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. 2019 Jul 3;218(8):2659–2676. doi: 10.1083/jcb.201811147

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© 2019 Lee et al.

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Figure 6. — Drg1 is required for Dvl–Daam1 signaling in MCCs. (A–C) The affinity of protein interactions among Drg1, Dvl2, and Daam1 were tested by IP with the adjusted expression levels of two proteins tagged with the same epitope while expressing the other binding partner tagged with a different epitope. The indicated mRNAs were injected at the one-cell stage, and co-IPs were performed using embryos at stage 12. (D) The Drg1–Daam1 interaction is disrupted by Dvl knockdown. Drg1-V5 and Daam1-GFP mRNAs were coinjected with Dvl2 and Dvl3 MOs and/or Dvl2-Flag (MO resistant) mRNA, and co-IPs were performed with embryos harvested at stage 20. The reduced interaction between Drg1 and Daam1 is restored by Dvl2-Flag expression. (E) mCherry-DAD and GFP-CLAMP mRNAs were coinjected with the indicated mRNAs and MOs, and MCCs of embryos at stage 25 were observed. The close location of mCherry-DAD to CLAMP-GFP was disrupted by Drg1 or Dvl knockdown in MCCs. Images were generated by maximum intensity projection of serial z-stack confocal images from surface to subapical regions (up to −2 µm). Scale bars, 5 µm. DD, dimerization domain; DID, diaphanous inhibitory domain; FH1, formin homology 1; FH2, formin homology 2; GBD, GTPase-binding domain.