PMC:3432770 / 22875-27241 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3432770","sourcedb":"PMC","sourceid":"3432770","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3432770","text":"Rpgrip1l acts in PCP by stabilizing dishevelled\nWe analyzed the functional interaction of rpgrip1l with several PCP genes and with inversin by performing combined knock-down in zebrafish embryos. Co-injection of low doses of Mo-Rpgrip1l with morpholinos specific for vangl2 (Mo-Vangl2), prickle1 (Mo-Pk1), for all three zebrafish dvl genes (Mo-3Dvl; Ségalen et al., 2010), or for inv (Mo-inv; Sayer et al., 2006) led to an increase in the severity of the axis elongation phenotype as compared with individual morphants (Fig. 4 A and Fig. S3, A–C). These results suggest either that Rpgrip1l and these proteins functionally interact during zebrafish CE, or that they act in parallel to promote common cellular processes.\nFigure 4. Rpgrip1l stabilizes dishevelled at the cilium base. (A) Diagram illustrating the functional interaction between Rpgrip1l and dishevelled in axis elongation. For each lane, the injected morpholinos are indicated on the right. n = 37 control, 29 Mo-Rpgrip1l–injected, 37 Mo-3Dvl–injected, and 24 co-injected embryos. The differences in the repartition in classes between batches of embryos injected with one morpholino only and the batch of co-injected embryos are significant (α \u003c 0.001, Khi2 test). (B) Western blots illustrating the amounts of Dvl2-Myc protein after injection of 15 pg Dvl2-Myc RNA per embryo at the 1-cell stage with or without co-injecting Mo-Rpgrip1l (0.8 mM). The diagrams illustrate the relative amounts of Dvl2-Myc in control and Mo-Rpgrip1l–injected embryos at the 80% epiboly and at the 12-s stages, in six independent experiments. Dvl amounts are in arbitrary units and the average amounts in control embryos are arbitrarily set at 1.0. (C) Diagram illustrating the axis elongation phenotype in embryos injected with Mo-Rpgrip1l and/or with Dsh-GFP RNA (12 ng/µl, corresponding to 12 pg/embryo). n = 23 control, 23 Dsh-GFP injected, 24 Mo-Rpgrip1l–injected, and 58 doubly injected embryos. Dsh-GFP mRNA (12 pg/embryo) significantly rescues the morphant phenotype (α \u003c 0.001, Khi2 test). (D and E) IF with an anti-GFP antibody to reveal the GFP tagged Dsh protein and with an anti-acetylated α-tubulin antibody (Ac Tub) to label cilia in embryos injected with Dsh-GFP (10 pg/embryo) with (E) or without (D, controls) Mo-Rpgrip1l (0.8 mM). (F and G) View of the floor plate after IF with anti-GFP, anti-βgal, and anti–Ac-Tub antibodies in 18-s stage embryos injected with DshGFP (10 pg/embryo) and nlsLacZ RNAs (60 pg/embryo) with (G) or without (F, controls) Mo-Rpgrip1l. anti-βgal staining in nuclei indicates that the corresponding cells have received injected RNA. (H) IF with anti–Ac-Tub, anti-GFP, and anti–γ-Tub antibodies in embryos injected with Dsh-GFP RNA alone. (I) IF with anti-GFP, anti-Myc, and anti–γ-Tub antibodies in embryos injected with Dsh-GFP (10 pg/embryo) and Rpgrip1l-Myc (7 pg/embryo) RNAs. Bars, 10 µm.Because dishevelled is a major actor of the Wnt pathways whose stability is modulated by the Rpgrip1l interactors nephrocystin-4 and inversin (Simons et al., 2005; Burcklé et al., 2011), we investigated a direct role of Rpgrip1l on its stability. For that purpose, we co-injected an RNA coding for a Myc-tagged form of Xenopus Dvl2 with Mo-Rpgrip1l into zebrafish embryos and analyzed by Western blots the amounts of Dvl2-Myc protein recovered at different stages (Fig. 4 B). The amounts of Dvl2-Myc were not significantly different between control and rpgrip1l morphants at 80% epiboly (8.5 hpf). However, at 12 s (15 hpf), rpgrip1l morphants displayed a 40% reduction in Dvl2-Myc levels compared with controls, suggesting that Rpgrip1l stabilized dishevelled. Dvl protein levels are known to be important for zebrafish CE (Angers et al., 2006) and the knock-down of all three zebrafish dvl genes led to defects in asymmetric basal body localization in floor plate cells (Fig. S3 D). To test whether Rpgrip1l function in PCP is mediated by dishevelled stabilization, we performed a rescue experiment by co-injecting Mo-Rpgrip1l together with an RNA coding for a GFP-tagged form of Drosophila dishevelled (Dsh-GFP). This led to a significant rescue of the CE phenotype (Fig. 4 C), as well as of the posterior localization of basal body in floor plate cells (Fig. 3 K). Together, these data show that in these two processes, Rpgrip1l acts at least in part by stabilizing dishevelled.","divisions":[{"label":"Title","span":{"begin":0,"end":47}},{"label":"Figure caption","span":{"begin":720,"end":2884}}],"tracks":[{"project":"NEUROSES","denotations":[{"id":"T307","span":{"begin":17,"end":20},"obj":"CHEBI_17642"},{"id":"T308","span":{"begin":112,"end":115},"obj":"CHEBI_17642"},{"id":"T309","span":{"begin":3906,"end":3909},"obj":"CHEBI_17642"},{"id":"T310","span":{"begin":112,"end":115},"obj":"CHEBI_8058"},{"id":"T311","span":{"begin":3906,"end":3909},"obj":"CHEBI_8058"},{"id":"T312","span":{"begin":64,"end":74},"obj":"PATO_0001510"},{"id":"T313","span":{"begin":212,"end":215},"obj":"PATO_0000471"},{"id":"T314","span":{"begin":413,"end":416},"obj":"CHEBI_43739"},{"id":"T315","span":{"begin":3782,"end":3785},"obj":"CHEBI_43739"},{"id":"T316","span":{"begin":4105,"end":4108},"obj":"CHEBI_43739"},{"id":"T317","span":{"begin":601,"end":609},"obj":"CHEBI_36080"},{"id":"T318","span":{"begin":3650,"end":3657},"obj":"CHEBI_36080"},{"id":"T319","span":{"begin":3096,"end":3100},"obj":"CHEBI_50906"},{"id":"T320","span":{"begin":3167,"end":3170},"obj":"CHEBI_33697"},{"id":"T321","span":{"begin":4030,"end":4033},"obj":"CHEBI_33697"},{"id":"T322","span":{"begin":3167,"end":3170},"obj":"CHEBI_18273"},{"id":"T323","span":{"begin":4030,"end":4033},"obj":"CHEBI_18273"},{"id":"T324","span":{"begin":3650,"end":3657},"obj":"CHEBI_16541"},{"id":"T325","span":{"begin":3800,"end":3810},"obj":"PATO_0000616"},{"id":"T326","span":{"begin":3894,"end":3902},"obj":"PATO_0000173"}],"attributes":[{"subj":"T307","pred":"source","obj":"NEUROSES"},{"subj":"T308","pred":"source","obj":"NEUROSES"},{"subj":"T309","pred":"source","obj":"NEUROSES"},{"subj":"T310","pred":"source","obj":"NEUROSES"},{"subj":"T311","pred":"source","obj":"NEUROSES"},{"subj":"T312","pred":"source","obj":"NEUROSES"},{"subj":"T313","pred":"source","obj":"NEUROSES"},{"subj":"T314","pred":"source","obj":"NEUROSES"},{"subj":"T315","pred":"source","obj":"NEUROSES"},{"subj":"T316","pred":"source","obj":"NEUROSES"},{"subj":"T317","pred":"source","obj":"NEUROSES"},{"subj":"T318","pred":"source","obj":"NEUROSES"},{"subj":"T319","pred":"source","obj":"NEUROSES"},{"subj":"T320","pred":"source","obj":"NEUROSES"},{"subj":"T321","pred":"source","obj":"NEUROSES"},{"subj":"T322","pred":"source","obj":"NEUROSES"},{"subj":"T323","pred":"source","obj":"NEUROSES"},{"subj":"T324","pred":"source","obj":"NEUROSES"},{"subj":"T325","pred":"source","obj":"NEUROSES"},{"subj":"T326","pred":"source","obj":"NEUROSES"}]},{"project":"2_test","denotations":[{"id":"22927466-21074723-56740295","span":{"begin":366,"end":370},"obj":"21074723"},{"id":"22927466-16682973-56740296","span":{"begin":407,"end":411},"obj":"16682973"},{"id":"22927466-15852005-56740297","span":{"begin":3042,"end":3046},"obj":"15852005"},{"id":"22927466-21498478-56740298","span":{"begin":3064,"end":3068},"obj":"21498478"},{"id":"22927466-16547521-56740299","span":{"begin":3724,"end":3728},"obj":"16547521"}],"attributes":[{"subj":"22927466-21074723-56740295","pred":"source","obj":"2_test"},{"subj":"22927466-16682973-56740296","pred":"source","obj":"2_test"},{"subj":"22927466-15852005-56740297","pred":"source","obj":"2_test"},{"subj":"22927466-21498478-56740298","pred":"source","obj":"2_test"},{"subj":"22927466-16547521-56740299","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"NEUROSES","color":"#ec93c9","default":true},{"id":"2_test","color":"#93e4ec"}]}]}}