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

Abstract

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 inv−/− and inv+/+ animals, we confirmed that both inv−/− and inv+/+ MEFs form primary cilia, and that Inversin localizes to the primary cilium in inv+/+ MEFs. In wound healing assays, inv−/− 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 inv−/− MEFs as compared to inv+/+ MEFs. Further, Dvl-1 and Dvl-3 localized to MEF primary cilia, and β-catenin/Wnt signaling was elevated in inv−/− MEFs, which moreover showed reduced ciliary localization of Dvl-3. Finally, inv−/− 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 inv−/− 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.

Cilia and coordination of signaling networks during heart development

“…32,35,197 However, many Wnt signaling components, such as Fz3, Dvl, and β-catenin, localize to the ciliary/centrosomal axis, and dysregulated Wnt signaling has been reported in cells and tissues with disrupted ciliogenesis or basal body integrity. [198][199][200] Moreover, in the mouse inner ear, establishment of PCP was shown to depend on the formation of functional primary cilia. 201 However, other studies in mice and zebrafish reveal no obvious canonical or non-canonical Wnt phenotypes in cilia mutants, 202,203 emphasizing the importance of further studies in this area.…”

“…Interestingly, NPHP2-4 and Ftm/Rpgrip1l/NPHP8 all seem to impair canonical Wnt-signaling while promoting non-canonical Wnt responses. 14,165,200,[207][208][209] As an example, NPHP2/inversin and the structurally related diversin in zebrafish are required for convergence extension movement in vertebrates, and depletion of either result in cardiac defects. 190,208 Together, these aspects suggest that inversin and other NPHPs are implicated in heart development by controlling Wnt signaling, but whether this is coordinated by cardiac primary cilia remains speculative at this point.…”

“…Because so many different signaling systems are associated with primary cilia, 20,21,31,171,172,175,200,[213][214][215][216] we suggest that primary cilia may function as signaling hubs in the spatiotemporal crosstalking between diverse signaling networks during heart development. Future work should therefore focus on how primary cilia are involved in the integration and cross-talking between different signaling networks and how this may impact on different stages of heart development.…”

Phosphorylation‐dependent Akt–Inversin interaction at the basal body of primary cilia

“…This result can be partly explained by altered Wnt signaling, which is known to control spindle orientation (Kikuchi et al , 2010). INVS is localized at the spindle poles during anaphase (Eley et al , 2004) where it controls the orientation of cell division, cell polarity, and cell migration (Simons & Walz, 2006; Veland et al , 2013; Werner et al , 2013). We postulate that loss of INVS phosphorylation possibly underlies the distorted lumen formation observed in NPHP2, perhaps due to disorientation of the spindle axis during metaphase.…”

Wnt Signaling Inhibits the Growth of Primary Cilia and Activates CyclinD1, Snail and VEGFA Expression in Breast Cancer Cell Line MDA-MB-231

“…Inversin, a ciliary protein, has significance in part through transcriptional regulation of genes involved in Wnt signaling and pathways that control cytoskeletal organization and ion transport (12). There is a growing body of evidence to support the notion that ciliary proteins are required not only for regulation of Wnt signaling, but also as downstream effectors of Wntsignaling (13). Reduced primary cilia expression has been observed in human cancers, such as pancreatic cancer, renal cell carcinoma, breast cancer, and melanoma.…”