Inversin/Nephrocystin-2 Is Required for Fibroblast Polarity and Directional Cell Migration

Abstract

<div><p>Inversin is a ciliary protein that critically regulates developmental processes and tissue homeostasis in vertebrates, partly through the degradation of Dishevelled (Dvl) proteins to coordinate Wnt signaling in planar cell polarity (PCP). Here, we investigated the role of Inversin in coordinating cell migration, which highly depends on polarity processes at the single-cell level, including the spatial and temporal organization of the cytoskeleton as well as expression and cellular localization of proteins in leading edge formation of migrating cells. Using cultures of mouse embryonic fibroblasts (MEFs) derived from <i>inv^−/−</i> and <i>inv^+/+</i> animals, we confirmed that both <i>inv^−/−</i> and <i>inv^+/+</i> MEFs form primary cilia, and that Inversin localizes to the primary cilium in <i>inv^+/+</i> MEFs. In wound healing assays, <i>inv^−/−</i> MEFs were severely compromised in their migratory ability and exhibited cytoskeletal rearrangements, including distorted lamellipodia formation and cilia orientation. Transcriptome analysis revealed dysregulation of Wnt signaling and of pathways regulating actin organization and focal adhesions in <i>inv^−/−</i> MEFs as compared to <i>inv^+/+</i> MEFs. Further, Dvl-1 and Dvl-3 localized to MEF primary cilia, and β-catenin/Wnt signaling was elevated in <i>inv^−/−</i> MEFs, which moreover showed reduced ciliary localization of Dvl-3. Finally, <i>inv^−/−</i> MEFs displayed dramatically altered activity and localization of RhoA, Rac1, and Cdc42 GTPases, and aberrant expression and targeting of the Na⁺/H⁺ exchanger NHE1 and ezrin/radixin/moesin (ERM) proteins to the edge of cells facing the wound. Phosphorylation of β-catenin at the ciliary base and formation of well-defined lamellipodia with localization and activation of ERM to the leading edge of migrating cells were restored in <i>inv^−/−</i> MEFs expressing Inv-GFP. Collectively, our findings point to the significance of Inversin in controlling cell migration processes, at least in part through transcriptional regulation of genes involved in Wnt signaling and pathways that control cytoskeletal organization and ion transport.</p> </div