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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-40 undergoes periodic clustering and disassembly in the absence of UNC-6. Anterior is left; ventral is down. (A) A schematic diagram shows five membrane portions of the ACs used for scoring F-actin localization. (B and C) Most ACs in unc-6 mutants (69%) had a single dominant F-actin patch (orange broken lines, visualized with the F-actin probe cdh-3 > mCherry::moeABD) polarized randomly toward one of the five divided portions (P > 0.1, χ2 test, n = 65 animals observed). (D) Time series of F-actin localization in the AC of an unc-6 mutant revealed cycling between multiple F-actin patches and a single cluster. Initially, multiple small F-actin patches (white and orange arrowheads) formed randomly. After 9 min, one patch (orange arrowheads) grew and became dominant. By 26 min, this single cluster began disassembling, and multiple new F-actin foci (orange broken lines) formed. The orange broken line in the first frame indicates the position of the basement membrane where integrin maintains a light band of F-actin in the AC. (E) The volume of individual F-actin patches over time in an unc-6 mutant. Each colored line represents an individual patch. Images above show snapshots at times marked by the vertical broken lines (patches are outlined by colors that correspond to the graph). (F) Similar analysis of four other ACs in unc-6 mutants. (G) Schematic of cluster oscillations in unc-6 mutants. (H) A 42-min time series shows that clusters of UNC-40::GFP (top, green) colocalized with F-actin (bottom, magenta). (I) The fluorescence intensity of UNC-40::GFP and colocalized F-actin during the life cycle of a representative F-actin patch in an unc-6 mutant animal. (J) UNC-40::GFP intensity at each time point (black dots) was plotted against colocalized F-actin patch intensity, revealing a tight correlation (measured using coefficient of determination R2) during cluster oscillations. Data were pooled from nine cluster life cycles in seven animals. Bars, 5 µm.
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fig4: UNC-40 undergoes periodic clustering and disassembly in the absence of UNC-6. Anterior is left; ventral is down. (A) A schematic diagram shows five membrane portions of the ACs used for scoring F-actin localization. (B and C) Most ACs in unc-6 mutants (69%) had a single dominant F-actin patch (orange broken lines, visualized with the F-actin probe cdh-3 > mCherry::moeABD) polarized randomly toward one of the five divided portions (P > 0.1, χ2 test, n = 65 animals observed). (D) Time series of F-actin localization in the AC of an unc-6 mutant revealed cycling between multiple F-actin patches and a single cluster. Initially, multiple small F-actin patches (white and orange arrowheads) formed randomly. After 9 min, one patch (orange arrowheads) grew and became dominant. By 26 min, this single cluster began disassembling, and multiple new F-actin foci (orange broken lines) formed. The orange broken line in the first frame indicates the position of the basement membrane where integrin maintains a light band of F-actin in the AC. (E) The volume of individual F-actin patches over time in an unc-6 mutant. Each colored line represents an individual patch. Images above show snapshots at times marked by the vertical broken lines (patches are outlined by colors that correspond to the graph). (F) Similar analysis of four other ACs in unc-6 mutants. (G) Schematic of cluster oscillations in unc-6 mutants. (H) A 42-min time series shows that clusters of UNC-40::GFP (top, green) colocalized with F-actin (bottom, magenta). (I) The fluorescence intensity of UNC-40::GFP and colocalized F-actin during the life cycle of a representative F-actin patch in an unc-6 mutant animal. (J) UNC-40::GFP intensity at each time point (black dots) was plotted against colocalized F-actin patch intensity, revealing a tight correlation (measured using coefficient of determination R2) during cluster oscillations. Data were pooled from nine cluster life cycles in seven animals. Bars, 5 µm.

Mentions: To further explore the UNC-6–independent activity and dynamics of UNC-40 in the AC, we first examined static images of the spatial distribution of ectopic F-actin (the output of UNC-40 activity) during the P6.p two-cell stage (an ∼1-h period) in unc-6 mutants. To facilitate analysis, we partitioned the AC’s apical and lateral cell membranes into five equally sized portions: anterior, posterior, left, right, and apical (Fig. 4 A). The basal cell membrane (in contact with the basement membrane) was not included, as it contained F-actin that was generated by integrin (see Fig. S2). Importantly, we found that 69% of the ACs (45 out of 65 ACs observed) had a single ectopic F-actin patch polarized within one of the five membrane portions. The location of the F-actin cluster was not biased to any of the five membrane domains (Fig. 4, B and C). Thus, in the absence of UNC-6, UNC-40 mediates randomly directed F-actin polarity within the AC, where we define random as unbiased F-actin patch localization in any one of the equally sized membrane regions.


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-40 undergoes periodic clustering and disassembly in the absence of UNC-6. Anterior is left; ventral is down. (A) A schematic diagram shows five membrane portions of the ACs used for scoring F-actin localization. (B and C) Most ACs in unc-6 mutants (69%) had a single dominant F-actin patch (orange broken lines, visualized with the F-actin probe cdh-3 > mCherry::moeABD) polarized randomly toward one of the five divided portions (P > 0.1, χ2 test, n = 65 animals observed). (D) Time series of F-actin localization in the AC of an unc-6 mutant revealed cycling between multiple F-actin patches and a single cluster. Initially, multiple small F-actin patches (white and orange arrowheads) formed randomly. After 9 min, one patch (orange arrowheads) grew and became dominant. By 26 min, this single cluster began disassembling, and multiple new F-actin foci (orange broken lines) formed. The orange broken line in the first frame indicates the position of the basement membrane where integrin maintains a light band of F-actin in the AC. (E) The volume of individual F-actin patches over time in an unc-6 mutant. Each colored line represents an individual patch. Images above show snapshots at times marked by the vertical broken lines (patches are outlined by colors that correspond to the graph). (F) Similar analysis of four other ACs in unc-6 mutants. (G) Schematic of cluster oscillations in unc-6 mutants. (H) A 42-min time series shows that clusters of UNC-40::GFP (top, green) colocalized with F-actin (bottom, magenta). (I) The fluorescence intensity of UNC-40::GFP and colocalized F-actin during the life cycle of a representative F-actin patch in an unc-6 mutant animal. (J) UNC-40::GFP intensity at each time point (black dots) was plotted against colocalized F-actin patch intensity, revealing a tight correlation (measured using coefficient of determination R2) during cluster oscillations. Data were pooled from nine cluster life cycles in seven animals. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig4: UNC-40 undergoes periodic clustering and disassembly in the absence of UNC-6. Anterior is left; ventral is down. (A) A schematic diagram shows five membrane portions of the ACs used for scoring F-actin localization. (B and C) Most ACs in unc-6 mutants (69%) had a single dominant F-actin patch (orange broken lines, visualized with the F-actin probe cdh-3 > mCherry::moeABD) polarized randomly toward one of the five divided portions (P > 0.1, χ2 test, n = 65 animals observed). (D) Time series of F-actin localization in the AC of an unc-6 mutant revealed cycling between multiple F-actin patches and a single cluster. Initially, multiple small F-actin patches (white and orange arrowheads) formed randomly. After 9 min, one patch (orange arrowheads) grew and became dominant. By 26 min, this single cluster began disassembling, and multiple new F-actin foci (orange broken lines) formed. The orange broken line in the first frame indicates the position of the basement membrane where integrin maintains a light band of F-actin in the AC. (E) The volume of individual F-actin patches over time in an unc-6 mutant. Each colored line represents an individual patch. Images above show snapshots at times marked by the vertical broken lines (patches are outlined by colors that correspond to the graph). (F) Similar analysis of four other ACs in unc-6 mutants. (G) Schematic of cluster oscillations in unc-6 mutants. (H) A 42-min time series shows that clusters of UNC-40::GFP (top, green) colocalized with F-actin (bottom, magenta). (I) The fluorescence intensity of UNC-40::GFP and colocalized F-actin during the life cycle of a representative F-actin patch in an unc-6 mutant animal. (J) UNC-40::GFP intensity at each time point (black dots) was plotted against colocalized F-actin patch intensity, revealing a tight correlation (measured using coefficient of determination R2) during cluster oscillations. Data were pooled from nine cluster life cycles in seven animals. Bars, 5 µm.
Mentions: To further explore the UNC-6–independent activity and dynamics of UNC-40 in the AC, we first examined static images of the spatial distribution of ectopic F-actin (the output of UNC-40 activity) during the P6.p two-cell stage (an ∼1-h period) in unc-6 mutants. To facilitate analysis, we partitioned the AC’s apical and lateral cell membranes into five equally sized portions: anterior, posterior, left, right, and apical (Fig. 4 A). The basal cell membrane (in contact with the basement membrane) was not included, as it contained F-actin that was generated by integrin (see Fig. S2). Importantly, we found that 69% of the ACs (45 out of 65 ACs observed) had a single ectopic F-actin patch polarized within one of the five membrane portions. The location of the F-actin cluster was not biased to any of the five membrane domains (Fig. 4, B and C). Thus, in the absence of UNC-6, UNC-40 mediates randomly directed F-actin polarity within the AC, where we define random as unbiased F-actin patch localization in any one of the equally sized membrane regions.

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