Connexin43 modulates cell polarity and directional cell migration by regulating microtubule dynamics.
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.
Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
Richard
R
Francis
Xin
X
Xu
Hyunsoo
H
Park
Chin-Jen
CJ
Wei
Stephen
S
Chang
Bishwanath
B
Chatterjee
Cecilia
C
Lo
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D016764:Cell Polarity; D018031:Connexin 43 / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D022101:Microtubule-Organizing Center / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D041681:NIH 3T3 Cells; D009432:Neural Crest / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D014945:Wound Healing
D018031:Connexin 43; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins