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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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MADD-2 promotes UNC-40 clustering and polarization toward UNC-6. Anterior is left; ventral is down. (A) MADD-2::GFP was colocalized with F-actin (visualized with mCherry::moeABD) at the AC’s invasive cell membrane in wild-type animals and in ectopic F-actin patches in unc-6 mutants, locations where UNC-40 resides (arrowheads; the location of basement membrane [BM] is indicated with broken lines; colocalization graphs on the right were measured along the yellow lines in the magnified insets below). (B) The dominant UNC-40–mediated F-actin patch polarized in random sections of the plasma membrane in the ACs of madd-2; unc-6 mutants (P > 0.1, χ2 test, n = 24 animals observed; see Fig. 4 C). (C) F-actin cluster formation (arrowheads) was slower before disassembly in a madd-2; unc-6 mutant (shown over a 79-min time lapse). (D) The volume of F-actin patch formation (red line, dominant patch) in the madd-2; unc-6 animal shown in C. (E) Similar analysis of three additional madd-2; unc-6 mutants. (F) UNC-40::GFP and F-actin colocalized (arrowheads) in madd-2; unc-6 mutants (top) and in animals with RNAi-induced loss of madd-2 (bottom). UNC-40::GFP and F-actin were mispolarized after loss of madd-2 (arrowheads, bottom). (G) Quantification of UNC-40 polarity in wild-type and madd-2 (RNAi)-treated animals (n > 20 for each stage per genotype; ***, P < 0.001, Student’s t test). Bars: (main panels) 5 µm; (magnified insets) 1 µm.
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fig7: MADD-2 promotes UNC-40 clustering and polarization toward UNC-6. Anterior is left; ventral is down. (A) MADD-2::GFP was colocalized with F-actin (visualized with mCherry::moeABD) at the AC’s invasive cell membrane in wild-type animals and in ectopic F-actin patches in unc-6 mutants, locations where UNC-40 resides (arrowheads; the location of basement membrane [BM] is indicated with broken lines; colocalization graphs on the right were measured along the yellow lines in the magnified insets below). (B) The dominant UNC-40–mediated F-actin patch polarized in random sections of the plasma membrane in the ACs of madd-2; unc-6 mutants (P > 0.1, χ2 test, n = 24 animals observed; see Fig. 4 C). (C) F-actin cluster formation (arrowheads) was slower before disassembly in a madd-2; unc-6 mutant (shown over a 79-min time lapse). (D) The volume of F-actin patch formation (red line, dominant patch) in the madd-2; unc-6 animal shown in C. (E) Similar analysis of three additional madd-2; unc-6 mutants. (F) UNC-40::GFP and F-actin colocalized (arrowheads) in madd-2; unc-6 mutants (top) and in animals with RNAi-induced loss of madd-2 (bottom). UNC-40::GFP and F-actin were mispolarized after loss of madd-2 (arrowheads, bottom). (G) Quantification of UNC-40 polarity in wild-type and madd-2 (RNAi)-treated animals (n > 20 for each stage per genotype; ***, P < 0.001, Student’s t test). Bars: (main panels) 5 µm; (magnified insets) 1 µm.

Mentions: To explore whether MADD-2 might mediate UNC-6–independent UNC-40 clustering, we first examined MADD-2 localization. Consistent with direct regulation of UNC-40, MADD-2::GFP was concentrated at regions of the cell where UNC-40 localizes: at the invasive cell membrane in wild-type animals and with ectopic patches of F-actin in unc-6 mutants (Fig. 7 A). To determine whether MADD-2 regulates UNC-6–independent UNC-40 clustering, we examined F-actin dynamics in unc-6 mutants harboring the putative madd-2– allele madd-2(ok2226) (Alexander et al., 2010). In madd-2(ok2226); unc-6(ev400) double mutant animals, dominant F-actin clusters still formed and were localized in random regions in the cell (Fig. 7, B and C). Furthermore, the F-actin patches reached a peak volume comparable to that observed in unc-6 mutants and disassembled at a similar rate (Table 1; Fig. 7, C–E). In madd-2(ok2226); unc-6(ev400) mutants, UNC-40 still colocalized with ectopic F-actin clusters (Fig. 7 F). Strikingly, however, madd-2(ok2226); unc-6(ev400) animals had a threefold decrease in the rate of cluster assembly (Fig. 7, C–E; and Table 1). In addition, the F-actin patches had a significantly longer lifetime before disassembly (>80 min versus ∼26 min in unc-6 mutants alone; Table 1). These results indicate that MADD-2 promotes UNC-6–independent UNC-40 cluster assembly. In addition, these observations suggest that the negative feedback mechanism that triggers cluster disassembly is induced at peak UNC-40 clustering.


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)

MADD-2 promotes UNC-40 clustering and polarization toward UNC-6. Anterior is left; ventral is down. (A) MADD-2::GFP was colocalized with F-actin (visualized with mCherry::moeABD) at the AC’s invasive cell membrane in wild-type animals and in ectopic F-actin patches in unc-6 mutants, locations where UNC-40 resides (arrowheads; the location of basement membrane [BM] is indicated with broken lines; colocalization graphs on the right were measured along the yellow lines in the magnified insets below). (B) The dominant UNC-40–mediated F-actin patch polarized in random sections of the plasma membrane in the ACs of madd-2; unc-6 mutants (P > 0.1, χ2 test, n = 24 animals observed; see Fig. 4 C). (C) F-actin cluster formation (arrowheads) was slower before disassembly in a madd-2; unc-6 mutant (shown over a 79-min time lapse). (D) The volume of F-actin patch formation (red line, dominant patch) in the madd-2; unc-6 animal shown in C. (E) Similar analysis of three additional madd-2; unc-6 mutants. (F) UNC-40::GFP and F-actin colocalized (arrowheads) in madd-2; unc-6 mutants (top) and in animals with RNAi-induced loss of madd-2 (bottom). UNC-40::GFP and F-actin were mispolarized after loss of madd-2 (arrowheads, bottom). (G) Quantification of UNC-40 polarity in wild-type and madd-2 (RNAi)-treated animals (n > 20 for each stage per genotype; ***, P < 0.001, Student’s t test). Bars: (main panels) 5 µm; (magnified insets) 1 µm.
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fig7: MADD-2 promotes UNC-40 clustering and polarization toward UNC-6. Anterior is left; ventral is down. (A) MADD-2::GFP was colocalized with F-actin (visualized with mCherry::moeABD) at the AC’s invasive cell membrane in wild-type animals and in ectopic F-actin patches in unc-6 mutants, locations where UNC-40 resides (arrowheads; the location of basement membrane [BM] is indicated with broken lines; colocalization graphs on the right were measured along the yellow lines in the magnified insets below). (B) The dominant UNC-40–mediated F-actin patch polarized in random sections of the plasma membrane in the ACs of madd-2; unc-6 mutants (P > 0.1, χ2 test, n = 24 animals observed; see Fig. 4 C). (C) F-actin cluster formation (arrowheads) was slower before disassembly in a madd-2; unc-6 mutant (shown over a 79-min time lapse). (D) The volume of F-actin patch formation (red line, dominant patch) in the madd-2; unc-6 animal shown in C. (E) Similar analysis of three additional madd-2; unc-6 mutants. (F) UNC-40::GFP and F-actin colocalized (arrowheads) in madd-2; unc-6 mutants (top) and in animals with RNAi-induced loss of madd-2 (bottom). UNC-40::GFP and F-actin were mispolarized after loss of madd-2 (arrowheads, bottom). (G) Quantification of UNC-40 polarity in wild-type and madd-2 (RNAi)-treated animals (n > 20 for each stage per genotype; ***, P < 0.001, Student’s t test). Bars: (main panels) 5 µm; (magnified insets) 1 µm.
Mentions: To explore whether MADD-2 might mediate UNC-6–independent UNC-40 clustering, we first examined MADD-2 localization. Consistent with direct regulation of UNC-40, MADD-2::GFP was concentrated at regions of the cell where UNC-40 localizes: at the invasive cell membrane in wild-type animals and with ectopic patches of F-actin in unc-6 mutants (Fig. 7 A). To determine whether MADD-2 regulates UNC-6–independent UNC-40 clustering, we examined F-actin dynamics in unc-6 mutants harboring the putative madd-2– allele madd-2(ok2226) (Alexander et al., 2010). In madd-2(ok2226); unc-6(ev400) double mutant animals, dominant F-actin clusters still formed and were localized in random regions in the cell (Fig. 7, B and C). Furthermore, the F-actin patches reached a peak volume comparable to that observed in unc-6 mutants and disassembled at a similar rate (Table 1; Fig. 7, C–E). In madd-2(ok2226); unc-6(ev400) mutants, UNC-40 still colocalized with ectopic F-actin clusters (Fig. 7 F). Strikingly, however, madd-2(ok2226); unc-6(ev400) animals had a threefold decrease in the rate of cluster assembly (Fig. 7, C–E; and Table 1). In addition, the F-actin patches had a significantly longer lifetime before disassembly (>80 min versus ∼26 min in unc-6 mutants alone; Table 1). These results indicate that MADD-2 promotes UNC-6–independent UNC-40 cluster assembly. In addition, these observations suggest that the negative feedback mechanism that triggers cluster disassembly is induced at peak UNC-40 clustering.

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