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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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UNC-6 stably polarizes UNC-40/F-actin oscillatory clustering. Anterior is left; ventral is down. (A) The time series shows F-actin polarity (orange arrowhead, grayscale) stabilized in the AC of an unc-6 mutant toward UNC-6 (zmp-5 > unc-6::nlg-1 TM::GFP; magenta), which is localized to dorsal uterine cell membranes (outlined with yellow lines; the asterisk marks a dorsal cell expressing UNC-6; the location of basement membrane [BM] is indicated with an orange line). (B) The red line in the graph represents the volume of the dominant F-actin patch in the time series shown above (the light green line is a small, transient F-actin patch localized away from the dominant patch). (C) Similar stabilization in four other cases. (D) The time series shows dynamic reorientation of F-actin polarity (grayscale) toward a changing UNC-6 source (magenta in top panels; outlined by broken lines in the bottom panels with spectral representation of fluorescence intensity). When the AC made contact with a new anterior source of UNC-6 (orange arrowheads), a polarized response was directed toward this new source, and polarity was lost on the posterior dorsal uterine cell as UNC-6 levels diminished (fading white arrowheads). See also Video 4. (E) A kymograph of the two outlined areas in the top of D from time 24 to 55 min of Video 4. (F) Quantification of UNC-6 (top) within the anterior domain and the associated increase in F-actin volume (bottom) during polarity reorientation after contact with this UNC-6 source (white boxed area in D). Bars: (A and D) 5 µm; (E) 0.5 µm.
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fig6: UNC-6 stably polarizes UNC-40/F-actin oscillatory clustering. Anterior is left; ventral is down. (A) The time series shows F-actin polarity (orange arrowhead, grayscale) stabilized in the AC of an unc-6 mutant toward UNC-6 (zmp-5 > unc-6::nlg-1 TM::GFP; magenta), which is localized to dorsal uterine cell membranes (outlined with yellow lines; the asterisk marks a dorsal cell expressing UNC-6; the location of basement membrane [BM] is indicated with an orange line). (B) The red line in the graph represents the volume of the dominant F-actin patch in the time series shown above (the light green line is a small, transient F-actin patch localized away from the dominant patch). (C) Similar stabilization in four other cases. (D) The time series shows dynamic reorientation of F-actin polarity (grayscale) toward a changing UNC-6 source (magenta in top panels; outlined by broken lines in the bottom panels with spectral representation of fluorescence intensity). When the AC made contact with a new anterior source of UNC-6 (orange arrowheads), a polarized response was directed toward this new source, and polarity was lost on the posterior dorsal uterine cell as UNC-6 levels diminished (fading white arrowheads). See also Video 4. (E) A kymograph of the two outlined areas in the top of D from time 24 to 55 min of Video 4. (F) Quantification of UNC-6 (top) within the anterior domain and the associated increase in F-actin volume (bottom) during polarity reorientation after contact with this UNC-6 source (white boxed area in D). Bars: (A and D) 5 µm; (E) 0.5 µm.

Mentions: We next sought to determine how UNC-6 might influence the observed ligand-independent UNC-40/F-actin clustering activity. We have previously shown that UNC-40 and F-actin are polarized to the invasive cell membrane of the AC in contact with the basement membrane (where UNC-6 is localized) for ∼5 h before AC invasion (Hagedorn et al., 2009; Ziel et al., 2009). We next wanted to determine whether UNC-6 was sufficient to orient and stabilize UNC-40. To test this, we expressed a membrane-tethered UNC-6::GFP protein in the dorsal uterine cells of an unc-6 mutant, thus presenting the AC with a localized source of UNC-6 opposite to the endogenous ventral presentation of UNC-6 in the basement membrane of wild-type animals (Fig. 5, A and B). This ectopic dorsal presentation of UNC-6 directed UNC-40 and F-actin clustering stably toward the AC’s apical cell membrane in contact with UNC-6 (Fig. 5, A–G). Time-lapse analysis of F-actin indicated that a constant UNC-6 source in dorsal uterine cells stabilized F-actin formation for the entire duration of time-lapse imaging (∼70 min; Fig. 6, A–C; Video 3; and Table 1). The volume of F-actin in these patches was equivalent to the peak volume in UNC-40 clusters in the absence of UNC-6 (Table 1). These results demonstrate that UNC-6 orients and stabilizes UNC-40 clustering and indicate that UNC-6–UNC-40 interactions must counter the negative feedback mechanism that disassembles UNC-40 clusters in the absence of UNC-6.


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)

UNC-6 stably polarizes UNC-40/F-actin oscillatory clustering. Anterior is left; ventral is down. (A) The time series shows F-actin polarity (orange arrowhead, grayscale) stabilized in the AC of an unc-6 mutant toward UNC-6 (zmp-5 > unc-6::nlg-1 TM::GFP; magenta), which is localized to dorsal uterine cell membranes (outlined with yellow lines; the asterisk marks a dorsal cell expressing UNC-6; the location of basement membrane [BM] is indicated with an orange line). (B) The red line in the graph represents the volume of the dominant F-actin patch in the time series shown above (the light green line is a small, transient F-actin patch localized away from the dominant patch). (C) Similar stabilization in four other cases. (D) The time series shows dynamic reorientation of F-actin polarity (grayscale) toward a changing UNC-6 source (magenta in top panels; outlined by broken lines in the bottom panels with spectral representation of fluorescence intensity). When the AC made contact with a new anterior source of UNC-6 (orange arrowheads), a polarized response was directed toward this new source, and polarity was lost on the posterior dorsal uterine cell as UNC-6 levels diminished (fading white arrowheads). See also Video 4. (E) A kymograph of the two outlined areas in the top of D from time 24 to 55 min of Video 4. (F) Quantification of UNC-6 (top) within the anterior domain and the associated increase in F-actin volume (bottom) during polarity reorientation after contact with this UNC-6 source (white boxed area in D). Bars: (A and D) 5 µm; (E) 0.5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4151141&req=5

fig6: UNC-6 stably polarizes UNC-40/F-actin oscillatory clustering. Anterior is left; ventral is down. (A) The time series shows F-actin polarity (orange arrowhead, grayscale) stabilized in the AC of an unc-6 mutant toward UNC-6 (zmp-5 > unc-6::nlg-1 TM::GFP; magenta), which is localized to dorsal uterine cell membranes (outlined with yellow lines; the asterisk marks a dorsal cell expressing UNC-6; the location of basement membrane [BM] is indicated with an orange line). (B) The red line in the graph represents the volume of the dominant F-actin patch in the time series shown above (the light green line is a small, transient F-actin patch localized away from the dominant patch). (C) Similar stabilization in four other cases. (D) The time series shows dynamic reorientation of F-actin polarity (grayscale) toward a changing UNC-6 source (magenta in top panels; outlined by broken lines in the bottom panels with spectral representation of fluorescence intensity). When the AC made contact with a new anterior source of UNC-6 (orange arrowheads), a polarized response was directed toward this new source, and polarity was lost on the posterior dorsal uterine cell as UNC-6 levels diminished (fading white arrowheads). See also Video 4. (E) A kymograph of the two outlined areas in the top of D from time 24 to 55 min of Video 4. (F) Quantification of UNC-6 (top) within the anterior domain and the associated increase in F-actin volume (bottom) during polarity reorientation after contact with this UNC-6 source (white boxed area in D). Bars: (A and D) 5 µm; (E) 0.5 µm.
Mentions: We next sought to determine how UNC-6 might influence the observed ligand-independent UNC-40/F-actin clustering activity. We have previously shown that UNC-40 and F-actin are polarized to the invasive cell membrane of the AC in contact with the basement membrane (where UNC-6 is localized) for ∼5 h before AC invasion (Hagedorn et al., 2009; Ziel et al., 2009). We next wanted to determine whether UNC-6 was sufficient to orient and stabilize UNC-40. To test this, we expressed a membrane-tethered UNC-6::GFP protein in the dorsal uterine cells of an unc-6 mutant, thus presenting the AC with a localized source of UNC-6 opposite to the endogenous ventral presentation of UNC-6 in the basement membrane of wild-type animals (Fig. 5, A and B). This ectopic dorsal presentation of UNC-6 directed UNC-40 and F-actin clustering stably toward the AC’s apical cell membrane in contact with UNC-6 (Fig. 5, A–G). Time-lapse analysis of F-actin indicated that a constant UNC-6 source in dorsal uterine cells stabilized F-actin formation for the entire duration of time-lapse imaging (∼70 min; Fig. 6, A–C; Video 3; and Table 1). The volume of F-actin in these patches was equivalent to the peak volume in UNC-40 clusters in the absence of UNC-6 (Table 1). These results demonstrate that UNC-6 orients and stabilizes UNC-40 clustering and indicate that UNC-6–UNC-40 interactions must counter the negative feedback mechanism that disassembles UNC-40 clusters in the absence of UNC-6.

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