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UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity.

Wang Z, Linden LM, Naegeli KM, Ziel JW, Chi Q, Hagedorn EJ, Savage NS, Sherwood DR - J. Cell Biol. (2014)

Bottom Line: By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin).Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering.We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.

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Affiliation: Department of Biology, Duke University, Durham, NC 27708.

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Mispolarized UNC-40 colocalizes with F-actin in the absence of UNC-6. Anterior is left; ventral is down. (A–C) All animals were examined at the P6.p two-cell stage. Shown are UNC-40::GFP and UNC-40(ΔFN4/5)::GFP (left), F-actin (middle), and an overlay (right). Magnification (below) and colocalization quantification graphs (far right) show extensive UNC-40 and F-actin overlapping localization in all cases (arrowheads; colocalization was measured along the yellow line in the insets from the outside to the inside of the cell, fluorescent intensity was plotted in arbitrary units, and distances are given in pixels). (D) A schematic diagram illustrates the structural domains of the wild-type UNC-40 protein and the UNC-40(ΔFN4/5) protein, an engineered form of UNC-40 lacking the putative UNC-6 binding site in the fourth and fifth FNIII domains. (E) In unc-40 mutants, UNC-40(ΔFN4/5)::GFP colocalized with F-actin in the apical and lateral membranes of the AC (arrowheads). Bars, 5 µm.
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fig2: Mispolarized UNC-40 colocalizes with F-actin in the absence of UNC-6. Anterior is left; ventral is down. (A–C) All animals were examined at the P6.p two-cell stage. Shown are UNC-40::GFP and UNC-40(ΔFN4/5)::GFP (left), F-actin (middle), and an overlay (right). Magnification (below) and colocalization quantification graphs (far right) show extensive UNC-40 and F-actin overlapping localization in all cases (arrowheads; colocalization was measured along the yellow line in the insets from the outside to the inside of the cell, fluorescent intensity was plotted in arbitrary units, and distances are given in pixels). (D) A schematic diagram illustrates the structural domains of the wild-type UNC-40 protein and the UNC-40(ΔFN4/5) protein, an engineered form of UNC-40 lacking the putative UNC-6 binding site in the fourth and fifth FNIII domains. (E) In unc-40 mutants, UNC-40(ΔFN4/5)::GFP colocalized with F-actin in the apical and lateral membranes of the AC (arrowheads). Bars, 5 µm.

Mentions: Considering the UNC-6–independent activity of UNC-40, we hypothesized that the ectopic F-actin found in unc-6 mutants might be directly organized by mislocalized, but active UNC-40. Supporting this idea, we found that F-actin patches were strongly colocalized with UNC-40 in both wild-type animals and unc-6 mutants (Fig. 2, A and B; and Fig. 3 D). In addition, a form of UNC-40 lacking the fourth and fifth FNIII repeats, which are necessary for UNC-6 binding (Geisbrecht et al., 2003; Kruger et al., 2004), was also active and mislocalized when expressed in both wild-type and unc-40 mutant ACs (Fig. 2, C–E; and Fig. 3 D). We also found that in the absence of UNC-6, F-actin patches colocalized tightly with membrane-localized clusters of UNC-40 downstream effectors (UNC-34, CED-10, MIG-2, and UNC-115; Fig. 3, A–D; Wang et al., 2014). This genetic, molecular, and colocalization analysis offers strong evidence that the UNC-40 receptor promotes F-actin formation in an UNC-6–independent manner.


UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity.

Wang Z, Linden LM, Naegeli KM, Ziel JW, Chi Q, Hagedorn EJ, Savage NS, Sherwood DR - J. Cell Biol. (2014)

Mispolarized UNC-40 colocalizes with F-actin in the absence of UNC-6. Anterior is left; ventral is down. (A–C) All animals were examined at the P6.p two-cell stage. Shown are UNC-40::GFP and UNC-40(ΔFN4/5)::GFP (left), F-actin (middle), and an overlay (right). Magnification (below) and colocalization quantification graphs (far right) show extensive UNC-40 and F-actin overlapping localization in all cases (arrowheads; colocalization was measured along the yellow line in the insets from the outside to the inside of the cell, fluorescent intensity was plotted in arbitrary units, and distances are given in pixels). (D) A schematic diagram illustrates the structural domains of the wild-type UNC-40 protein and the UNC-40(ΔFN4/5) protein, an engineered form of UNC-40 lacking the putative UNC-6 binding site in the fourth and fifth FNIII domains. (E) In unc-40 mutants, UNC-40(ΔFN4/5)::GFP colocalized with F-actin in the apical and lateral membranes of the AC (arrowheads). Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4151141&req=5

fig2: Mispolarized UNC-40 colocalizes with F-actin in the absence of UNC-6. Anterior is left; ventral is down. (A–C) All animals were examined at the P6.p two-cell stage. Shown are UNC-40::GFP and UNC-40(ΔFN4/5)::GFP (left), F-actin (middle), and an overlay (right). Magnification (below) and colocalization quantification graphs (far right) show extensive UNC-40 and F-actin overlapping localization in all cases (arrowheads; colocalization was measured along the yellow line in the insets from the outside to the inside of the cell, fluorescent intensity was plotted in arbitrary units, and distances are given in pixels). (D) A schematic diagram illustrates the structural domains of the wild-type UNC-40 protein and the UNC-40(ΔFN4/5) protein, an engineered form of UNC-40 lacking the putative UNC-6 binding site in the fourth and fifth FNIII domains. (E) In unc-40 mutants, UNC-40(ΔFN4/5)::GFP colocalized with F-actin in the apical and lateral membranes of the AC (arrowheads). Bars, 5 µm.
Mentions: Considering the UNC-6–independent activity of UNC-40, we hypothesized that the ectopic F-actin found in unc-6 mutants might be directly organized by mislocalized, but active UNC-40. Supporting this idea, we found that F-actin patches were strongly colocalized with UNC-40 in both wild-type animals and unc-6 mutants (Fig. 2, A and B; and Fig. 3 D). In addition, a form of UNC-40 lacking the fourth and fifth FNIII repeats, which are necessary for UNC-6 binding (Geisbrecht et al., 2003; Kruger et al., 2004), was also active and mislocalized when expressed in both wild-type and unc-40 mutant ACs (Fig. 2, C–E; and Fig. 3 D). We also found that in the absence of UNC-6, F-actin patches colocalized tightly with membrane-localized clusters of UNC-40 downstream effectors (UNC-34, CED-10, MIG-2, and UNC-115; Fig. 3, A–D; Wang et al., 2014). This genetic, molecular, and colocalization analysis offers strong evidence that the UNC-40 receptor promotes F-actin formation in an UNC-6–independent manner.

Bottom Line: By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin).Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering.We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Duke University, Durham, NC 27708.

Show MeSH
Related in: MedlinePlus