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Goode

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  • 1. Cytoskeleton Journal Club June 13, 2012 Presented by Suk Namgoong
  • 2. Adenomanous Polypolis Coli (APC)• Main component of Wnt Signaling Pathway• In the absence of bounded Wnt on Wnt receptor, it forms ‘destruction complex’ for β-catanin, - mediates proteolytic degradation of β-catenin• By the bound of wnt into wnt receptor Frizzled, APC release β-catenin and β- catenin was translocated into nucleus and ctivated various Wnt target genes (Development, Morphogenesis, Cancers..)• Tumor Suppressor Gene - Mutations on APC gene cause a hereditary cancer syndrome called Familiar Adenomanous Polypolis (FAP) - Loss of APC functions by mutations has been found frequently in colorectal cancers
  • 3. APC directly nucleates actin assembly via C-terminal Basic Domain© 2010 Okada et al. Okada K et al. J Cell Biol 2010;189:1087-1096
  • 4. ANS : Actin nucleation sequencesNo Sequence Homology with known actin binding domain/motifsIt is dimer in solutio
  • 5. mDia1 and APC-B synergize to promote actin assembly in the combined presence of profilin and capping protein. Okada K et al. J Cell Biol 2010;189:1087-1096© 2010 Okada et al.
  • 6. Two different binding mode of Actin nucleatorsArp2/3 complex stay on the ‘pointed end’ (slow growing end) of actin filamentsFormins remains processively bound on ‘barbed end’ (fast growing end) How about APC? From which side of actin filaments it initiate actin nucleation?
  • 7. Synergistic actin nucleations by APC and mDia1 (Cappuccino) Yeast Bnr1 – bud6 During the nucleation, Formins and other nucleation factors interact each other and synergize nucleations. But what will happen after nucleation? Do formins and other nucleators dissociate after nucleation? or formins carry all other stuffs along with elongation? (not likely, but we don‟t know yet)
  • 8. Fluorescence Probe Labeling with SNAP-tag (O6-alkylguanine-DNA alkyltransferase) 54 9 SNAP FH1 FH2 SNAP APC (Basic) 64 7 Oregon-Green Labeled Actin 64 7 PEG A Laser excitation
  • 9. Immobilized SNAP-APC Stepwise Photobleaching Addition of LatBCan generate actin filaments Inhibit new filament generation(APC is stay bound on pointed end)
  • 10. Bulk Pyrene Actin Assay Stepwise photobleaching for the determination of Oligomeric state in single molecules
  • 11. Complex Formations of mDia1, APC-C, OregonGreen-Actin (LatB treated)
  • 12. Interaction via C-terminal region ofmDia1
  • 13. Namgoong et al., Nature Struct Mol. Biol. 2011I feel De ja vu….. 
  • 14. APC VopLAPC stays bound on the pointedend of actin Filaments VopL is dissociated from the filament end and „spit out‟ new filaments continuously vs “Rocket Launcher” “Machine Gun”

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