GJA1 depletion causes ciliary defects and abnormal laterality

Abstract

Gap junction protein alpha 1 (GJA1), also known as Connexin 43 (CX43), is the most common and a major subunit of the gap junction complex. In the cytoplasm, the C-terminal domain of GJA1 protein regulates the cytoskeletal network, including actin and tubulin for cell protrusions, migration, and polarity. But the mechanisms and roles of GJA1 protein in the formation and function of cilia is yet to be determined. Cilia are a microtubule-based cellular organelle and play crucial roles in embryonic development and physiological maintenance of human body. Disruption of cilia formation and function is known to cause syndromic disorder, ciliopathy, in human. In this study, we examined knockdown phenotypes in Xenopus and human RPE1 cells and found that, by regulating ciliogenesis, GJA1 gene is critical during early development. GJA1 protein is localized not only at the gap junction, but also at the epithelial ciliary axoneme of multiciliated cells in Xenopus embryos and the pericentriolar matrix around primary cilium of human RPE1 cells. Dominant negative mutant-mediated dysfunction of gja1 protein caused severe malformation of motile cilia formation while the basal bodies are normally formed and apically localized. Further analysis revealed that, morpholino mediated-knockdown of gja1 disrupted normal ciliogenesis in Xenopus multiciliated cells and siRNA mediated-knockdown of GJA1 decreased the number of primary cilium in human RPE1 cells. Moreover, knockdown of gja1 in gastrocoel roof plate (GRP) showed decreased GRP cilium length, which caused reversed or disrupted left-right asymmetry in embryonic development. These finding suggest that GJA1 is necessary for proper ciliogenesis as it affects ciliary axoneme formation and assembly.