Transcript What are Tetrahymena? - Department of Biological Sciences

What are Tetrahymena?
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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
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How do they respond to external GTP and ATP?
Tetrahymena responses to a GTP gradient
(2ul of 10mM GTP added on the right)
Why Avoid External ATP
and GTP?
ATP and GTP are Depolarizing
Chemorepellents.
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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?
Approaches for Studying ATP and GTP
Responses in Tetrahymena
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Behavioral bioassays
Intracellular electrophysiology
External 32P-ATP and 32P-GTP binding
External photoaffinity labeling
Pharmacology
Genome database mining
Forward Genetics: Behavioral mutant screens
Reverse Genetics: Gene knockouts
Proteomics: Identification of gene product
functions in the transduction pathways
What is the Behavioral
Bioassay?
Single cell added to solution containing ATP or GTP
Cells show “avoiding reactions”
(AR)
Cells in micropipet
Different cell added to control solution
No AR, straight swimming
Cells in micropipet
Tetrahymena Behavioral
Responses to ATP and GTP
What are the
Electrophysiological Responses
to ATP and GTP?
Electrophysiological Responses
of Tetrahymena to Repellents
Transient receptor potentials last about 1 minute. Graded action potentials seen as fast spikes
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
No Cross-Adaptation between ATP
and GTP Responses
How Can We Assay External ATP
and GTP Binding?
Does this binding change during adaptation?
Adaptation Decreases In Vivo
32
Radioactive P-GTP Binding
Non-adapted
De-adapted
Adapted
Same thing happens with
ATP too
32P Azido ATP
and GTP
Photoaffinity Labeling
ATP
GTP
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
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?)
• 32P-photoaffintity labeling shows 58kD ATP binding
protein and 48kD GTP binding protein
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 ectoATPases (44% similarity)
• Many signal transduction genes present (G-proteins,
protein kinase C, tyrosine kinase, calmodulin, cAMPdependent protein kinase, etc.)
What Stuctural Information
Can be Predicted from
Database Sequences?
Hydropathy Plot and Transmembrane
Regions of rat P2Y2 Receptor (P49651)
Looks like a 7-transmembrane receptor
Hyropathy Plot and Transmemebrane
Regions of Tetrahymena TP2Y
Looks like a 7-transmembrane receptor
Predicted Transmembrane
Domains of Rat P2Y2 and TP2Y
Rat
P2Y2
Tetrahymena
TP2Y
Although different amino acid sequences, they look structurally quite similar
Structure Determines Function
Example: Structure of
ATP
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