1. What are Tetrahymena? Eukaryotic, single cell, ciliated, motile About 50 to 70um long Excitable (action potentials) Model sensory cell (chemosensory, thermosensory, mechanosensory) Grow to 500,000 cells/ml as clonal, axenic cultures Electrophysiology, biochemistry, behavior and molecular biology well described Genome sequenced, knockouts How do they respond to external GTP and ATP?

2. Tetrahymena responses to a GTP gradient (2ul of 10mM GTP added on the right)

3. Why Avoid External ATP and GTP?

4. ATP and GTP are Depolarizing Chemorepellents. They are supposed to be INSIDE cells They are released during cell death and lysis Therefore, they represent lysis of nearby cells They are cytoplasmic indicators “Blood-in-the-water” signals for dangerous situations (worth avoiding) Negative necrotaxis (necrophobiac) Choices: Avoid, adapt or die How do you study these responses in Tetrahymena?

5. Approaches for Studying ATP and GTP Responses in Tetrahymena 1.Behavioral bioassays 2.Intracellular electrophysiology 3.External 32 P-ATP and 32 P-GTP binding 4.External photoaffinity labeling 5.Pharmacology 6.Genome database mining 7.Forward Genetics: Behavioral mutant screens 8.Reverse Genetics: Gene knockouts 9.Proteomics: Identification of gene product functions in the transduction pathways

6. What is the Behavioral Bioassay?

7. Single cell added to solution containing ATP or GTP Different cell added to control solution Cells in micropipet Cells show “avoiding reactions” (AR) No AR, straight swimming

8. Tetrahymena Behavioral Responses to ATP and GTP

9. What are the Electrophysiological Responses to ATP and GTP?

10. Electrophysiological Responses of Tetrahymena to Repellents Transient receptor potentials last about 1 minute. Graded action potentials seen as fast spikes

11. Do Tetrahymena adapt to ATP and GTP? Chemosensory adaptation is a decrease in responsiveness to a ligand as a function of time of exposure to that stimulus

12. No Cross-Adaptation between ATP and GTP Responses

13. How Can We Assay External ATP and GTP Binding? Does this binding change during adaptation?

14. Adaptation Decreases In Vivo Radioactive 32 P-GTP Binding Non-adapted De-adapted Adapted Same thing happens with ATP too

15. 32 P Azido ATP and GTP Photoaffinity Labeling ATPGTP An externally facing ATP-binding protein was separated by standard SDS-PAGE and visualized by autoradiography. A GTP-binding protein band was separated by 2D SDS-PAGE and also visualized by autoradiography

16. Evidence for Different ATP and GTP Receptors Cold GTP doesn’t compete with hot ATP for binding (and vice-versa) No cross-adaptation (behavior and binding) ATP responses are inhibited by pertussis toxin, calphostin C and Rp-cAMPS but not GTP responses The ATP receptor may be metabotropic (P2Y-like?) and the GTP receptor ionotropic (novel?) 32 P-photoaffintity labeling shows 58kD ATP binding protein and 48kD GTP binding protein

17. Tetrahymena Genome Database Comparisons Highest homolgies found to P2Y type mammalian (58% similarity) and 7-transmembrane receptor from Arabidopsis (42% similarity) High homology of ecto-ATPase to mammalian ecto- ATPases (44% similarity) Many signal transduction genes present (G-proteins, protein kinase C, tyrosine kinase, calmodulin, cAMP- dependent protein kinase, etc.)

18. What Stuctural Information Can be Predicted from Database Sequences?

19. Hydropathy Plot and Transmembrane Regions of rat P2Y2 Receptor ( P49651) Looks like a 7-transmembrane receptor

20. Hyropathy Plot and Transmemebrane Regions of Tetrahymena TP2Y Looks like a 7-transmembrane receptor

21. Predicted Transmembrane Domains of Rat P2Y2 and TP2Y Rat P2Y2 Tetrahymena TP2Y Although different amino acid sequences, they look structurally quite similar

22. Structure Determines Function Example: Structure of ATP

24. End