Spatially-Resolved Proteomic Analysis of the Lens Extracellular Diffusion Barrier

Purpose The presence of a physical barrier to molecular diffusion through lenticular extracellular space has been repeatedly detected in multiple species. This extracellular diffusion barrier has been proposed to restrict the movement of solutes into the lens and to direct nutrients into the lens core via the sutures at both poles. The purpose of this study is to characterize the molecular components that could contribute to the formation of this barrier. Methods Three distinct regions in the bovine lens cortex were captured by laser capture microdissection guided by dye penetration. Proteins were digested by endoproteinase Lys C and trypsin. Mass spectrometry-based quantitative proteomic analysis followed by gene ontology (GO) and protein-protein interaction network analysis was performed. Results Dye penetration showed that lens fiber cells first shrink the extracellular spaces of the broad sides of fiber cells followed by closure of the extracellular space between narrow sides at normalized lens distance (r/a) of 0.9. Accompanying the closure of extracellular space of the broad sides, dramatic proteomic changes were detected including up-regulation of several cell junctional proteins. AQP0 and its interacting partners ERM proteins were among a few proteins that were upregulated accompanying the closure of extracellular space of the narrow sides suggesting a particularly important role for the major lens membrane protein AQP0 in controlling the narrowing of the extracellular spaces between lens fiber cells. The results also provided important information related to biological processes that occur during fiber cell differentiation such as organelle degradation, cytoskeletal remodeling and GSH synthesis. Conclusions The formation of lens extracellular diffusion barrier is accompanied by significant membrane and cytoskeletal protein remodeling.