Lee M et al. (2019), Developmentally regulated GTP-binding protein 1...

XB-ART-56106

J Cell Biol 2019 Aug 05;2188:2659-2676. doi: 10.1083/jcb.201811147.

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Developmentally regulated GTP-binding protein 1 modulates ciliogenesis via an interaction with Dishevelled.

Lee M , Hwang YS , Yoon J , Sun J , Harned A , Nagashima K , Daar IO .

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Cilia are critical for proper embryonic development and maintaining homeostasis. Although extensively studied, there are still significant gaps regarding the proteins involved in regulating ciliogenesis. Using the Xenopus laevis embryo, we show that Dishevelled (Dvl), a key Wnt signaling scaffold that is critical to proper ciliogenesis, interacts with Drg1 (developmentally regulated GTP-binding protein 1). The loss of Drg1 or disruption of the interaction with Dvl reduces the length and number of cilia and displays defects in basal body migration and docking to the apical surface of multiciliated cells (MCCs). Moreover, Drg1 morphants display abnormal rotational polarity of basal bodies and a decrease in apical actin and RhoA activity that can be attributed to disruption of the protein complex between Dvl and Daam1, as well as between Daam1 and RhoA. These results support the concept that the Drg1-Dvl interaction regulates apical actin polymerization and stability in MCCs. Thus, Drg1 is a newly identified partner of Dvl in regulating ciliogenesis.

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Species referenced: Xenopus laevis
Genes referenced: daam1 drg1 drg2 dvl1 dvl2 dvl3 fmn1 isyna1 limk1 mcc rho rhoa tal1
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	Figure 1. Drg1 associates with Dvl. (A) Exogenous Drg1 interacts with Dvl2. Dvl2-HA and Drg1-V5 mRNAs were injected in Xenopus embryos, and reciprocal co-IPs were performed. 2.5% of input was loaded. (B) Endogenous Drg1 associates with Dvl2. Using lysates from HT-29 and LS174T colon cancer cells, endogenous co-IPs were performed by precipitating endogenous Dvl2. 2.5% of input was loaded. IgG pull-down is a negative control. (C) Drg1 interacts with Dvl2 and Dvl3, but not Dvl1. The indicated Dvl-HA and Drg1-V5 mRNA were coinjected, and co-IPs were conducted by pull-down in both directions. 2.5% of input was loaded. (D) Drg1 interaction domain mapping of Dvl2. The DEP+C region of Dvl2 is necessary and sufficient to interact with Drg1. The indicated WT and deletion mutants of Dvl2-HA and Drg1-V5 mRNAs were expressed in Xenopus embryos, and co-IPs were performed in both directions. 2.5% of input was loaded. (E) Dvl2 interaction domain mapping of Drg1. WT and the indicated deletion mutants of Drg1-V5 and Dvl2-HA were expressed in Xenopus embryos, and co-IPs were conducted in both directions. Amino acids between 329 and 344 of Drg1 are required for the association with Dvl2. 2.5% of input was loaded. (F) Dvl2-HA associates with Drg1-GST in vitro. 1 µg of the purified recombinant proteins was used for the in vitro binding assay. The asterisk marks a dimerized form of isolated TGS domain. 10% of input was loaded. (G) The subcellular localization of Drg1 in an MCC. Drg1-GFP (green) and centrin-RFP (red, basal body marker) mRNAs were coinjected into the marginal region of both ventral blastomeres at the four-cell stage. The embryos were observed at stage 25. Images were generated by maximum intensity projection from serial z-stack images. Scale bar, 5 µm.
	Figure 2. The Drg1–Dvl interaction is important for ciliogenesis in MCCs and the GRP. (A) Drg1 knockdown causes a significant reduction of acetylated tubulin on the epidermis of tadpoles, and the expression of WT Drg1, but not the Δ329–344 mutant, rescues the knockdown phenotype. A cocktail of synthetic mRNAs and morpholinos (7.5 ng each) was injected into both ventral blastomeres at the four-cell stage of embryos. The injected embryos were fixed at stage 27. For immunostaining, anti-acetylated tubulin antibody was used to visualize multicilia (red), and membrane-GFP (green) was used as a tracer. Relative acetylated tubulin intensity is quantified (image n = 45 from 15 embryos for each condition). **, P < 0.0001, one-way ANOVA; scale bars, 100 µm. Error bars indicate ± SD. (B) Scanning EM of embryonic epidermis confirms Drg1 knockdown causes ciliogenesis defects. 7.5 ng of control and Drg1 morpholinos was injected into both marginal ventral blastomeres at the four-cell stage, and embryos were fixed at stage 31. Scale bars, 12 µm. (C) Quantification of cilia length in MCCs. Data are plotted as the mean, with error bars representing SD. Measured cilia, n > 100; embryos per group, n = 3; , P < 0.001; , P = 0.012, one-way ANOVA. (D) Quantification of cilia number per MCC, n = 10; embryos per group, n = 3; **, P < 0.0001, one-way ANOVA. Error bars indicate ± SD. (E) The area of epidermal MCCs of stage 27 embryos; MCCs, n = 100; embryos per group, n = 10; two-tailed unpaired t test. Error bars indicate ± SD. (F) Quantification of the fluid flow with green fluorescent beads (Videos 1, 2, and 3). Velocity of five beads along the body axis was manually measured. Measured bead velocity per embryo, n = 5; embryos (stage 27–28) per group, n = 10; stage 27 embryos; , P < 0.0001, one-way ANOVA. Error bars indicate ± SD. (G) Drg1 knockdown decreases the length of cilia in the GRP. The indicated mRNAs and MOs were injected to both marginal dorsal blastomeres at the four-cell stage, and embryos were harvested at stage 18. Dissected GRPs were fixed, followed by immunostaining using acetylated tubulin antibody (red) and anti-GFP antibody (green). Mem-GFP is used as a tracer. Scale bars, 30 µm. (H) Quantification of cilia length in the GRP, measured cilia per group; n > 900, dissected GRPs per group; n = 10, , P < 0.0001, one-way ANOVA. Error bars indicate ± SD. (I) Quantification of cilia number on GRP; the ratio of the number of ciliated cells expressing GFP to total GRP cells expressing GFP in experimental groups is normalized to the control and expressed as a relative value to the control (set as 1). GRPs per group; n = 10, **, P < 0.0001, one-way ANOVA. Error bars indicate ± SD. a.u., arbitrary units; n.s., not significant.
	Figure 3. Drg1 is required for planar polarization and apical docking of basal bodies in MCCs. (A) Planar polarization defect in Drg1 morphants. CLAMP-GFP (rootlet marker) and centrin-RFP (basal body marker) were coinjected with the mixture of the indicated MOs and mRNAs. Areas enclosed by the red box are magnified in the insets, and arrows indicate the orientation of basal body/rootlets in MCCs. The circular plots depict polarity of basal body/rootlets in MCCs. Each arrow indicates the mean polar direction of basal body/rootlets in a single MCC. Arrow length depicts 1 minus circular variance of basal body/rootlet orientation around the mean. Different colored arrows represent data from different embryos. Scale bars, 5 µm. (B) Basal body docking defect at the apical regions of MCCs upon Drg1 knockdown. CLAMP-GFP and centrin-RFP mRNAs were coinjected with the cocktail of the indicated MOs and mRNAs. Serial z-stack confocal images were projected in the y-z plane. Arrowheads indicate the position of MCC apical surface. Scale bars, 3 µm. (C) Basal body docking defect at the MCC apical region upon Drg1 knockdown. CLAMP-GFP (green) and membrane-RFP (red) mRNAs were injected with the indicated morpholinos and mRNAs. Top panels indicate apical region of MCCs and bottom panels 2 µm below the apical region. Scale bars, 5 µm.
	Figure 4. Drg1–Dvl interaction modulates apical actin enrichment in MCCs through Rho activation. (A) Apical actin meshwork decreases upon Drg1 knockdown. The loss of apical actin signal is rescued by expression of Drg1 WT, but not by the Drg1 Δ329–344 mutant. The embryos were injected with the indicated MOs and mRNAs, fixed at stage 25, and stained with phalloidin (red) to visualize cortical F-actin in apical region of MCCs. GFP-CLAMP (green) marks rootlets. **, P < 0.0001, one-way ANOVA; n = 65. Scale bars, 5 µm. Error bars indicate ± SD. (B) Drg1 knockdown decreases Rho activity. Synthetic mRNAs of RBD-GFP (green) and centrin-RFP (red) were coinjected with the indicated MOs and mRNAs, and embryos were fixed at stage 25. Fluorescence intensity of experimental groups is normalized to the control MCCs and expressed as a percentage relative to control. Expression level of RBD-GFP was tested by Western blot. Images are generated by maximum intensity projection of serial z-stack confocal images from apical (0 µm) to subapical region (−2.5 µm). , P < 0.0001, one-way ANOVA; n = 49. Scale bars, 5 µm. Error bars indicate ± SD. (C and D) Phosphorylation of Rho downstream targets is decreased by Drg1 knockdown. The embryos injected with the indicated MOs and mRNAs were stained for phospho-cofilin (C) and phospho-LIMK1 (D). Statistical analysis by one-way ANOVA with post hoc Turkey’s multiple range tests. All results are derived from three independent experiments. **, P < 0.0001, one-way ANOVA; n = 42 (C), 40 (D). Error bars represent mean ± SD. Scale bars, 30 µm. a.u., arbitrary units; n.s., not significant.
	Figure 5. Drg1 knockdown has a mild effect on Dvl localization to the basal body region in MCCs. (A) Drg1 colocalizes with the Dvl2 C terminus in MCCs. The mRNAs of the Dvl2 C-terminal fragment tagged with mCherry and Drg1-GFP were coinjected into both marginal ventral blastomeres of four-cell-stage embryos and fixed at stage 25. Images are generated by maximum intensity projection of serial z-stack confocal images. Scale bar, 10 µm. (B) The C-terminus of Dvl2 associates with Drg1. The indicated HA-tagged Dvl2 mutants were coexpressed with Drg1-Flag, and co-IP was performed. (C) Drg1 knockdown diminishes Dvl2 C-terminal localization to the basal body area. mRNAs of Dvl2-c-term-GFP and centrin-RFP (basal body marker) were coinjected with the indicated MOs and mRNAs. The expression level of Dvl2-c-term-GFP was tested by Western blot. Embryos were fixed at stage 25–27. Serial z-stack images were generated. A maximum intensity projection image is shown. Scale bars, 5 µm.
	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.
	Figure 7. Drg1 modulation of Daam1 activity is critical for multiciliation. (A) Drg1 knockdown reduces the Dvl2–Daam1 interaction. The cocktail of the indicated mRNAs and MOs was injected into embryos, and co-IPs were conducted with embryos harvested at stage 20. MO-resistant Drg1-V5 expression rescues decreased the Dvl2–Daam1 interaction upon Drg1 knockdown. (B) Drg1 knockdown decreases the Daam1–RhoA interaction. The indicated mRNAs and morpholinos were coinjected into embryos, which were collected at stage 20 for co-IP analysis. (C) C-Daam1 expression rescues reduced acetylated tubulin staining upon Drg1 knockdown. The indicated mRNAs and MOs were injected to two ventral blastomeres at the four-cell stage. For C-Daam1 expression, 2.5 pg of HA-tagged C-Daam1 encoding plasmid DNAs were injected. The embryos fixed at stage 27 were used for immunostaining. Expression levels of Daam1-WT and C-Daam1 were tested by Western blot. Relative acetylated tubulin intensity is quantified (image n = 40 from 15 embryos for each condition). **, P < 0.0001, one-way ANOVA. Scale bars, 50 µm. (D) C-Daam1 expression restores the formation of the apical actin meshwork that was decreased upon Drg1 knockdown. The indicated mRNAs and MOs were coinjected into two ventral blastomeres of four-cell-stage embryos and fixed at stage 27 for phalloidin and acetylated tubulin staining. **, P < 0.0001, one-way ANOVA; n = 40. Scale bars, 5 µm. Error bars indicate ± SD. n.s., not significant.

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