Abstract
Purpose :
Connexin-forming gap junctions play an important role in maintaining lens transparency and hemeostasis. However, the roles of connexins during lens development are not fully understood. Recently, the studies from our laboratory has shown that Cx50, in addition to forming gap junction channels, exhibits a new role in mediating cell-cell adhesion function. Here, we tested the hypothesis that Cx50 E2 domain was responsible for this cell adhesion function using both in vitro and in vivo appraoches.
Methods :
Adhesion assay was conducted using chick embryo fibroblast (CEF) cells infected with recombinant RCAS(A) retrovirus containing FLAG-tagged wild-type Cx50, Cx46, Cx43 or Cx50 mutants. To determine whether Cx50 E2 domain mediates lens development, we generated GST fusion protein containing E2 domain of Cx50 (GST-Cx50E2), or GST as a control and injected them to embryonic day 3 chick lens.
Results :
We showed that the expression of Cx50, but not Cx43 and Cx46 promoted cell adhesion. This function appeared to be unrelated to its role in forming gap junction channels. Moreover, the cell adhesion function was attenuated in cell expressing Cx50 chimeric mutant with the replacement of Cx50 E2 domain with either Cx43 or Cx46. Conversely, Cx43 or Cx46 chimeric mutant with Cx50 E2 domain showed comparable cell adhesion function as Cx50. We identified six amino acid residues in the E2 domain unique to Cx50 primarily responsible for this cell adhesive function. To assess the role of E2-mediated adhesion role in vivo, we developed an in vivo assay by injecting a GST-fusion protein that contained Cx50 E2 domain and has previously shown to inhibit Cx50 mediates cell adhesion. The histochemical data showed that the treatment of this fusion protein impaired chicken lens nuclear degradation, suggesting delayed lens development.
Conclusions :
This study shows that Cx50, unlike two other lens connexins, Cx43 and Cx46, mediates cell adhesion function through unique amino acid residues of its second extracellular loop domain. Moreover, Cx50 E2 domain plays a critical role for lens nuclear degradation during lens development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.