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

Dictyostelium cAMP Chemosensory Signal Transduction Pathway b g Downstream Effectors Chemotaxis cAMP cAR1 (GPCR) Heterotrimeric G Protein

Loss of Fluorescence Resonance Energy Transfer (FRET) can be used to Monitor G Protein Activation Science. 2001 Mar 23;291(5512):2408-11 CFP YFP α2 β CFP YFP γ Energy Transfer Yellow (FRET) Emission cAMP Cyan Excitation Cyan Emission

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Model of Gα2 Recruitment b g a2 GTP GDP Activation of Effectors cAMP cAR Back to Receptor

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

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