1. RIC-8 is Required for GPR-1/2 Dependent G  Function During Asymmetric Division of C.elegans embryos Colombo K., Johnston C.A., McCudden C.R., Sidervoski D.P. and Grönczy P. Cell, Vol. 119, 219-230 Colombo K., Johnston C.A., McCudden C.R., Sidervoski D.P. and Grönczy P. Cell, Vol. 119, 219-230

2. Background  G-Proteins are heterotrimeric protein complexs…G  + (G  +G  ) = G protein  Ligand binding to GCPR activates G- proteins…  G-Proteins are heterotrimeric protein complexs…G  + (G  +G  ) = G protein  Ligand binding to GCPR activates G- proteins…

3. G-protein coupled receptors   

4. Heterotrimeric G protein cycle GEF (ligand-bound receptor) GAP (RGS) “ON” “OFF”        ©2002 Lee Bardwell

5. What does G do next ?    Adenylate Cyclase ©2002 Lee Bardwell

6. Background  G-Proteins are heterotrimeric protein complexs…G  + (G  +G  ) = G protein  Ligand binding to GCPR activates G- proteins…  Many types of G-proteins made of different sub-unit compositions all with different effectors.  Interested in GOA-1 and GPA-16  G-Proteins are heterotrimeric protein complexs…G  + (G  +G  ) = G protein  Ligand binding to GCPR activates G- proteins…  Many types of G-proteins made of different sub-unit compositions all with different effectors.  Interested in GOA-1 and GPA-16

7. In One Cell embryos…it works much differently Experimental evidence has shown that …. 1) G-pro’s activate without GCPR 2) Single cell cleavage involves a GoLoco protein GPR1/2- a GDI The hypothesis is that.. G  -GDP mediates spindle elongation not G  -GTP Two G  proteins… GOA-1 and GPA-16

8. Experimental Question and Significance  What are the mechanisms regulating G-protein mediated pulling forces from Spindles  Do other proteins get involved?  What are the mechanisms regulating G-protein mediated pulling forces from Spindles  Do other proteins get involved? RIC-8 -Thought to positively regulate G  activity -Mutants show abnormal spindle formation

9. The story…  Where is Ric-8?  RIC-8 Functions?  What are RIC-8 and GPR-1/2  Role of G  Subunits?  Functional model  Where is Ric-8?  RIC-8 Functions?  What are RIC-8 and GPR-1/2  Role of G  Subunits?  Functional model

10. Results…RIC-8 distribution RIC-8 = Red  Tubulin = Green DNA= Blue

11. RIC-8 distribution Mutant strains don’t produce null ric-8 embryos ric-8 RNAi gives a more intense knock down

12. RIC-8 distribution Interfering with goa-1 and gpa-16 doesn’t effect RIC-8 distribution. Conclusion: RIC-8 is primarily found in Cytoplasm a bit in the cortex and peri-nuclear area

13. Results….RIC-8 required for asymmetric division Ric-8 RNAi + mutant equals strongest effect

14. Movies… 1.Wild type first cell division 2.Ric-8 RNAi first cell division

15. ric-8 is required for pulling forces Focus on the loss of asymmetry…not so much The overall reduction

16. RIC-8 interacts with GOA-1 and GPA-16…In vitro Ric-8(md303) = AA substitution at conserved residue (A275E) Ric-8(md1909)= deletion of AA b/w 193-249 Constructs didn’t bind either Intragenic revertant ric-8(md1712 md303) rescued binding

17. RIC-8 interacts with GOA-1 and GPA-16…In vivo Coimmunoprecipitation Lack of RIC-8 GOA-1 Coprecip. In Mutant strains Why no binding to to GPA-1/2?? Where is GPA-16??

18. RIC-8 is a GEF…GPR-1/2 a GDI Biochemical GTP S assay RIC-8 prefers GOA-1-GDP Increase in RIC-8 = Increase In GTP S binding…. RIC-8 is a GEF RIC-8 increases the G-Protein Cycle GPR-1/2 inhibits RIC-8 Association with GOA-1

19. HOW are a GEF and a GDI working together to activate a G  subunit?

20. RIC-8 necessary for GOA-1/ GPR1/2 interaction GPR-1/2 GOA-1 association Is not required for RIC-8 binding To GOA-1… THE REVERSE RIC-8 GOA-1 binding is required for GPR-1/2 binding To GOA-1 RIC-8 acts on GOA-1 before GPR-1/2

21. GDI affects the GEF Kinetics Now this graph makes sense…

22. Role of G 

23. G  inactivation alleviates RIC-8 requirement RIC-8 works by increasing the rate of G-pro cycle Knocking down the G  is going to do the same thing More free G 

24. G  inactivation alleviates RIC-8 requirement Epistasis assay GPB function is Epistatic to RIC-8

25. The final model

26. Some Questions  What happened to GPA-16?  How does the RGS in the model fit in your experimental data?  Why can’t GPR-1/2 act on G  -GDP as soon as it is dissociated from G-protein complex?  What happened to GPA-16?  How does the RGS in the model fit in your experimental data?  Why can’t GPR-1/2 act on G  -GDP as soon as it is dissociated from G-protein complex?

27. Question But they are both Asymmetric?