UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity.
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.
Affiliation: Department of Biology, Duke University, Durham, NC 27708.Show MeSH
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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.
Affiliation: Department of Biology, Duke University, Durham, NC 27708.