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, Colombo K., Johnston C.A., McCudden C.R., Sidervoski D.P. and Grönczy P. Cell, Vol. 119,

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…

G-protein coupled receptors   

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

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

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

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

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

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

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

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

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

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

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

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

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 Constructs didn’t bind either Intragenic revertant ric-8(md1712 md303) rescued binding

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??

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

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

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

GDI affects the GEF Kinetics Now this graph makes sense…

Role of G 

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 

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

The final model

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?

Question But they are both Asymmetric?