| dc.description.abstract | Aquaporin-5 (AQP5) regulates osmotic balance and transparency of the ocular lens through facilitated diffusion of water and represents an integral component of the lens microcirculation system, which convects nutrients to and waste from fiber cells in the avascular lens. Regulatory mechanisms which underpin lenticular AQP5 function are imprecisely understood. In this study, the bovine lens was used as a model to define AQP5 spatial expression, phospho-Thr259 spatial expression, subcellular localization, putative protein-protein interactions, and a potential mechanism of AQP5 trafficking. Immunofluorescence was performed on bovine lens cryosections targeting AQP5, AQP5 phospho-T259, and organelle marker proteins. PI3K-Akt-mTOR signal pathway protein expression in bovine lenses was analyzed via Western blotting, and ex vivo cultured bovine lenses were treated with bafilomycin A1 to determine changes in AQP5 plasma membrane expression. Lastly, putative AQP5 interacting partners were identified via AQP5 co-immunoprecipitation experiments coupled with LC-MS/MS analysis. Immunofluorescence revealed ubiquitous AQP5 expression in the bovine lens and that AQP5-containing cytoplasmic vesicles in bovine lens fiber cells are TOMM20-positive and increasingly become LC3B-positive and LIMP-2 positive with fiber cell differentiation. PI3K/Akt/mTOR signaling pathway protein expression decreases with bovine lens fiber cell differentiation consistent with upregulation of autophagic induction. Bafilomycin A1 treatment reduces AQP5 plasma membrane expression in bovine lens fiber cells suggesting the importance of autophagosome-lysosome fusion for AQP5 plasma membrane trafficking. AQP5 pT259 spatial expression resembles unphosphorylated AQP5 spatial expression throughout the bovine lens aside from the inner cortex and outer nucleus, where AQP5 pT259 is cytoplasmic. Additionally, AQP5 putatively interacts with multiple protein regulators of autophagic induction in bovine lens cortical fiber cells including seryl-tRNA synthetase, serine/threonine-protein kinase 24, sorting nexin 8, RPN2, and DDOST. Our data suggest that AQP5 associates with mitochondria then trafficks to the plasma membrane through autolysosome-associated unconventional protein secretion in bovine lens fiber cells. Putative autophagy-associated, AQP5 interacting proteins are consistent with AQP5 subcellular localization and a potential regulatory relationship between AQP5 and autophagic induction in the bovine lens. AQP5 C-terminal Thr259 phosphorylation may modulate AQP5 protein-protein interactions as aquaporin C-terminal phosphorylation is generally inhibitory towards aquaporin protein-protein interactions. | |
