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Connexin43 modulates cell polarity and directional cell migration by regulating microtubule dynamics.

ABSTRACT: Knockout mice deficient in the gap junction gene connexin43 exhibit developmental anomalies associated with abnormal neural crest, primordial germ cell, and proepicardial cell migration. These migration defects are due to a loss of directional cell movement, and are associated with abnormal actin stress fiber organization and a loss of polarized cell morphology. To elucidate the mechanism by which Cx43 regulates cell polarity, we used a wound closure assays with mouse embryonic fibroblasts (MEFs) to examine polarized cell morphology and directional cell movement. Studies using embryonic fibroblasts from Cx43 knockout (Cx43KO) mice showed Cx43 deficiency caused cell polarity defects as characterized by a failure of the Golgi apparatus and the microtubule organizing center to reorient with the direction of wound closure. Actin stress fibers at the wound edge also failed to appropriately align, and stabilized microtubule (Glu-tubulin) levels were markedly reduced. Forced expression of Cx43 with deletion of its tubulin-binding domain (Cx43dT) in both wildtype MEFs and neural crest cell explants recapitulated the cell migration defects seen in Cx43KO cells. However, forced expression of Cx43 with point mutation causing gap junction channel closure had no effect on cell motility. TIRF imaging revealed increased microtubule instability in Cx43KO cells, and microtubule targeting of membrane localized Cx43 was reduced with expression of Cx43dT construct in wildtype cells. Together, these findings suggest the essential role of Cx43 gap junctions in development is mediated by regulation of the tubulin cytoskeleton and cell polarity by Cx43 via a nonchannel function.

SUBMITTER: Francis R

PROVIDER: S-EPMC3194834 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Connexin43 modulates cell polarity and directional cell migration by regulating microtubule dynamics.

Francis Richard R Xu Xin X Park Hyunsoo H Wei Chin-Jen CJ Chang Stephen S Chatterjee Bishwanath B Lo Cecilia C

PloS one 20111014 10

Knockout mice deficient in the gap junction gene connexin43 exhibit developmental anomalies associated with abnormal neural crest, primordial germ cell, and proepicardial cell migration. These migration defects are due to a loss of directional cell movement, and are associated with abnormal actin stress fiber organization and a loss of polarized cell morphology. To elucidate the mechanism by which Cx43 regulates cell polarity, we used a wound closure assays with mouse embryonic fibroblasts (MEFs ...[more]

PMID: 22022608

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Project description: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.

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Connexin43 modulates cell polarity and directional cell migration by regulating microtubule dynamics.

Publications

Connexin43 modulates cell polarity and directional cell migration by regulating microtubule dynamics.

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