL-2 receptor on T cells and stimulates T cell proliferation

Crabtree, 1999

The drop in ER Ca2+ stimulated Ca 2+ entry into the cell via CRAC Phospholipase C is activated by T cell receptor activation also

Crabtree, 1999

TATA IL-2 Gene NF-ATNFIL-2D (OCT?) NFIL-2C (NF-KB) NFIL-2B NFIL-2A (OCT?) mRNA START ATG DNA Binding Proteins NFAT Z Construct lacZ TK Promoter Hygromycin resistance gene Trimer of the NF-AT Binding Site -70 NF-AT IL-2 TATA ATG Your B3Z cells have been transfected with the lacZ reporter

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 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 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 “CO 2 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-flask B3Z cells Most cell types

Hemacytometer: Use 10X objective Count the number of cells in squares 1-4; determine average # cells/square Average # cells/square X 10 4 = # 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 5 th for the M/W section and Thurs Dec 6 th for the T/Th section