Limits...
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.

View Article: PubMed Central - HTML - PubMed

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

Show MeSH

Related in: MedlinePlus

F-actin colocalizes with UNC-40 effectors 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. UNC-40 effectors (left), F-actin (middle), overlay (right), and magnifications (below) are shown. Colocalization graphs (far right) show areas of colocalization (arrowheads; measured along yellow lines in insets; fluorescent intensity is plotted in arbitrary units and distances are given in pixels). In unc-6 mutants, the UNC-40 effectors, GFP::UNC-34 (Ena/VASP), GFP::CED-10 (Rac), and GFP::UNC-115 (abLIM; green), were all colocalized (arrowheads) with F-actin (magenta) at the AC’s apical and lateral membranes. (D) The percentage of the F-actin patches that were associated with the patches of UNC-40::GFP, UNC-40(ΔFN4/5)::GFP, GFP::UNC-34, GFP::CED-10, GFP::MIG-2 (Rac), and GFP::UNC-115 (n ≥ 15 for each stage per genotype). Error bars indicate ± SEM. No significant differences (P > 0.05, Student’s t test) compared with wild-type UNC-40/F-actin were observed. Bars: (main panels) 5 µm; (magnified insets) 0.5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: F-actin colocalizes with UNC-40 effectors 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. UNC-40 effectors (left), F-actin (middle), overlay (right), and magnifications (below) are shown. Colocalization graphs (far right) show areas of colocalization (arrowheads; measured along yellow lines in insets; fluorescent intensity is plotted in arbitrary units and distances are given in pixels). In unc-6 mutants, the UNC-40 effectors, GFP::UNC-34 (Ena/VASP), GFP::CED-10 (Rac), and GFP::UNC-115 (abLIM; green), were all colocalized (arrowheads) with F-actin (magenta) at the AC’s apical and lateral membranes. (D) The percentage of the F-actin patches that were associated with the patches of UNC-40::GFP, UNC-40(ΔFN4/5)::GFP, GFP::UNC-34, GFP::CED-10, GFP::MIG-2 (Rac), and GFP::UNC-115 (n ≥ 15 for each stage per genotype). Error bars indicate ± SEM. No significant differences (P > 0.05, Student’s t test) compared with wild-type UNC-40/F-actin were observed. Bars: (main panels) 5 µm; (magnified insets) 0.5 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)

F-actin colocalizes with UNC-40 effectors 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. UNC-40 effectors (left), F-actin (middle), overlay (right), and magnifications (below) are shown. Colocalization graphs (far right) show areas of colocalization (arrowheads; measured along yellow lines in insets; fluorescent intensity is plotted in arbitrary units and distances are given in pixels). In unc-6 mutants, the UNC-40 effectors, GFP::UNC-34 (Ena/VASP), GFP::CED-10 (Rac), and GFP::UNC-115 (abLIM; green), were all colocalized (arrowheads) with F-actin (magenta) at the AC’s apical and lateral membranes. (D) The percentage of the F-actin patches that were associated with the patches of UNC-40::GFP, UNC-40(ΔFN4/5)::GFP, GFP::UNC-34, GFP::CED-10, GFP::MIG-2 (Rac), and GFP::UNC-115 (n ≥ 15 for each stage per genotype). Error bars indicate ± SEM. No significant differences (P > 0.05, Student’s t test) compared with wild-type UNC-40/F-actin were observed. Bars: (main panels) 5 µm; (magnified insets) 0.5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: F-actin colocalizes with UNC-40 effectors 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. UNC-40 effectors (left), F-actin (middle), overlay (right), and magnifications (below) are shown. Colocalization graphs (far right) show areas of colocalization (arrowheads; measured along yellow lines in insets; fluorescent intensity is plotted in arbitrary units and distances are given in pixels). In unc-6 mutants, the UNC-40 effectors, GFP::UNC-34 (Ena/VASP), GFP::CED-10 (Rac), and GFP::UNC-115 (abLIM; green), were all colocalized (arrowheads) with F-actin (magenta) at the AC’s apical and lateral membranes. (D) The percentage of the F-actin patches that were associated with the patches of UNC-40::GFP, UNC-40(ΔFN4/5)::GFP, GFP::UNC-34, GFP::CED-10, GFP::MIG-2 (Rac), and GFP::UNC-115 (n ≥ 15 for each stage per genotype). Error bars indicate ± SEM. No significant differences (P > 0.05, Student’s t test) compared with wild-type UNC-40/F-actin were observed. Bars: (main panels) 5 µm; (magnified insets) 0.5 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