Abstract
Purpose :
Aquaporin-5 (AQP5) is the second most abundant aquaporin in ocular lens fiber cells and its localization and thus fiber cell permeability can be regulated. Previously we have shown that in bovine outer cortical lens fiber cells, AQP5 localizes to cytoplasmic spheroidal, tubular structures that co-stain with autophagosomal and mitochondrial markers. The purpose of this work is to define the mechanism that trafficks AQP5 from cytoplasmic structures to the plasma membrane.
Methods :
High resolution immunofluorescence (IF) was conducted on cryosections from cultured bovine lenses with zonules intact and treated with or without bafilomycin A1, an autophagosome/lysosome fusion inhibitor. AQP5, autophagosome marker LC3B, and lysosome marker LIMP2 were imaged by confocal microscopy. Quantitation of cytoplasmic and plasma membrane localized AQP5 was carried by image segmentation and assessing fluorescence intensity in subcellular regions using Nikon NIS Elements software. Transmission electron microscopy was also used to characterize subcellular structures and fusion of cytoplasmic structures to plasma membranes.
Results :
In the bovine lens outer cortex, AQP5 was entirely localized to cytoplasmic structures that were mitochondrial marker TOMM20-positive. Little overlap between LC3B or LIMP2 signals and these AQP5-containing structures was observed in this region. In the transition region between the outer cortex and inner cortex, AQP5 structures co-stained with autophagosomal marker LC3B and LIMP2 and appeared to fuse with the plasma membrane. In the inner cortex, AQP5 was completely localized to the plasma membrane. LIMP2 was detected in the plasma membrane in the inner cortex as well. Upon treatment with bafilomycin A1, plasma membrane AQP5 signal was reduced in the outer cortex/inner cortex transition region and cytoplasmic AQP5 structures appear to increase in number.
Conclusions :
Bovine AQP5 appears to traffic to the plasma membrane in the outer cortex/inner cortex transition region via an unconventional lysosomal trafficking mechanism that involves fusion of autolysosomes with the plasma membrane. Future experiments will focus on defining the signals that regulate AQP5 trafficking.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.