UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity.
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.
Affiliation: Department of Biology, Duke University, Durham, NC 27708.
- Caenorhabditis elegans/cytology*/metabolism
- Caenorhabditis elegans Proteins/metabolism*/physiology*
- Cell Adhesion Molecules/metabolism*
- Nerve Tissue Proteins/physiology*
- Cell Polarity
- Intracellular Signaling Peptides and Proteins/metabolism
- Protein Multimerization
- Protein Stability
- Protein Transport
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fig1: UNC-40 is mispolarized and active in the absence of UNC-6. Anterior is left; ventral is down. (A) AC invasion in C. elegans (top, schematic; green, differential interference contrast [DIC] microscopy with basement membrane marker laminin::GFP; bottom, overlay). During the L2/L3 molt (left), the AC (bottom, arrow) is attached to the basement membrane (BM; arrowhead) over the P6.p vulval precursor cell (bracket outlines nucleus, bottom). UNC-6 (netrin; top, blue arrows) secreted from the ventral nerve cord (VNC) polarizes UNC-40 (DCC; orange ovals) and F-actin (magenta) to the AC’s basal, invasive cell membrane. After P6.p divides (middle, P6.p two-cell stage), a protrusion breaches (bottom, arrowhead) and then removes basement membrane, and moves between the central P6.p granddaughter cells (right, P6.p four-cell stage). (B–F) Fluorescence (left), corresponding dense F-actin network (isosurface, middle), and overlay (right). (B) In wild-type animals, F-actin (visualized with cdh-3 > mCherry::moeABD) was polarized to the basal membrane (arrowhead). (C) In unc-6 mutants, F-actin was mislocalized to the AC’s apical and lateral membranes (arrowhead). (D) In unc-40 mutants, F-actin volume was reduced but polarized (arrowhead). (E) In unc-6; unc-40 double mutants, F-actin was reduced but polarized (arrowhead), comparable to unc-40. (F) In unc-6; unc-40; qyIs68[cdh-3 > unc-40::GFP] animals, F-actin was mislocalized (arrowheads), resembling unc-6. (G and H) The total volume of F-actin and the percentage that localized apicolaterally, respectively, at the P6.p four-cell stage (n ≥ 15 per genotype). *, P < 0.05; **, P < 0.01; ***, P < 0.001. N.S., no significant difference (P > 0.05, Student’s t test). Error bars indicate ± SEM. Significant differences relative to wild-type are indicated. Bars, 5 µm.
The C. elegans anchor cell (AC) is a specialized gonadal cell that polarizes toward and then invades through the basement membrane separating the uterine and vulval epithelium to initiate uterine–vulval attachment (Fig. 1 A; Sherwood and Sternberg, 2003; Hagedorn and Sherwood, 2011; Ihara et al., 2011; Kelley et al., 2014). UNC-6 (netrin) and UNC-40 (DCC) are crucial mediators of AC polarization. The receptor UNC-40 is enriched at the AC’s invasive cell membrane, where it directs the formation of an invasive protrusion that breaches the basement membrane (Ziel et al., 2009; Hagedorn et al., 2013). UNC-40 polarization relies on UNC-6 (netrin), which is secreted from the ventral nerve cord and accumulates in the basement membrane in contact with the invasive cell membrane of the AC (Ziel et al., 2009). Loss of unc-6 perturbs invasion and results in UNC-40 and F-actin regulators mislocalizing to all regions of the AC’s plasma membrane.