Spatially Resolved Proteomics Reveals Lens Suture-Related Cell–Cell Junctional Protein Distributions

Abstract

Purpose Lens transparency relies on the precise organization of lens fiber cells. The formation of the highly ordered lens architecture results from not only cell–cell adhesion along the lateral interfaces, but also from proper organization of fiber cells tips at lens sutures. Little is known about the cell adhesion between fiber tips at the sutures. The purpose of this study is to map suture-specific protein distributions. Methods Tissue sections were obtained from fresh frozen bovine lenses and washes were performed to remove soluble proteins and to retain membrane and membrane associated proteins. Imaging mass spectrometry (IMS) combined with on-tissue trypsin digestion was used to visualize protein spatial distributions. Sutures and adjacent regions were captured by laser capture microdissection and samples were digested by trypsin. Proteins were analyzed by liquid chromatography tandem MS and quantified by label-free quantification. Protein spatial distributions were confirmed by immunofluorescence. Results IMS results showed enrichment of adherens junction proteins cadherin-2 and armadillo repeat gene deleted in velo–cardio–facial syndrome (ARVCF) in both anterior and posterior sutures of bovine lenses. Liquid chromatography tandem MS confirmed higher expression of cadherin-2 and ARVCF and other adherens junction proteins including catenin α2 (CTNNA2) and catenin β1 (CTNNB1) in sutures. In contrast, IMS indicated low expression of gap junction protein connexin 50 and connexin 46 in the suture regions. The localization of cadherin-2 and connexin 50 was confirmed by immunofluorescence. Conclusions The complementary expression of adherens junction proteins and gap junction proteins in lens suture regions implicates adherens junctions in fiber cell tip adhesion and in maintaining the integrity of the lens.