Lens aquaporin-5 inserts into bovine fiber cell plasma membranes through mitochondria-associated lysosome secretion

Abstract

PURPOSE: To spatially map aquaporin-5 (AQP5) expression in 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, LIMP-2, and connexin-50 antibodies and the fluorescent lipid membrane dye CM-DiI. AQP5 plasma membrane insertion was defined via line expression profile analysis. Transmission electron microscopy (TEM) was performed on bovine lens tissue sections to define cytoplasmic organelle identity, morphology, and subcellular localization in cortical fiber cells. Bovine lenses were treated with 10 nM bafilomycin A1 or 0.1% dimethyl sulfoxide vehicle control in ex vivo culture to determine changes in AQP5 plasma membrane expression. RESULTS. Immunofluorescence analysis revealed cytoplasmic AQP5 expression in bovine lens epithelial cells and differentiating fiber cells. In the bovine lens cortex, complete AQP5 plasma membrane insertion occurs at r/a 0.951 + 0.005. AQP5-containing cytoplasmic vesicles are spheroidal, tubular in morphology, express TOMM20, and contain LC3B and LIMP-2 as fiber cells mature. TEM analysis revealed spheroidal, tubular autophagosomes, autolysosomes, and lysosomes with degrading mitochondria. AQP5-containing cytoplasmic vesicles and autolysosomes dock and fuse with the plasma membrane. Bafiloymcin A1 treatment reduced AQP5 plasma membrane expression by 27%. CONCLUSIONS: AQP5 localizes to spheroidal, tubular cytoplasmic vesicles in the differentiating bovine lens fiber cells. During fiber cell differentiation, these vesicles incorporate LC3B and fuse with LIMP-2-positive lysosomes. AQP5 trafficking to the plasma membrane occurs through lysosome secretion as a novel mechanism of AQP5 trafficking.

Proteome Remodeling of the Eye Lens at 50 Years Identified with Data-Independent Acquisition

“…Though structurally similar to AQP0, AQP5 has approximately 20-fold higher water permeability than AQP0 (79). While AQP0 is consistently localized in the plasma membrane, AQP5 originates in lysosomal structures and is translocated to the plasma membrane as a part of maturation and in response to of osmotic stress (12, 19). Unlike AQP0, which does not undergo any significant change in abundance with the proteome remodeling event, AQP5 undergoes a significant decrease in abundance with age in the inner nucleus (Supplemental Figure S9).…”

“…The resulting potential, coupled to aquaporin water transport and gap junction intercellular contacts leads to a net current of metabolite convection into the lens at the anterior and posterior poles (15,16)(Figure 1B). Although unshown in Figure 1, it is also hypothesized that transient receptor potential cation channel subfamily V members 1 and 4 (TRPV1/4) mediate fiber cell osmolarity by regulation of Na/K ATPase activity and Aquaporin-5 trafficking to the plasma membrane (12,(17)(18)(19).…”

Lens Aquaporins in Health and Disease: Location is Everything!

“…In contrast, AQP5, a close homologue of AQP2, appears to traffic by a different mechanism from that of AQP0 ( Gletten et al, 2022 ). AQP5 displays a cell-dependent localization pattern where it is predominantly found in the cytoplasm of differentiating fiber cells and in the plasma membrane of mature fiber cells ( Grey et al, 2013 ; Petrova et al, 2015 ; Gletten et al, 2022 ).…”

“…In contrast, AQP5, a close homologue of AQP2, appears to traffic by a different mechanism from that of AQP0 ( Gletten et al, 2022 ). AQP5 displays a cell-dependent localization pattern where it is predominantly found in the cytoplasm of differentiating fiber cells and in the plasma membrane of mature fiber cells ( Grey et al, 2013 ; Petrova et al, 2015 ; Gletten et al, 2022 ). Within individual fiber cells of the same lens region, differences in the trafficking of AQP5 to the apical and basal membrane tips of fiber cells, corresponding to the anterior and posterior sutures, was observed ( Petrova et al, 2020 ).…”