Surprisingly, we observed that Cx50, by itself, significantly increased cell adhesion, and also acted in either a homotypic or heterotypic manner (Fig.?1CCE). suggest that in addition to forming space junction channels, Cx50 functions as an adhesive molecule that is crucial in maintaining lens fiber integrity and epithelial-fiber differentiation. Introduction Space junctions that connect the cytoplasm of adjacent cells and permit passage of metabolites, ions and second messengers play essential functions in lens homeostasis and transparency. Space junctions are created by a family of membrane proteins called connexins1, which have four conserved transmembrane and two extracellular loop (E) domains and, a variable intracellular loop (IL) and a C-terminal (CT) domains. Three major connexins have been recognized in the vertebrate lens; Cx43, Cx46 and Cx50. Mutations of Cx46 and Cx50 genes are the most common causes of congenital cataracts in humans. Similar lens phenotypes ware reported in connexin-deficient or mutation murine models2, 3. Our previous studies have shown that Cx50, but not Cx46 or Cx43, associates Autophinib with aquaporin 0 (AQP0), the most abundant membrane protein in the differentiating, but not mature lens fibers4. This conversation promotes space junctional channel activity5, and the IL domain name of Cx50 and the CT domain name of AQP0 directly interact with each other6. The lens is an avascular organ, which is usually created by an anterior epithelial cell layer and highly differentiated fiber cells. Epithelial cells located at the lens equator differentiate to lens fiber cells, which gradually drop their intracellular nuclei and organelles in lens development. During this process, mature lens fibers accumulate high concentrations of AQP0, crystallins, Cx46 and Cx50. Because of the lack of vasculature, the lens is dependent upon an extensive network of space junction intercellular communication to maintain lens homeostasis7. AQP0, also known as major intrinsic protein (MIP), is the most abundant membrane protein expressed in lens fibers. However, unlike other users of aquaporin family, water permeability of mammalian AQP0 is usually amazingly low, estimated to be 40-times lower than that of the AQP1 channel in lens anterior epithelial cells8, while zebrafish AQP0 has high water permeability much like mammalian AQP19. Besides functioning as a water channel, AQP0 plays a crucial structural role as an adhesion molecule in mediating the formation of thin junctions between lens fibers10C13. In addition, AQP0 interacts with several proteins, such as calmodulin14, intermediate filament proteins filensin and CP4915, as well as -crystallins16, 17. Although connexin molecules have been implied to be involved in facilitating cell-cell conversation due to their formation Autophinib of space junctions between adjacent cells, there is a scarcity of knowledge with regards to the direct cell adhesive function of connexins. In this study, we show that Cx50, unlike two other lens connexins, Cx43 and Cx46, mediates cell adhesion function through its second extracellular loop domain name. Moreover, the cell-cell adhesion mediated by Cx50 plays a critical role for lens epithelial-fiber cell differentiation. Results Cx50 Exhibits Cell-cell Adhesion Function and Enhances the Adhesive Capability of AQP0 We have shown that Cx50 conversation with Autophinib AQP0 enhances space junctional coupling5, 6. To explore if Cx50 has any effect on the cell adhesion function of AQP0, we conducted a cell adhesion assay using chicken embryonic fibroblast (CEF) cells, a cell collection deficient in lens connexins and AQP018, and cannot form functional IKK-gamma (phospho-Ser376) antibody space junction channels between themselves and between parental CEF and the CEF expressing exogenous Cx50 (Fig.?S1). Exogenous Cx50 and AQP0 were expressed in CEF cells via retroviral contamination (Fig.?1A). The cell adhesion assay was then performed by parachuting Dil-labeled donor cells to the confluent recipient cells as illustrated in Fig.?1B. We expressed Cx50 and/or AQP0 in various combinations in donor and receipt cells. As compared to CEF cells only (C) and RCAS(A) vehicle (V) controls, the presence of AQP0 significantly increased the number of adherent cells when it was expressed in both donor and recipient cells (homotypic) (Fig.?1C) as well as when it was only present in either recipient or donor cells (heterotypic) (Fig.?1D and E). Similarly, co-expression of AQP0 with Cx50 further enhanced the numbers of adherent cells when expressed in a heterotypic or homotypic manner (Fig.?1CCE). Surprisingly, Autophinib we observed that Cx50, by itself, significantly increased cell adhesion, and also acted in either a homotypic or heterotypic manner (Fig.?1CCE). Autophinib There is no statistical difference when.