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.
Highlights
Inversin (Inv or Nephrocystin-2) is encoded by the inversion of embryo turning gene [1,2,3] and was first discovered for its role during mammalian embryonic development in establishment of left-right asymmetry [4], which is reversed in inv2/2 mice with a homozygous deletion of exons 4–12, rendering only the first three exons transcribed [5]
We show that Inversin, Dvl-1 and Dvl-3 localize to mouse embryonic fibroblasts (MEFs) primary cilia, and that canonical Wnt signaling is elevated in inv2/2 MEFs, which show reduced ciliary localization of Dvl-3
Inv2/2 MEFs are severely compromised in their ability to migrate and this is associated with defective formation of leading edge lamellipodia
Summary
Inversin (Inv or Nephrocystin-2) is encoded by the inversion of embryo turning (invs) gene [1,2,3] and was first discovered for its role during mammalian embryonic development in establishment of left-right asymmetry [4], which is reversed (situs inversus) in inv2/2 mice with a homozygous deletion of exons 4–12, rendering only the first three exons transcribed [5]. INVS was identified as the gene encoding Nephrocystin-2 (Nphp2) that is mutated in the recessive cystic kidney disease nephronophthisis type 2/infantile nephronophthisis [2], which, to a variable extent, is accompanied by situs inversus and other phenotypic traits of the inv2/2 mouse and retinitis pigmentosa [10,11]. Defects in the formation or function of primary cilia lead to a series of genetic disorders and diseases commonly known as ciliopathies, including laterality defects, congenital heart disease, cystic kidney diseases and retinitis pigmentosa [18,19]. Inversin interacts with other Nphp proteins to form complexes with Meckel-Gruber syndrome (MKS) and Joubert syndrome (JBS) proteins that control trafficking and signaling properties of the primary cilium [21,22,23,24]
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