The human lens must maintain transparency over many decades and, to do so without a blood supply, the lens maintains a fluid microcirculation system (MCS) to deliver nutrients throughout the tissue. Although the influx of water, ions, and nutrients occurs at the anterior and posterior poles via the lens sutures and fluid outflow occurs toward the equatorial efflux zone, the molecular details of how the MCS is established and maintained with age are not well understood. It is, however, agreed that spatial differences in the expression and functionality of channels and transporters are critical to the generation of ion and fluid circulation. In this application we focus on lens aquaporins (AQPs) that we hypothesize play important roles in generating the lens MCS by establishing differences in water permeability in influx, outflow, and efflux zones. Aquaporin-0 (AQP0), the most abundant lens membrane protein, has reported roles in lens fiber cell adhesion, in basal water permeability, and in fiber cell organization and, as such, is vital for the development and maintenance of lens transparency. A second aquaporin, AQP5, is also present in lens fiber cells and can act as a regulated water channel since mechanical tension can alter AQP5 subcellular localization in fiber cells of the anterior pole and equator to presumably dynamically regulate water permeability. The long-term goal of our research is to understand how lens protein modifications that occur during development, aging, and cataractogenesis help establish or alter the MCS thereby leading to lens transparency or opacification. In the context of the microcirculation system, we hypothesize that AQP posttranslational modifications and AQP-lipid interactions are important molecular mechanisms used to modulate AQP functionality in the influx, outflow, and efflux zones. To test our hypotheses, we will employ advanced quantitative proteomics, native mass spectrometry, multi-modal imaging methods, and water permeability assays to obtain a molecular level understanding of how the structures and functions of lens AQPs change in influx, outflow, and efflux zones. Further, we will examine changes in AQP modifications and subcellular localization in organ cultured lenses exposed to stimuli that either dynamically regulate lens water transport or compromise the ability of the MCS to maintain lens transparency. This suite of methods will be applied to lens to test three hypotheses: 1) that water influx into the lens is increased by AQP5 membrane trafficking in the anterior suture region of the lens, 2) that AQPs direct intracellular water flow toward the lens equator in the outflow zone, and 3) that AQP5 trafficking to the membrane in the efflux zone dynamically regulates the MCS in response to mechanical tension, and oxidative or osmotic stress; stresses that mimic age related nuclear and diabetic cataract, respectively. We expect to provide a new mechanistic understanding of the roles of AQPs and their m...