1. Jennifer Colby Research Advisor: Dr. Chris Janetopoulos
Visualization of Heterotrimeric G Protein Activation in Living Dictyostelium discoideum
Jennifer Colby
Research Advisor: Dr. Chris Janetopoulos

2. Dictyostelium cAMP Chemosensory Signal Transduction Pathwayb g
Downstream Effectors
Chemotaxis
cAMP
cAR1 (GPCR)
Heterotrimeric G Protein

3. Loss of Fluorescence Resonance Energy Transfer (FRET) can be used to Monitor G Protein Activation
Science Mar 23;291(5512):
CFP
YFP α2 β
CFP
YFP γ
Energy Transfer
Yellow (FRET)
Emission
cAMP
Cyan Excitation
Cyan Emission

4. Total Internal Reflection Fluorescence (TIRF) Microscopy
George, American Laboratory (2004).

5. Loss of FRET can be Visualized with TIRF Microscopy
Gα2-CFP Gβ-YFP Cells Stimulated with cAMP
CFP Emission
+ cAMP
FRET Emission
+ cAMP
N = 60 cells

6. Increase in CFP Emission/Decrease in FRET Emission is Specific to cAMP
Addition of Buffer to Gα2-CFP Gβ-YFP Cells
N = 60 cells

7. YFP Emission in Gβ-YFP Cells
cAMP Does not Induce an Increase in YFP Fluorescence in Cells Expressing Only Gβ-YFP
YFP Emission in Gβ-YFP Cells
Gβ-YFP Emission
+ cAMP
N = 120 cells

8. YFP Emission in Gγ-YFP Cells
cAMP Does not Induce an Increase in YFP Fluorescence in Cells Expressing Only Gγ-YFP
YFP Emission in Gγ-YFP Cells
N = 60 cells

9. CFP Emission in Gα2-CFP Cells
cAMP Induces a Sustained Increase in CFP Fluorescence in Cells Expressing Only Gα2-CFP
CFP Emission in Gα2-CFP Cells
Gα2-CFP Emission
+ cAMP
+ Buffer
N = 60 cells

10. Why Does the Amount of Gα2-CFP Fluorescence Increase upon cAMP Addition?
Changes in cell shape
More membrane in contact with the coverglass could result in increased Gα2-CFP fluorescence

11. YFP Emission in cAR1-YFP Cells
YFP Emission from Membrane Bound cAMP Receptor, cAR1-YFP, Does not Increase upon cAMP Stimulation
YFP Emission in cAR1-YFP Cells
N = 60 cells

12. Inhibition of F-actin Polymerization Does Not Inhibit Increase in CFP Emission in Gα2-CFP Cells
+ cAMP
+ Buffer

13. Why Does the Amount of Gα2-CFP Fluorescence Increase upon cAMP Addition?
Changes in cell shape
Fluorophore (CFP) itself
All negative results have been obtained with YFP tagged proteins but single positive result was obtained with CFP tagged proteins

14. CFP Emission in Gβ-CFP Cells Does Not Increase Upon cAMP Stimulation
Gβ-CFP Emission
+ cAMP

15. Why Does the Amount of Gα2-CFP Fluorescence Increase upon cAMP Addition?
Changes in cell shape
Fluorophore (CFP) itself
Artifact of TIRF microscopy
An increase has not been reported when the FRET assay was used with confocal or epifluorescence microscopy

16. Increase in CFP Emission in Gα2-CFP Cells is Visible with Epifluorescence Microscopy
Gα2-CFP Emission
+ cAMP
0 seconds
7.5 seconds
15 seconds
22.5 seconds
30 seconds
Gα2-CFP Emission
+ cAMP
+ Buffer

17. Why Does the Amount of Gα2-CFP Fluorescence Increase upon cAMP Addition?
Changes in cell shape
Fluorophore (CFP) itself
Artifact of TIRF microscopy
Recruitment of Gα2-CFP to the plasma membrane
Recruitment of protein in response to cAMP explains initial increase in CFP fluorescence
Continuous cycling of subunits between cytosol and plasma membrane explains reduced photobleaching rate

18. Summary
Gα2-CFP emission at the plasma membrane increases to ~10% above the initial value in response to cAMP
The initial increase is sustained as long as cAMP is present
The increase is correlated with a change in Gα2-CFP protein localization
The increase is independent of:
f-actin and changes in cell shape
fluorophore choice
specific microscopy technique
We propose that Gα2 is recruited to, and cycles at, the plasma membrane in response to cAMP stimulus

19. Model of Gα2 Recruitmentb g a2
GTP
GDP
Activation of Effectors
cAMP
cAR
Back to Receptor

20. Future Directions Duration and [cAMP] sensitivity of recruitment
In vitro reconstitutions
Co-IPs
Investigation of the folic acid chemosensory system and Gα4

21. Acknowledgements Dr. Carrie Elzie Miss Sophie Chen Mr. Morgan Sammons
Dr. John Wikswo and SyBBURE
Dr. Chris Janetopoulos