Lens Aquaporin-5 Inserts Into Bovine Fiber Cell Plasma Membranes Via Unconventional Protein Secretion

PURPOSE: To spatially map aquaporin-5 (AQP5) expression in the bovine lens, molecularly characterize cytoplasmic AQP5-containing vesicles in the outer cortex, and elucidate AQP5 membrane trafficking mechanisms. METHODS: Immunofluorescence was performed on bovine lens cryosections using AQP5, TOMM20, COX IV, calnexin, LC3B, Sec22β, LIMP-2, and connexin 50 antibodies and the membrane dye CM-DiI. AQP5 plasma membrane insertion was defined via line expression profile analysis. Transmission electron microscopy (TEM) was performed on bovine lens sections to examine cytoplasmic organelle morphology and subcellular localization in cortical fiber cells. Bovine lenses were treated with 10-nM bafilomycin A1 or 0.1% dimethyl sulfoxide vehicle control for 24 hours in ex vivo culture to determine changes in AQP5 plasma membrane expression. RESULTS: Immunofluorescence analysis revealed cytoplasmic AQP5 expression in lens epithelial cells and differentiating fiber cells. In the lens cortex, complete AQP5 plasma membrane insertion occurs at r/a = 0.951 ± 0.005. AQP5-containing cytoplasmic vesicles are spheroidal in morphology with linear extensions, express TOMM20, and contain LC3B and LIMP-2, but not Sec22β, as fiber cells mature. TEM analysis revealed complex vesicular assemblies with congruent subcellular localization to AQP5-containing cytoplasmic vesicles. AQP5-containing cytoplasmic vesicles appear to dock with the plasma membrane. Bafilomycin A1 treatment reduced AQP5 plasma membrane expression by 27%. CONCLUSIONS: AQP5 localizes to spheroidal, linear cytoplasmic vesicles in the differentiating bovine lens fiber cells. During fiber cell differentiation, these vesicles incorporate LC3B and presumably fuse with LIMP-2–positive lysosomes. Our data suggest that AQP5 to the plasma membrane through lysosome-associated unconventional protein secretion, a novel mechanism of AQP5 trafficking.