Wallkamm V et al. (2014), Live imaging of Xwnt5A-ROR2 complexes.

XB-ART-49498

PLoS One 2014 Jan 01;910:e109428. doi: 10.1371/journal.pone.0109428.

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Live imaging of Xwnt5A-ROR2 complexes.

Wallkamm V , Dörlich R , Rahm K , Klessing T , Nienhaus GU , Wedlich D , Gradl D .

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Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner.

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Species referenced: Xenopus
Genes referenced: atf2 gap43 gnl3 ror2 wnt11 wnt5a wnt8a
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	Figure 2. Long-range Wnt-5A inhibits the ATF2-Luc reporter.A) For studying long-range signaling, Xwnt5A-EGFP transfected HEK293 cells seeded on thinCerts TC chambers were transferred on cells transfected with the ATF2-Luc reporter and cultivated for additional 24 hours. To investigate paracrine signaling, cells transfected with Xwnt5A-EGFP were co-cultured with cells transfected with the ATF2-Luc reporter for 24 hours. To analyze both autocrine and paracrine signaling, the reporter and the Wnt ligand were cotransfected. B) Activation of the non-canonical Wnt reporter ATF2-Luc by co-transfected Wnt8, Wnt11 and Wnt5A. C) Activation of the non-canonical Wnt reporter ATF2-Luc by long-range Wnt8, Wnt11 and Wnt5A in a two-chamber assay. D) Xwnt5A-EGFP triggered ATF2-Luc activation depends on endocytosis. Wnt5A induced ATF2-Luc activation (cotransfection) was inhibited by addition of 5 Âµg/ml chlorpromazine (Chlo) and 150 ÂµM genistein (Gen) 24 h before the measurement. Given are the mean values Â± standard errors and the p-values according to Student's t-test, ns: not significant, n gives the number of transfections.
	Figure 3. Fluorescence intensity correlation functions.A) Auto- and dual-focus cross-correlation functions in the red channel show ROR2-mCherry diffusion within the plasma membrane. B) Auto- and dual-focus cross-correlation functions in the green channel show the mobility of Xwnt5A-EGFP at the membrane. C) Cross-correlation functions of the green and red channels, reveal concerted diffusion and, therefore, binding of Xwnt5A-EGFP to ROR2-mCherry.
	Figure 4. Xwnt5A-EGFP but not ROR2-mCherry clusters in membranes of DMZ cells.A) 150 pg of Xwnt5A-EGFP mRNA were co-injected with 125 pg membrane-anchored (growth-associated protein 43 (Gap43))-mCherry as a lineage tracer in the two dorsal blastomeres of eight-cell stage embryos. At stage 10.25, dorsal marginal zones were explanted and analyzed for subcellular localization of the Xwnt5A-EGFP protein. Prominent Xwnt5A-EGFP clusters localized at the membrane of DMZ cells at the onset (stage 10.5) and end (stage 12) of gastrulation. Shown are snapshots of movies S1 and S2. B) 40 pg ROR2-mCherry were injected into the two dorsal blastomeres of eight-cell stage embryos. ROR2-mCherry is homogenously distributed at the membrane of DMZ explants throughout gastrulation. Shown are snapshots of movies S3 and S4.
	Figure 5. Xwnt5A induces clustering of ROR2.A) 150 pg of Xwnt5A-EGFP mRNA were co-injected with 40 pg of the ROR2-mCherry in the two dorsal blastomeres of eight-cell stage embryos. At stage 10.25 dorsal marginal zones were explanted and analyzed for subcellular localization of the Xwnt5A-EGFP and ROR2-mCherry. Prominent Xwnt5A-EGFP clusters co-localize with ROR2-mCherry clusters at the membrane of DMZ cells. These clusters are found at the onset (stage 10.5) and at the end (stage 12) of gastrulation. Shown are snapshots of movies S5 and S6. B) 150 pg of Xwnt5A-EGFP mRNA were co-injected with 1.6 pmol Xwnt-5A specific antisense morpholino and 125 pg Gap43-mCherry. Also in these Xwnt-5A-depleted explants Xwnt5A-EGFP clustered in the membrane.
	Figure 1. Xwnt5A-EGFP can compensate for the loss of endogenous Xwnt5A.A) An anti-EGFP reactive protein of the expected size is produced by embryos injected with Xwnt5A-EGFP. 300 pg Xwnt5A-EGFP mRNA were injected into each blastomere of two-cell stage embryos. NOP lysates corresponding to one half embryo were separated on an 8% SDS PAGE, transferred onto nitrocellulose and incubated with an anti-EGFP and anti-tubulin antibody. B) 1.6 pmol Xwnt5A morpholino (5AMo) were injected together with the indicated amounts of Xwnt5A-EGFP mRNA in the two dorsal blastomeres of eight-cell stage embryos. At stage 10.25, dorsal marginal zones were explanted and analyzed for convergence and extension. C) Quantification of elongation and constriction phenotypes. Given is the portion of embryos with impaired constriction (left image) and impaired elongation (right image) of Xwnt5A morphants (Wnt5A_Mo) and morphants co-injected with the indicated amount of Xwnt5A-EGFP mRNA. N: number of independent experiments, n: number of analyzed explants. : p<0.05, : p<0.01, **: p<0.001 according to Fisher's exact test.

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