Aquaporin 0 plays a pivotal role in refractive index gradient development in mammalian eye lens to prevent spherical aberration

Aquaporin 0 (AQP0) is a transmembrane channel that constitutes ∼45% of the total membrane protein of the fiber cells in mammalian lens. It is critical for lens transparency and homeostasis as mutations and knockout cause autosomal dominant lens cataract. AQP0 functions as a water channel and as a cell-to-cell adhesion (CTCA) molecule in the lens. Our recent in vitro studies showed that the CTCA function of AQP0 could be crucial to establish lens refractive index gradient (RING). However, there is a lack of in vivo data to corroborate the role of AQP0 as a fiber CTCA molecule which is critical for creating lens RING. The present investigation is undertaken to gather in vivo evidence for the involvement of AQP0 in developing lens RING. Lenses of wild type (WT) mouse, AQP0 knockout (heterozygous, AQP0^+/−) and AQP0 knockout lens transgenically expressing AQP1 (heterozygous AQP0^+/−/AQP1^+/−) mouse models were used for the study. Data on AQP0 protein profile of intact and N- and/or C-terminal cleaved AQP0 in the lens by MALDI-TOF mass spectrometry and SDS–PAGE revealed that outer cortex fiber cells have only intact AQP0 of ∼28 kDa, inner cortical and outer nuclear fiber cells have both intact and cleaved forms, and inner nuclear fiber cells have only cleaved forms (∼26–24 kDa). Knocking out of 50% of AQP0 protein caused light scattering, spherical aberration (SA) and cataract. Restoring the lost fiber cell membrane water permeability (P_f) by transgene AQP1 did not reinstate complete lens transparency and the mouse lenses showed light scattering and SA. Transmission and scanning electron micrographs of lenses of both mouse models showed increased extracellular space between fiber cells. Water content determination study showed increase in water in the lenses of these mouse models. In summary, lens transparency, CTCA and compact packing of fiber cells were affected due to the loss of 50% AQP0 leading to larger extracellular space, more water content and SA, possibly due to alteration in RING. To our knowledge, this is the first report identifying the role of AQP0 in RING development to ward off lens SA during focusing.     

Aquaporin 5 knockout mouse lens develops hyperglycemic cataract

The scope of this investigation was to understand the role of aquaporin 5 (AQP5) for maintaining lens transparency and homeostasis. Studies were conducted using lenses of wild-type (WT) and AQP5 knockout (AQP5-KO) mice. Immunofluorescent staining verified AQP5 expression in WT lens sections and lack of expression in the knockout. In vivo and ex vivo, AQP5-KO lenses resembled WT lenses in morphology and transparency. Therefore, we subjected the lenses ex vivo under normal (5.6 mM glucose) and hyperglycemic (55.6 mM glucose) conditions to test for cataract formation. Twenty-four hours after incubation in hyperglycemic culture medium, AQP5-KO lenses showed mild opacification which was accelerated several fold at 48 h; in contrast, WT lenses remained clear even after 48 h of hyperglycemic treatment. AQP5-KO lenses displayed osmotic swelling due to increase in water content. Cellular contents began to leak into the culture medium after 48 h. We reason that water influx through glucose transporters and glucose cotransporters into the cells could mainly be responsible for creating hyperglycemic osmotic swelling; absence of AQP5 in fiber cells appears to cause lack of required water efflux, challenging cell volume regulation and adding to osmotic swelling. This study reveals that AQP5 could play a critical role in lens microcirculation for maintaining transparency and homeostasis, especially by providing protection under stressful conditions. To the best of our knowledge, this is the first report providing evidence that AQP5 facilitates maintenance of lens transparency and homeostasis by regulating osmotic swelling caused by glucose transporters and cotransporters under hyperglycemic stressful conditions.     

Aquaporin-0 membrane junctions form upon proteolytic cleavage

Aquaporin-0 (AQP0), previously known as major intrinsic protein (MIP), is the only water pore protein expressed in lens fiber cells. AQP0 is highly specific to lens fiber cells and constitutes the most abundant intrinsic membrane protein in these cells. The protein is initially expressed as a full-length protein in young fiber cells in the lens cortex, but becomes increasingly cleaved in the lens core region. Reconstitution of AQP0 isolated from the core of sheep lenses containing a proportion of truncated protein, produced double-layered two-dimensional (2D) crystals, which displayed the same dimensions as the thin 11 nm lens fiber cell junctions, which are prominent in the lens core. In contrast reconstitution of full-length AQP0 isolated from the lens cortex reproducibly yielded single-layered 2D crystals. We present electron diffraction patterns and projection maps of both crystal types. We show that cleavage of the intracellular C terminus enhances the adhesive properties of the extracellular surface of AQP0, indicating a conformational change in the molecule. This change of function of AQP0 from a water pore in the cortex to an adhesion molecule in the lens core constitutes another manifestation of the gene sharing concept originally proposed on the basis of the dual function of crystallins.     

Intact and N- or C-terminal end truncated AQP0 function as open water channels and cell-to-cell adhesion proteins: End truncation could be a prelude for adjusting the refractive index of the lens to prevent spherical aberration

Background

Investigate the impact of natural N- or C-terminal post-translational truncations of lens mature fiber cell Aquaporin 0 (AQP0) on water permeability (Pw) and cell-to-cell adhesion (CTCA) functions.

Methods

The following deletions/truncations were created by site-directed mutagenesis (designations in parentheses): Amino acid residues (AA) 2–6 (AQP0-N-del-2-6), AA235–263 (AQP0-1-234), AA239–263 (AQP0-1-238), AA244–263 (AQP0-1-243), AA247–263 (AQP0-1-246), AA250–263 (AQP0-1-249) and AA260–263 (AQP0-1-259). Protein expression was studied using immunostaining, fluorescent tags and organelle-specific markers. Pw was tested by expressing the respective complementary ribonucleic acid (cRNA) in Xenopus oocytes and conducting osmotic swelling assay. CTCA was assessed by transfecting intact or mutant AQP0 into adhesion-deficient L-cells and performing cell aggregation and adhesion assays.

Results

AQP0-1-234 and AQP0-1-238 did not traffic to the plasma membrane. Trafficking of AQP0-N-del-2-6 and AQP0-1-243 was reduced causing decreased membrane Pw and CTCA. AQP0-1-246, AQP0-1-249 and AQP0-1-259 mutants trafficked properly and functioned normally. Pw and CTCA functions of the mutants were directly proportional to the respective amount of AQP0 expressed at the plasma membrane and remained comparable to those of intact AQP0 (AQP0-1-263).

Conclusions

Post-translational truncation of N- or C-terminal end amino acids does not alter the basal water permeability of AQP0 or its adhesive functions. AQP0 may play a role in adjusting the refractive index to prevent spherical aberration in the constantly growing lens.

General significance

Similar studies can be extended to other lens proteins which undergo post-translational truncations to find out how they assist the lens to maintain transparency and homeostasis for proper focusing of objects on to the retina.     

The Effects of GPX-1 Knockout on Membrane Transport and Intracellular Homeostasis in the Lens

Glutathione peroxidase-1 (GPX-1) is an enzyme that protects the lens against H₂O₂-mediated oxidative damage. The purpose of the present study was to determine the effects of GPX-1 knockout (KO) on lens transport and intracellular homeostasis. To investigate these lenses we used (1) whole lens impedance studies to measure membrane conductance, resting voltage and fiber cell gap junction coupling conductance; (2) osmotic swelling of fiber cell membrane vesicles to determine water permeability; and (3) injection of Fura2 and Na⁺-binding benzofuran isophthalate (SBFI) into fiber cells to measure [Ca²⁺] _i and [Na⁺] _i , respectively, in intact lenses. These approaches were used to compare wild-type (WT) and GPX-1 KO lenses from mice around 2 months of age. There were no significant differences in clarity, size, resting voltage, membrane conductance or fiber cell membrane water permeability between WT and GPX-1 KO lenses. However, in GPX-1 KO lenses, coupling conductance was 72% of normal in the outer shell of differentiating fibers and 45% of normal in the inner core of mature fibers. Quantitative Western blots showed that GPX-1 KO lenses had about 50% as much labeled Cx46 and Cx50 protein as WT, whereas they had equivalent labeled AQP0 protein as WT. Both Ca²⁺ and Na⁺ accumulated significantly in the core of GPX-1 KO lenses. In summary, the major effect on lens transport of GPX-1 KO was a reduction in gap junction coupling conductance. This reduction affected the lens normal circulation by causing [Na⁺] _i and [Ca²⁺] _i to increase, which could increase cataract susceptibility in GPX-1 KO lenses.     

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

Aquaporin 0 (AQP0) is a lens-specific protein comprising more than 30% of lens membrane protein content and is a member of the aquaporin family. Water permeates through AQP0 much more slowly than other aquaporin family members, and other compounds, such as glycerol, also permeate AQP0. In the lens, ascorbic acid (AA) is found at high concentrations, protecting the lens from photochemical events such as photo-oxidation. The aim of the present study was to clarify the function of AQP0. Mouse fibroblast L-cells stably expressing AQP0 were established and incubated in medium containing AA, and intracellular AA levels were measured by high-performance liquid chromatography (HPLC) and 2,6-dichlorophenol-indophenol (DCPIP) analysis. Intracellular AA levels in AQP0-expressing cells quickly rose and reached saturation 10 min after incubation in medium containing 1000 μM AA. In contrast, AA levels in cells slowly decreased when AA was washed out from the medium. Cells overexpressing AQP0 increased the cellular uptake of AA in a time- and concentration-dependent manner. These data suggest that AA as well as water permeates AQP0.AQP0 expression on Xenopus oocyte membranes was achieved by the injection of AQP0 cRNA into oocytes that were incubated in medium containing AA. Intracellular AA levels were then measured by HPLC. AA uptake was demonstrated in the AQP0-expressing oocytes and was shown to quickly reach saturation. Intracellular AA concentration in oocytes increased in a time- and concentration-dependent manner.The data in the present study show that AA permeates AQP0, reveal the role of AQP0 in AA permeability ex vivo, and also indicate that there is a difference between the import and export of AA via AQP0. These findings suggest that AQP0 plays an important role in controlling lens AA content.     

Luminostat operation: a tool to maximize microalgae photosynthetic efficiency in photobioreactors during the daily light cycle?

The luminostat regime has been proposed as a way to maximize light absorption and thus to increase the microalgae photosynthetic efficiency within photobioreactors. In this study, simulated outdoor light conditions were applied to a lab-scale photobioreactor in order to evaluate the luminostat control under varying light conditions. The photon flux density leaving the reactor (PFD_out) was varied from 4 to 20 μmol photons m⁻² s⁻¹and the productivity and photosynthetic efficiency of Chlorella sorokiniana were assessed.Maximal volumetric productivity (1.22 g kg⁻¹ d⁻¹) and biomass yield on PAR photons (400-700 nm) absorbed (1.27 g mol⁻¹) were found when PFD_out was maintained between 4 and 6 μmol photons m⁻² s⁻¹. The resultant photosynthetic efficiency was comparable to that already reported in a chemostat-controlled reactor. A strict luminostat regime could not be maintained under varying light conditions. Further modifications to the luminostat control are required before application under outdoor conditions.     

Endogenous bioelectric currents promote differentiation of the mammalian lens

The functional roles of bioelectrical signals (ES) created by the flow of specific ions at the mammalian lens equator are poorly understood. We detected that mature, denucleated lens fibers expressed high levels of the α1 and β1 subunits of Na⁺/K⁺‐ATPase (ATP1A1 and ATP1B1 of the sodium pump) and had a hyperpolarized membrane potential difference (V_mem). In contrast, differentiating, nucleated lens fiber cells had little ATP1A1 and ATP1B1 and a depolarized V_mem. Mimicking the natural equatorial ES with an applied electrical field (EF) induced a striking reorientation of lens epithelial cells to lie perpendicular to the direction of the EF. An EF also promoted the expression of β‐crystallin, aquaporin‐0 (AQP0) and the Beaded Filament Structural Protein 2 (BFSP2) in lens epithelial cells (LECs), all of which are hallmarks of differentiation. In addition, applied EF activated the AKT and CDC2 and inhibition of AKT reduced the activation of CDC2. Our results indicate that the endogenous bioelectrical signal at the lens equator promotes differentiation of LECs into denucleated lens fiber cells via depolarization of V_mem. Development of methods and devices of EF application or amplification in vivo may supply a novel treatment for lens diseases and even promote regeneration of a complete new lens following cataract surgery.     

Increased dietary cholesterol promotes enhanced mutagenesis in DNA polymerase kappa-deficient mice

DNA polymerase kappa (Polκ) bypasses planar polycyclic N²-guanine adducts in an error-free manner. Cholesterol derivatives may interact with DNA to form similarly bulky lesions. In accordance, these studies examined whether increased mutagenesis of DNA accompanies hypercholesterolemia in Polk^−/− mice. These mice also carried apoE gene knockouts to ensure increased levels of plasma cholesterol following exposure to a high cholesterol diet. The mice carried a reporter transgene (the λ-phage cII gene) for subsequent quantitative analysis of mutagenesis in various tissues. We observed significantly increased mutation frequencies in several organs of apoE^−/−Polk^−/− mice following a high cholesterol diet, compared to those remaining on a standard diet. Regardless of dietary regime, the mutation frequency in many organs was significantly higher in apoE^−/−Polk^−/− than in apoE^−/−Polk^+/+ mice. As expected for polycyclic guanine adducts, the mutations mainly consisted of G:C transversions. The life expectancy of apoE^−/−Polk^−/− mice maintained on a high cholesterol diet was reduced compared to apoE^−/−Polk^+/+ mice. Overall, this study demonstrates a role for Polκ in bypass of cholesterol-induced guanine lesions.     

P2X7 receptor mediated phosphorylation of p38MAP kinase in the hippocampus

This study was designed to explore the effect of P2X7 receptor (P2X7R) activation on the expression of p38 MAP kinase (p38 MAPK) enzyme in hippocampal slices of wild-type (WT) and P2X7R^−/− mice using the Western blot technique and to clarify its role in P2X7 receptor mediated [³H]glutamate release. ATP (1 mM) and the P2X7R agonist BzATP (100 μM) significantly increased p38 MAPK phosphorylation in WT mice, and these effects were absent in the hippocampal slices of P2X7R^−/− mice. Both ATP- and BzATP-induced p38 MAPK phosphorylations were sensitive to the p38 MAP kinase inhibitor, SB203580 (1 μM). ATP elicited [³H]glutamate release from hippocampal slices, which was significantly attenuated by SB203580 (1 μM) but not by the extracellular signal-regulated kinase (ERK1/2) inhibitor, PD098095 (10 μM). Consequently, we suggest that P2X7Rs and p38 MAPK are involved in the stimulatory effect of ATP on glutamate release in the hippocampal slices of WT mice.     

Mutations at key pore-lining positions differentiate the water permeability of fish lens aquaporin from other vertebrates

Aquaporin-0 (AQP0) is the major integral membrane protein of lens fiber cell and helps to maintain lens transparency by mediating inter-cell adhesion. To shed light on the unexpected higher water transport efficiency of killifish AQP0 as compared to mammalian orthologues, we performed a comparative analysis of all available AQP0 sequences and built 3D-models for representatives of different vertebrate classes.The analysis shows that air-living organisms evolved specific mutations at pore-lining positions to modulate the AQP0 water transport efficiency while maintaining the correct tertiary/quaternary arrangement to allow the formation of “thin junctions” between lens fiber cells. We conclude that the low permeability of mammalian AQP0 is required not to promote cell adhesion, but to modulate the water balance in a dry environment.     

Irgm 1参与动脉粥样硬化斑块的形成

目的:探讨免疫相关GTP酶1(Irgm 1)对小鼠血管动脉粥样硬化(AS)斑块形成的影响。方法:高脂饲料喂养野生型(WT)、ApoE~(-/-)Irgm 1~(+/+)和ApoE~(-/-)Irgm1~(+/-)小鼠3个月,建立AS模型;取小鼠主动脉弓,免疫荧光染色方法观察WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1的表达情况及部位;Western blot方法检测WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1蛋白表达情况;Q-PCR方法检测WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1 m RNA表达情况;油红O染色观察ApoE~(-/-)Irgm1~(+/+)和ApoE~(-/-)Irgm1~(+/-)小鼠血管AS斑块形成情况;结果:与WT组相比,ApoE~(-/-)Irgm 1~(+/+)组小鼠主动脉弓AS斑块中Irgm 1+细胞明显增多,Irgm 1+细胞主要位于血管AS斑块的表面;与WT组相比,ApoE~(-/-)Irgm 1~(+/+)组小鼠血管AS斑块中Irgm 1蛋白表达显著增多(P0.001),Irgm 1 m RNA表达显著增多(P0.01);与ApoE~(-/-)Irgm1~(+/-)组相比,ApoE~(-/-)Irgm1~(+/+)组小鼠主动脉弓AS斑块面积显著增大(P0.01);结论:Irgm 1能够促进血管AS斑块的形成。     

IRBIT: A regulator of ion channels and ion transporters

IRBIT (also called AHCYL1) was originally identified as a binding protein of the intracellular Ca^2 + channel inositol 1,4,5-trisphosphate (IP₃) receptor and functions as an inhibitory regulator of this receptor. Unexpectedly, many functions have subsequently been identified for IRBIT including the activation of multiple ion channels and ion transporters, such as the Na⁺/HCO₃⁻ co-transporter NBCe1-B, the Na⁺/H⁺ exchanger NHE3, the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR), and the Cl⁻/HCO₃⁻ exchanger Slc26a6. The characteristic serine-rich region in IRBIT plays a critical role in the functions of this protein. In this review, we describe the evolution, domain structure, expression pattern, and physiological roles of IRBIT and discuss the potential molecular mechanisms underlying the coordinated regulation of these diverse ion channels/transporters through IRBIT. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

AQP0-LTR of the Cat mouse alters water permeability and calcium regulation of wild type AQP0

Aquaporin 0 (AQP0) is the major intrinsic protein of the lens and its water permeability can be modulated by changes in pH and Ca²⁺. The Cataract Fraser (Cat^Fr) mouse accumulates an aberrant AQP0 (AQP0-LTR) in sub-cellular compartments resulting in a congenital cataract. We investigated the interference of AQP0-LTR with normal function of AQP0 in three systems. First, we created a transgenic mouse expressing AQP0 and AQP0-LTR in the lens. Expression of AQP0 did not prevent the congenital cataract but improved the size and transparency of the lens. Second, we measured water permeability of AQP0 co-expressed with AQP0-LTR in Xenopus oocytes. A low expression level of AQP0-LTR decreased the water permeability of AQP0, and a high expression level eliminated its calcium regulation. Third, we studied trafficking of AQP0 and AQP0-LTR in transfected lens epithelial cells. At low expression level, AQP0-LTR migrated with AQP0 toward the cell membrane, but at high expression level, it accumulated in sub-cellular compartments. The deleterious effect of AQP0-LTR on lens development may be explained by lowering water permeability and abolishing calcium regulation of AQP0. This study provides the first evidence that calcium regulation of AQP0 water permeability may be crucial for maintaining normal lens homeostasis and development.     

Crystal structure of a complex between the phosphorelay protein YPD1 and the response regulator domain of SLN1 bound to a phosphoryl analog

The crystal structure of the yeast SLN1 response regulator (RR) domain bound to both a phosphoryl analog [beryllium fluoride (BeF₃ ⁻)] and Mg^2 +, in complex with its downstream phosphorelay signaling partner YPD1, has been determined at a resolution of 1.70 Å. Comparisons between the BeF₃ ⁻-activated complex and the unliganded (or apo) complex determined previously reveal modest but important differences. The SLN1-R1·Mg^2 +·BeF₃ ⁻ structure from the complex provides evidence for the first time that the mechanism of phosphorylation-induced activation is highly conserved between bacterial RR domains and this example from a eukaryotic organism. Residues in and around the active site undergo slight rearrangements in order to form bonds with the essential divalent cation and fluorine atoms of BeF₃ ⁻. Two conserved switch-like residues (Thr1173 and Phe1192) occupy distinctly different positions in the apo versus BeF₃ ⁻-bound structures, consistent with the “Y-T” coupling mechanism proposed for the activation of CheY and other bacterial RRs. Several loop regions and the α4-β5-α5 surface of the SLN1-R1 domain undergo subtle conformational changes (∼ 1-3 Å displacements relative to the apo structure) that lead to significant changes in terms of contacts that are formed with YPD1. Detailed structural comparisons of protein-protein interactions in the apo and BeF₃ ⁻-bound complexes suggest at least a two-state equilibrium model for the formation of a transient encounter complex, in which phosphorylation of the RR promotes the formation of a phosphotransfer-competent complex. In the BeF₃ ⁻-activated complex, the position of His64 from YPD1 needs to be within ideal distance of and in near-linear geometry with Asp1144 from the SLN1-R1 domain for phosphotransfer to occur. The ground-state structure presented here suggests that phosphoryl transfer will likely proceed through an associative mechanism involving the formation of a pentacoordinate phosphorus intermediate.     

Horizontal or vertical photobioreactors? How to improve microalgae photosynthetic efficiency

The productivity of a vertical outdoor photobioreactor was quantitatively assessed and compared to a horizontal reactor. Daily light cycles in southern Spain were simulated and applied to grow the microalgae Chlorella sorokiniana in a flat panel photobioreactor.The maximal irradiance around noon differs from 400 μmol photons m⁻² s⁻¹ in the vertical position to 1800 μmol photons m⁻² s⁻¹ in the horizontal position. The highest volumetric productivity was achieved in the simulated horizontal position, 4 g kg culture⁻¹ d⁻¹. The highest photosynthetic efficiency was found for the vertical simulation, 1.3 g of biomass produced per mol of PAR photons supplied, which compares favorably to the horizontal position (0.85 g mol⁻¹) and to the theoretical maximal yield (1.8 g mol⁻¹). These results prove that productivity per unit of ground area could be greatly enhanced by placing the photobioreactors vertically.     

Gene expression of LOX-1, VCAM-1, and ICAM-1 in pre-atherosclerotic mice

To identify markers of the earliest stage of atherosclerosis, endothelial dysfunction, we evaluated the gene expression of lectin-like oxidized-low-density-lipoprotein receptor-1 (LOX-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in very young pre-atherosclerotic mice. Furthermore, the plasma levels of the soluble VCAM-1 and ICAM-1 were compared to the gene expression profiles. Gene expressions of LOX-1 and VCAM-1 were up-regulated in young apoE^−/− mice, and thus, it seems probable that these genes play a role in pre-atherosclerosis. Contrarily, the gene expression profile of ICAM-1 did not show any apparent differences between the groups, questioning the involvement of this molecule in the early development of atherosclerosis. Plasma levels of sVCAM-1 and sICAM-1 were similar in all mice and did not correlate with the vascular gene expression of the corresponding genes. It therefore seems likely that these circulating markers are not suited to detect early atherosclerosis.     

Anilinonaphthalenesulfonate fluorescence changes induced by non-enzymatic generation of membrane potential in mitochondria and submitochondrial particles

Changes in the fluorescence of 1-anilino-8-naphthalenesulfonate (ANS⁻) accompanying non-enzymatic generation of the membrane potential in mitochondria and sonicated submitochondrial particles have been demonstrated. Generation of the membrane potential was induced by addition of an ionophore (valinomycin for K⁺, or tetrachlorotri-fluoromethylbenzimidazole for H⁺) under conditions where there existed K⁺ (or H⁺) gradients across the mitochondrial membrane. The ANS⁻ fluorescence decreased when the mitochondrial (or particle) interior became more negative, and increased when it became more positive. Collapse of the membrane potential reversed the ANS⁻ responses. A hypothesis is put forward to explain the energy-dependent ANS⁻ responses in mitochondria and particles by the membrane potential-induced redistribution of ANS⁻ between the membrane and water phases.     

Differentiation-dependent modification and subcellular distribution of aquaporin-0 suggests multiple functional roles in the rat lens

Using immunohistochemistry and mass spectrometry, differentiation-dependent changes in the subcellular distribution and processing of aquaporin-0 (AQP0) have been mapped in the rat lens. Sections labelled with C-terminal tail AQP0 antibodies yielded two concentric rings of labelling with minimal signal in the lens core. The rings were separated by a transient zone of decreased labelling located prior to the transition of differentiating fiber (DF) cells into mature denucleated fiber (MF) cells. Mass spectrometry showed that the loss of core labelling was due to AQP0 cleavage, while the transient loss of labelling was more likely caused by masking of the antibody epitope. AQP0 subcellular distribution changed with radial distance into the lens. In peripheral DF cells, AQP0 was found throughout both broad and narrow side membranes. In deeper-lying DF cells, AQP0 aggregated into plaque-like structures located on the broad sides. This shift occurred prior to the transient loss of AQP0 signal, and coincided with formation of broad-side membrane invaginations between adjacent fiber cells to which filensin, a known binding partner of AQP0, was also localized. After nuclei loss, AQP0 was once again distributed throughout MF cell membranes. In the absence of protein synthesis, the observed subcellular redistribution of AQP0 in DF and subsequent cleavage of AQP0 in MF are suggestive of a switch in the function of AQP0 from a water channel to a junctional protein.