Adherens Junctions and Spectrin-Associated Proteins Function Interdependently to Stabilize Fiber Cell Membranes and Preserve Lens Transparency

Purpose: Cortical cataracts develop in mice in the absence of Arvcf, a protein that serves as a key component of the adherens junctional complex (AJC) in lens cells. This study aimed to mechanistically probe the proteomic complex associated with Arvcf to identify novel mechanisms underlying cortical cataract formation. Methods: Arvcf associated proteins were identified by coimmunoprecipitation of P30 mouse lens lysates using liquid chromatography mass-spectroscopy/mass-spectroscopy, gene ontology analysis, and western blot. Immunofluorescent staining was also performed in Arvcf or ankyrin-B mutant mouse lenses to assess protein expression, association, and junctional localization. Results: In addition to members of the AJC, proteomic analysis revealed that Arvcf associates with several spectrin-associated proteins. Ankyrin-B, NrCAM, and β-spectrin. These findings were verified by quantitative western blots and colocalization analysis. Although spectrin-associated protein expression in Arvcf mutants was unaffected, a significant reduction in their localization specifically at radially orientated cell membranes was observed. Importantly, this reduction correlates with the exact location of cortical opacities and membrane fusion events that abnormally occur between neighboring fiber cells Arvcf-deficient lenses. Finally, Arvcf and N-cadherin localization is altered by the absence of ankyrin-B in lens fiber cells, suggesting that stabilization of the AJC and the spectrin-associated complex of proteins are interdependent. Conclusions: Together, this data supports a novel role for Arvcf linking adherens junctions to the spectrin-associated cytoskeleton. This association is required to stabilize fiber cell membranes, limit cell fusion events, and maintain transparency in the cortical regions of the lens.