Fiber Cell Membranes Research Articles

Zebrafish Optical Development Requires Regulated Water Permeability by Aquaporin 0.

Optical development of the zebrafish eye relies on the movement of the highly refractive lens nucleus from an anterior to a central location in the optical axis during development. We have shown that this mechanism in turn depends on the function of Aquaporin 0a (Aqp0a), a multifunctional and extremely abundant protein in lens fiber cell membranes. Here, we probe the specific cellular functions necessary for rescuing lens nucleus centralization defects in aqp0a-/- null mutants by stable overexpression of an Aqp0 orthologue from a killifish, MIPfun. We test in vivo requirements for lens transparency and nucleus centralization of MIPfun for auto-adhesion, water permeability (Pf), and Pf sensitivity to regulation by Ca2+ or pH by overexpression of MIPfun mutants previously shown to have defects in these functions in vitro or in silico. Water permeability of MIPfun is essential for rescuing lens transparency and nucleus centralization defects, whereas auto-adhesion is not. Furthermore, water permeability regulation by Ca2+ and pH appear residue-dependent, because some Ca2+-insensitive mutants fail to rescue, and pH-insensitive mutants only partially rescue defects. MIPfun lacking Pf sensitivity to both, Ca2+ and pH, also fails to rescue lens nucleus centralization. This study shows that regulation of water permeability by Aqp0 plays a key role in the centralization of the zebrafish lens nucleus, providing the first direct evidence for water transport in this aspect of optical development.

Verification of the gene and protein expression of the aquaglyceroporin AQP3 in the mammalian lens

Transport of water is critical for maintaining the transparency of the avascular lens, and the lens is known to express at least five distinctly different water channels from the Aquaporin (AQP) family of proteins. In this study we report on the identification of a sixth lens AQP, AQP3 an aquaglyceroporin, which in addition to water also transports glycerol and H2O2. AQP3 was identified at the transcript level and protein levels using RT-PCR and Western blotting, respectively, in the mouse, rat, bovine and human lens, showing that its expression is conserved in the mammalian lens. Western blotting showed AQP3 in the lens exists as 25 kDa non-glycosylated and 37 kDa glycosylated monomeric forms in all lens species. To identify the regions in the lens where AQP3 is expressed Western blotting was repeated using epithelial, outer cortical and inner cortical/core fractions isolated from the mouse lens. AQP3 was found in all lens regions, with the highest signal of non-glycosylated AQP3 being found in the epithelium. While in the inner cortex/core region AQP3 signal was not only lower but was predominately from the glycosylated form of AQP3. Immunolabelling of lens sections with AQP3 antibodies confirmed that AQP3 is found in all regions of the adult mouse, and also revealed that the subcellular distribution of AQP3 changes as a function of fiber cell differentiation. In epithelial and peripheral fiber cells of the outer cortex AQP3 labelling was predominately associated with membrane vesicles in the cytoplasm, but in the deeper regions of the lens AQP3 labelling was associated with the plasma membranes of fiber cells located in the inner cortex and core of the lens. To determine how this adult pattern of AQP3 subcellular distribution was established, immunolabelling for AQP3 was performed on embryonic and postnatal lenses. AQP3 expression was first detected on embryonic day (E) 11 in the membranes of primary fiber cells that have started to elongate and fill the lumen of the lens vesicle, while later at E16 the AQP3 labelling in the primary fiber cells had shifted to a predominately cytoplasmic location. In the following postnatal (P) stages of lens growth at P3 and P6, AQP3 labelling remained cytoplasmic across all regions of the lens and it was not until P15 when the pattern of localisation of AQP3 changed to an adult distribution with cytoplasmic labelling detected in the outer cortex and membrane localisation detected in the inner cortex and core of the lens. Comparison of the AQP3 labelling pattern to those obtained previously for AQP0 and AQP5 showed that the subcellular distribution was more similar to AQP5 than AQP0, but there were still significant differences that suggest AQP3 may have unique roles in the maintenance of lens transparency.

Cell surface plasticity in response to shape change in the whole organism

Plasma membrane rupture can result in catastrophic cell death. The skeletal muscle fiber plasma membrane, the sarcolemma, provides an extreme example of a membrane subject to mechanical stress since these cells specifically evolved to generate contraction and movement. A quantitative model correlating ultrastructural remodeling of surface architecture with tissue changes invivo is required to understand how membrane domains contribute to the shape changes associated with tissue deformation in whole animals. We and others have shown that loss of caveolae, small invaginations of the plasma membrane particularly prevalent in the muscle sarcolemma, renders the plasma membrane more susceptible to rupture during stretch.1,2,3 While it is thought that caveolae are able to flatten and be absorbed into the bulk membrane to buffer local membrane expansion, a direct demonstration of this model invivo has been unachievable since it would require measurement of caveolae at the nanoscale combined with detailed whole-animal morphometrics under conditions of perturbation. Here, we describe the development and application of the "active trapping model" where embryonic zebrafish are immobilized in a curved state that mimics natural body axis curvature during an escape response. The model is amenable to multiscale, multimodal imaging including high-resolution whole-animal three-dimensional quantitative electron microscopy. Using the active trapping model, we demonstrate the essential role of caveolae in maintaining sarcolemmal integrity and quantify the specific contribution of caveolar-derived membrane to surface expansion. We show that caveolae directly contribute to an increase in plasma membrane surface area under physiologically relevant membrane deformation conditions.

Modulation of Membrane Trafficking of AQP5 in the Lens in Response to Changes in Zonular Tension Is Mediated by the Mechanosensitive Channel TRPV1.

In mice, the contraction of the ciliary muscle via the administration of pilocarpine reduces the zonular tension applied to the lens and activates the TRPV1-mediated arm of a dual feedback system that regulates the lens' hydrostatic pressure gradient. In the rat lens, this pilocarpine-induced reduction in zonular tension also causes the water channel AQP5 to be removed from the membranes of fiber cells located in the anterior influx and equatorial efflux zones. Here, we determined whether this pilocarpine-induced membrane trafficking of AQP5 is also regulated by the activation of TRPV1. Using microelectrode-based methods to measure surface pressure, we found that pilocarpine also increased pressure in the rat lenses via the activation of TRPV1, while pilocarpine-induced removal of AQP5 from the membrane observed using immunolabelling was abolished by pre-incubation of the lenses with a TRPV1 inhibitor. In contrast, mimicking the actions of pilocarpine by blocking TRPV4 and then activating TRPV1 resulted in sustained increase in pressure and the removal of AQP5 from the anterior influx and equatorial efflux zones. These results show that the removal of AQP5 in response to a decrease in zonular tension is mediated by TRPV1 and suggest that regional changes to PH2O contribute to lens hydrostatic pressure gradient regulation.

Charged multivesicular body protein 4b forms complexes with gap junction proteins during lens fiber cell differentiation.

Charged multivesicular body protein 4b (CHMP4B) is a core sub-unit of the endosomal sorting complex required for transport III (ESCRT-III) machinery that serves myriad remodeling and scission processes of biological membranes. Mutation of the human CHMP4B gene underlies rare forms of early-onset lens opacities or cataracts, and CHMP4B is required for lens growth and differentiation in mice. Here, we determine the sub-cellular distribution of CHMP4B in the lens and uncover a novel association with gap junction alpha-3 protein (GJA3) or connexin 46 (Cx46) and GJA8 or Cx50. Immunofluorescence confocal microscopy revealed that CHMP4B localized to cell membranes of elongated fiber cells in the outer cortex of the lens-where large gap junction plaques begin to form-particularly, on the broad faces of these flattened hexagon-like cells in cross-section. Dual immunofluorescence imaging showed that CHMP4B co-localized with gap junction plaques containing Cx46 and/or Cx50. When combined with the in situ proximity ligation assay, immunofluorescence confocal imaging indicated that CHMP4B lay in close physical proximity to Cx46 and Cx50. In Cx46-knockout (Cx46-KO) lenses, CHMP4B-membrane distribution was similar to that of wild-type, whereas, in Cx50-KO lenses, CHMP4B localization to fiber cell membranes was lost. Immunoprecipitation and immunoblotting analyses revealed that CHMP4B formed complexes with Cx46 and Cx50 in vitro. Collectively, our data suggest that CHMP4B forms plasma membrane complexes, either directly and/or indirectly, with gap junction proteins Cx46 and Cx50 that are often associated with "ball-and-socket" double-membrane junctions during lens fiber cell differentiation.

Oxidation of active cysteines mediates protein aggregation of S10R, the cataract-associated mutant of mouse GammaB-crystallin.

The Ser10 to Arg mutation in mouse γB-crystallin (MGB) has been associated with protein aggregation, dense nuclear opacity, and the degeneration of fiber cells in the lens core. Overexpression of the gap junction protein, connexin 46 (Cx46), was found to suppress the nuclear opacity and restore normal cell-cell contact. However, the molecular basis for the protein aggregation and related downstream effects were not evident from these studies. Here, we provide a comparison of the structures and solution properties of wild type MGB and the S10R mutant in vitro and show that, even though the mutation does not directly involve cysteine residues, some cysteines in the mutant protein are activated, leading to the enhanced formation of intermolecular disulfide-crosslinked protein aggregates relative to the wild-type. This occurs even as the protein structure is essentially unaltered. Thus, the primary event is enhanced protein aggregation due to the disulfide crosslinking of the mutant protein. We suggest that these aggregates eventually get deposited on fiber cell membranes. Since the gap junction protein, Cx46 is involved in the transport of reduced glutathione, we posit that these deposits interfere in Cx46-mediated glutathione transport and facilitate the oxidative stress-mediated downstream changes. Overexpression of Cx46 suppresses such oxidative aggregation. These studies provide a plausible explanation for the protein aggregation and other changes that accompany this mutation. If indeed cysteine oxidation is the primary event for protein aggregation also in vivo, then the S10R mutant mouse, which is currently available, could serve as a viable animal model for human age-onset cataract.

Measurements of basal d-glucose transport through GLUT1 across the intact plasma membrane of isolated segments from single fast- and slow-twitch skeletal muscle fibres ofrat.

AimTo develop a method for direct measurement of the fluorescent d‐glucose analogue 2‐NBDG transport across the plasma membrane of single skeletal muscle fibres and derive the theoretical framework for determining the kinetic parameters for d‐glucose transport under basal conditions.MethodsA novel method is described for measuring free 2‐NBDG transport across plasma membrane of single rat muscle fibres at rest. The 2‐NBDG uptake was >90% suppressed by 100 µM cytochalasin B in both fast‐twitch and slow‐twitch fibres, indicating that the 2‐NBDG transport is GLUT‐mediated. Fibres were identified as fast‐twitch or slow‐twitch based on the differential sensitivity of their contractile apparatus to Sr2+.ResultsThe time course of 2‐NBDG uptake in the presence of 50 µM 2‐NBDG follows a one‐phase exponential plateau curve and is faster in fast‐twitch (rate constant 0.053 ± 0.0024 s‐1) than in slow‐twitch fibres (rate constant 0.031 ± 0.0021 s‐1). The rate constants were markedly reduced in the presence of 20 mM d‐glucose to 0.0082 ± 0.0004 s‐1 and 0.0056 ± 0.0002 s‐1 in fast‐twitch and slow‐twitch fibres respectively. 2‐NBDG transport was asymmetric, consistent with GLUT1 being the major functional GLUT isoform transporting 2‐NBDG in muscle fibres at rest. The parameters describing the transport kinetics for both 2‐NBDG and d‐glucose (dissociation constants, Michaelis–Menten constants, maximal rates of uptake and outflow) were calculated from the measurements made with 2‐NBDG.ConclusionFree 2‐NBDG and d‐glucose transport across the plasma membrane of single rat muscle fibres at rest is fast, conclusively showing that the rate‐limiting step in d‐glucose uptake in skeletal muscle is not necessarily the GLUT‐mediated transport of d‐glucose.

EphA2 Affects Development of the Eye Lens Nucleus and the Gradient of Refractive Index.

PurposeOur studies in mouse eye lenses demonstrate that ephrin-A5 and EphA2 are needed for normal epithelial cells and lens transparency. We sought to determine whether EphA2 and ephrin-A5 are important for lens morphometrics, nucleus formation, and refractive index.MethodsWe performed tissue morphometric measurements, electron microscopy, Western blots, and interferometric measurements using an X-ray synchrotron beam source to measure the gradient of refractive index (GRIN) to compare mouse lenses with genetic disruption of EphA2 or ephrin-A5.ResultsMorphometric analysis revealed that although there is no change in the overall lens volume, there is a change in lens shape in both EphA2−/− lenses and ephrin-A5−/− lenses. Surprisingly, EphA2−/− lenses had small and soft lens nuclei different from hard lens nuclei of control lenses. SEM images revealed changes in cell morphology of EphA2−/− fiber cells close to the center of the lens. Inner EphA2−/− lens fibers had more pronounced tongue-and-groove interdigitations and formed globular membrane morphology only in the deepest layers of the lens nucleus. We did not observe nuclear defects in ephrin-A5−/− lenses. There was an overall decrease in magnitude of refractive index across EphA2−/− lenses, which is most pronounced in the nucleus.ConclusionsThis work reveals that Eph-ephrin signaling plays a role in fiber cell maturation, nuclear compaction, and lens shape. Loss of EphA2 disrupts the nuclear compaction resulting in a small lens nucleus. Our data suggest that Eph-ephrin signaling may be required for fiber cell membrane reorganization and compaction and for establishing a normal GRIN.

Eph-ephrin Signaling Affects Eye Lens Fiber Cell Intracellular Voltage and Membrane Conductance.

The avascular eye lens generates its own microcirculation that is required for maintaining lifelong lens transparency. The microcirculation relies on sodium ion flux, an extensive network of gap junction (GJ) plaques between lens fiber cells and transmembrane water channels. Disruption of connexin proteins, the building blocks of GJs, or aquaporins, which make up water and adhesion channels, lead to lens opacification or cataracts. Recent studies have revealed that disruption of Eph-ephrin signaling, in particular the receptor EphA2 and the ligand ephrin-A5, in humans and mice lead to congenital and age-related cataracts. We investigated whether changes in lens transparency in EphA2 or ephrin-A5 knockout (–/–) mice is related to changes in GJ coupling and lens fluid and ion homeostasis. Immunostaining revealed changes in connexin 50 (Cx50) subcellular localization in EphA2–/– peripheral lens fibers and alteration in aquaporin 0 (Aqp0) staining patterns in ephrin-A5–/– and EphA2–/– inner mature fiber cells. Surprisingly, there was no obvious change in GJ coupling in knockout lenses. However, there were changes in fiber cell membrane conductance and intracellular voltage in knockout lenses from 3-month-old mice. These knockout lenses displayed decreased conductance of mature fiber membranes and were hyperpolarized compared to control lenses. This is the first demonstration that the membrane conductance of lens fibers can be regulated. Together these data suggest that EphA2 may be needed for normal Cx50 localization to the cell membrane and that conductance of lens fiber cells requires normal Eph-ephrin signaling and water channel localization.

Regulation of the Membrane Trafficking of the Mechanosensitive Ion Channels TRPV1 and TRPV4 by Zonular Tension, Osmotic Stress and Activators in the Mouse Lens.

Lens water transport generates a hydrostatic pressure gradient that is regulated by a dual-feedback system that utilizes the mechanosensitive transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, to sense changes in mechanical tension and extracellular osmolarity. Here, we investigate whether the modulation of TRPV1 or TRPV4 activity dynamically affects their membrane trafficking. Mouse lenses were incubated in either pilocarpine or tropicamide to alter zonular tension, exposed to osmotic stress, or the TRPV1 and TRPV4 activators capsaicin andGSK1016790A (GSK101), and the effect on the TRPV1 and TRPV4 membrane trafficking in peripheral fiber cells visualized using confocal microscopy. Decreases in zonular tension caused the removal of TRPV4 from the membrane of peripheral fiber cells. Hypotonic challenge had no effect on TRPV1, but increased the membrane localization of TRPV4. Hypertonic challenge caused the insertion of TRPV1 and the removal of TRPV4 from the membranes of peripheral fiber cells. Capsaicin caused an increase in TRPV4 membrane localization, but had no effect on TRPV1; while GSK101 decreased the membrane localization of TRPV4 and increased the membrane localization of TRPV1. These reciprocal changes in TRPV1/4 membrane localization are consistent with the channels acting as mechanosensitive transducers of a dual-feedback pathway that regulates lens water transport.

Visualization of the Skeletal Muscle Stem Cell Niche in Fiber Bundles.

Skeletal muscle stem cells (MuSCs) reside in a complex niche composed of the muscle fiber plasma membrane and the laminin‐rich basal lamina surrounded by the microvasculature, as well as different supportive cell types such as fibro‐adipogenic progenitors residing in the interstitial extracellular matrix. Within the first few hours after tissue damage, MuSCs undergo cytoskeletal rearrangements and transcriptional changes that prime the cells for activation. Due to their time‐consuming nature, enzymatic methods for liberation of single muscle fibers with fully quiescent MuSCs are challenging. Moreover, during enzymatic digestion, important niche components including the microvasculature and the collagenous interstitial matrix are destroyed. Here, we provide a method for the visualization of MuSCs on muscle fibers in their intact niche. Our method relies on mechanical teasing of fiber bundles from fixed skeletal muscles. We demonstrate that teased muscle fiber bundles allow the investigator to capture a representative snapshot of the MuSC niche in skeletal muscle, and outline how stem cell morphology and different microenvironmental components can be visualized. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of fiber bundles Basic Protocol 2: Immunofluorescence staining of MuSCs on fiber bundles Support Protocol: Preparation of Sylgard dishes

Mutation of the EPHA2 Tyrosine-Kinase Domain Dysregulates Cell Pattern Formation and Cytoskeletal Gene Expression in the Lens.

Genetic variations in ephrin type-A receptor 2 (EPHA2) have been associated with inherited and age-related forms of cataract in humans. Here, we have characterized the eye lens phenotype and transcript profile of germline Epha2 knock-in mutant mice homozygous for either a missense variant associated with age-related cataract in humans (Epha2-Q722) or a novel insertion-deletion mutation (Epha2-indel722) that were both located within the tyrosine-kinase domain of EPHA2. Confocal imaging of ex vivo lenses from Epha2-indel722 mice on a fluorescent reporter background revealed misalignment of epithelial-to-fiber cell meridional-rows at the lens equator and severe disturbance of Y-suture formation at the lens poles, whereas Epha2-Q722 lenses displayed mild disturbance of posterior sutures. Immunofluorescent labeling showed that EPHA2 was localized to radial columns of hexagonal fiber cell membranes in Epha2-Q722 lenses, whereas Epha2-indel722 lenses displayed disorganized radial cell columns and cytoplasmic retention of EPHA2. Immunoprecipitation/blotting studies indicated that EPHA2 formed strong complexes with Src kinase and was mostly serine phosphorylated in the lens. RNA sequencing analysis revealed differential expression of several cytoskeleton-associated genes in Epha2-mutant and Epha2-null lenses including shared downregulation of Lgsn and Clic5. Collectively, our data suggest that mutations within the tyrosine-kinase domain of EPHA2 result in lens cell patterning defects and dysregulated expression of several cytoskeleton-associated proteins.

Deletion of beaded filament proteins or the C-terminal end of Aquaporin 0 causes analogous abnormal distortion aberrations in mouse lens

Lens-specific beaded filament (BF) proteins CP49 and filensin interact with the C-terminus of the water channel protein Aquaporin 0 (AQP0). Previously we have reported that a C-terminally end-deleted AQP0-expressing transgenic mouse model AQP0ΔC/ΔC developed abnormal optical aberrations in the lens. This investigation was undertaken to find out whether the total loss of the BF structural proteins alter the optical properties of the lens and cause optical aberrations similar to those in AQP0ΔC/ΔC lenses; also, to map the changes in the optical quality as a function of age in the single or double BF protein knockouts as well as to assess whether there is any significant change in the water channel function of AQP0 in these knockouts. A double knockout mouse (2xKO) model for CP49 and filensin was developed by crossing CP49-KO and filensin-KO mice. Wild type, CP49-KO, filensin-KO, and 2xKO lenses at different ages, and AQP0ΔC/ΔC lenses at postnatal day-17 were imaged through the optical axis and compared for optical quality and focusing property. All three knockout models showed loss of transparency, and development of abnormal optical distortion aberration similar to that in AQP0ΔC/ΔC. Copper grid focusing by the lenses at 6, 9 and 12 months of age showed an increase in aberrations as age advanced. With progression in age, the grid images produced by the lenses of all KO models showed a transition from a positive barrel distortion aberration to a pincushion distortion aberration with the formation of three distinct aberration zones similar to those produced by AQP0ΔC/ΔC lenses. Water permeability of fiber cell membrane vesicles prepared from CP49-KO, filensin-KO and 2xKO models, measured using the osmotic shrinking method, remained similar to that of the wild type without any statistically significant alteration (P > 0.05). Western blotting and quantification revealed the expression of comparable quantities of AQP0 in all three BF protein KOs. Our study reveals that loss of single or both beaded filament proteins significantly affect lens refractive index gradient, transparency and focusing ability in an age-dependent manner and the interaction of BF proteins with AQP0 is critical for the proper functioning of the lens. The presence of BF proteins is necessary to prevent abnormal optical aberrations and maintain homeostasis in the aging lens.

Morphological and Nanomechanical Properties of Model of Animal and Human Eye Lens Lipid Membranes

The four major phospholipids found in eye lens membrane are phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Lens lipid composition of the fiber-cell membrane significantly changes with species. In lower-age animals, phosphatidylcholine (PC) is dominant, whereas, in humans, sphingomyelin (SM) is more dominant. We have used atomic force microscopy (AFM) to study morphological and nanomechanical properties of the lens lipid membrane models from mouse, pig, and humans. Small unilamellar vesicles (SUVs) were prepared from the mixture of four phospholipids (PC, SM, PS, and PE) that resemble mouse, pig, and human phospholipid composition lens membranes. The SUVs phospholipids mixtures were prepared with 0 mol% and 50 mol% of cholesterol. The topographical image and the mechanical properties of animal and human lens model membranes were accessed in a supported lipid membrane (SLM) formed on mica. The topographical images of phospholipid mixtures with 0 mol% cholesterol for both animal and human lens membrane models exhibited coexisting solid-ordered and liquid-disordered phase, where the proportion of solid-ordered phase increases with an increase in SM content in phospholipid mixture. The height difference between the two phases was ∼ 1nm. However, in the presence of 50 mol% of cholesterol, the membrane exhibited only a liquid-ordered phase, both for animal and human phospholipid mixtures. The breakthrough force on the SLM of the animals and human lens model membrane obtained with 50 mol% of cholesterol follow the trends: F (human) > F (pig) > F (mouse). Similarly, the membrane area compressibility modulus (Ka) of the membrane with 50 mol% cholesterol was accessed using the force curve's elastic regime. We found: Ka (human) > Ka (pig) > Ka (mouse). Our results show that phospholipid composition plays a significant role in modulating membrane mechanical properties.

Mutation of the TRPM3 cation channel underlies progressive cataract development and lens calcification associated with pro-fibrotic and immune cell responses.

Transient-receptor-potential cation channel, subfamily M, member 3 (TRPM3) serves as a polymodal calcium sensor in diverse mammalian cell-types. Mutation of the human TRPM3 gene (TRPM3) has been linked with inherited forms of early-onset cataract with or without other eye abnormalities. Here, we have characterized the ocular phenotypes of germline "knock-in" mice that harbor a human cataract-associated isoleucine-to-methionine mutation (p.I65M) in TRPM3 (Trpm3-mutant) compared with germline "knock-out" mice that functionally lack TRPM3 (Trpm3-null). Despite strong expression of Trpm3 in lens epithelial cells, neither heterozygous (Trpm3+/- ) nor homozygous (Trpm3-/- ) Trpm3-null mice developed cataract; however, the latter exhibited a mild impairment of lens growth. In contrast, homozygous Trpm3-M/M mutants developed severe, progressive, anterior pyramid-like cataract with microphthalmia, whereas heterozygous Trpm3-I/M and hemizygous Trpm3-M/- mutants developed anterior pyramidal cataract with delayed onset and progression-consistent with a semi-dominant lens phenotype. Histochemical staining revealed abnormal accumulation of calcium phosphate-like deposits and collagen fibrils in Trpm3-mutant lenses and immunoblotting detected increased αII-spectrin cleavage products consistent with calpain hyper-activation. Immunofluorescent confocal microscopy of Trpm3-M/M mutant lenses revealed fiber cell membrane degeneration that was accompanied by accumulation of alpha-smooth muscle actin positive (α-SMA+ve) myofibroblast-like cells and macrosialin positive (CD68+ve) macrophage-like cells. Collectively, our mouse model data support an ocular disease association for TRPM3 in humans and suggest that (1) Trpm3 deficiency impaired lens growth but not lens transparency and (2) Trpm3 dysfunction resulted in progressive lens degeneration and calcification coupled with pro-fibrotic (α-SMA+ve) and immune (CD68+ve) cell responses.

Formation of cholesterol Bilayer Domains Precedes Formation of Cholesterol Crystals in Membranes Made of the Major Phospholipids of Human Eye Lens Fiber Cell Plasma Membranes

ABSTRACTPurpose/Aim: The goal of this study is to reveal how age-related changes in phospholipid (PL) composition in the fiber cell plasma membranes of the human eye lens affect the cholesterol (Chol) content at which Chol bilayer domains (CBDs) and Chol crystals start to form.Materials and Methods: Saturation-recovery electron paramagnetic resonance with spin-labeled cholesterol analogs and differential scanning calorimetry were used to determine the Chol contents at which CBDs and cholesterol crystals, respectively, start to form in in membranes made of the major PL constituents of the plasma membrane of the human eye lens fiber cells. To preserve compositional homogeneity throughout the membrane suspension, the lipid multilamellar dispersions investigated in this work were prepared using a rapid solvent exchange method. The cholesterol content changed from 0 to 75 mol%.Results: The saturation recovery electron paramagnetic resonance results show that CBDs start to form at 33, 50, 46, and 48 mol% Chol in the phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin bilayers, respectively. The differential scanning calorimetry results show that Chol crystals start to form at 50, 66, 70, and 66 mol% Chol in the phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin bilayers, respectively.Conclusions: These results, as well those of our previous studies, indicate that the formation of CBDs precedes the formation of Chol crystals in all of the studied systems, and the appearance of each depends on the type of PL forming the bilayer. These findings contribute to a better understanding of the molecular mechanisms involved in the regulation of Chol-dependent processes in eye lens fiber cell membranes.

Verification and spatial mapping of TRPV1 and TRPV4 expression in the embryonic and adult mouse lens

The transient receptor protein vanilloid channels, TRPV1 and TRPV4, have recently been shown to be mechanosensors in the ocular lens that act to transduce physical changes in lens volume and internal hydrostatic pressure into the activation of signalling pathways in lens epithelial cells. These pathways in turn regulate ion and water transport to ensure that the optical properties of the lens remain constant. Despite the functional evidence that implicate the roles of TRPV1 and TRPV4 in the lens, their respective cellular expression patterns in the different regions of the lens has to date not been fully characterised. Using Western blotting we have confirmed that TRPV1 and TRPV4 are expressed throughout all regions (epithelium, outer cortex, inner cortex/core) of the adult mouse lens. Subsequent immunolabeling of lens cryosections confirmed that TRPV1 and TRPV4 are expressed throughout all regions of the lens, but revealed differentiation-dependent differences in the subcellular expression of the two channels in the different regions. In the epithelium and outer cortex, intense TRPV1 and TRPV4 labeling was predominately associated with the cytoplasm. In a discrete zone in the inner cortex, labeling for both proteins was greatly diminished, but could be enhanced by incubating sections with the detergent Triton X-100 to reveal TRPV1 and TRPV4 labelling that was associated with the membrane. This suggests that in this region of the lens there is a potential interacting protein that masks the binding of the TRPV1 and TRPV4 antibodies to their respective epitopes in the lens inner cortex. In the core of the lens, which contains the embryonic nucleus, TRPV1 and TRPV4 labelling was associated exclusively with fibre cell membranes. This labelling in the lens core of the adult mouse lens appeared to originate in early development as a similar membrane labelling was observed at embryonic day 10 (E10) of the cells in the lens vesicle that subsequently forms the embryonic nucleus in the adult lens. During subsequent stages of embryonic development TRPV1 and TRPV4 remained membranous in the inner cortex and core, while showing labelling that was associated with the cytoplasm in the superficial outer cortical region. The extent of cytoplasmic labelling for TRPV4, but not TRPV1, in this cortical region could however be dynamically regulated by cutting the zonules that normally attach the lens to the ciliary body. We have shown an early onset and continuous expression of TRPV1 and TRPV4 across all lens regions, and that TRPV4 can be dynamically trafficked into the membranes of differentiating fibre cells, results that suggests that these mechanosensitive channels may also be functionally active in lens fibre cells.

A predominant form of C-terminally end-cleaved AQP0 functions as an open water channel and an adhesion protein in AQP0ΔC/ΔC mouse lens

The purpose of this investigation was to find out whether C-terminally end-cleaved aquaporin 0 (AQP0), that is present predominantly in the lens mature fiber cells of the WT, functions as a water channel and a cell-to-cell adhesion (CTCA) protein in a knockin (KI) mouse model (AQP0ΔC/ΔC) that does not express intact AQP0. A genetically engineered KI mouse model, AQP0ΔC/ΔC, expressing only end-cleaved AQP0 was developed. This model expresses 1–246 amino acids of AQP0, instead of the full length 1–263 amino acids. Lens transparency of postnatal day 10 (P10) was analyzed qualitatively by dark field imaging. WT, AQP0+/− and AQP0+/ΔC lenses were transparent; AQP0−/− and AQP0ΔC/ΔC mouse lenses displayed loss of transparency. Lens fiber cell membrane vesicles (FCMVs) were prepared from wild type (WT), AQP0 heterozygous (AQP0+/−), AQP0 knockout (AQP0−/−), AQP0+/ΔC and AQP0ΔC/ΔC; water permeability (Pf) was measured using the osmotic shrinking method. CTCA assay was performed using adhesion-deficient L-cells and FCMVs prepared from the abovementioned genotypes. FCMVs of AQP0+/− and AQP0−/− showed a statistically significant reduction (P < 0.001) in Pf and CTCA compared to those of WT. AQP0+/ΔC and AQP0ΔC/ΔC FCMVs exhibited no statistically significant alteration (P > 0.05) in Pf compared to those of WT. However, CTCA of AQP0+/ΔC AQP0ΔC/ΔC FCMVs was significantly higher (P < 0.001) than that of WT FCMVs. Our experiments clearly show that C-terminally end-cleaved AQP0 can function both as a water channel and a CTCA molecule in the lens fiber cell membranes. Also, end-truncation plays an important role in increasing the CTCA between fiber cells.

Tropomyosin 3.5 protects the F-actin networks required for tissue biomechanical properties.

Tropomyosins (Tpms) stabilize F-actin and regulate interactions with other actin-binding proteins. The eye lens changes shape in order to focus light to transmit a clear image, and thus lens organ function is tied to its biomechanical properties, presenting an opportunity to study Tpm functions in tissue mechanics. Mouse lenses contain Tpm3.5 (also known as TM5NM5), a previously unstudied isoform encoded by Tpm3, which is associated with F-actin on lens fiber cell membranes. Decreased levels of Tpm3.5 lead to softer and less mechanically resilient lenses that are unable to resume their original shape after compression. While cell organization and morphology appear unaffected, Tmod1 dissociates from the membrane in Tpm3.5-deficient lens fiber cells resulting in reorganization of the spectrin-F-actin and α-actinin-F-actin networks at the membrane. These rearranged F-actin networks appear to be less able to support mechanical load and resilience, leading to an overall change in tissue mechanical properties. This is the first in vivo evidence that a Tpm protein is essential for cell biomechanical stability in a load-bearing non-muscle tissue, and indicates that Tpm3.5 protects mechanically stable, load-bearing F-actin in vivoThis article has an associated First Person interview with the first author of the paper.

Molecular mechanism of Aquaporin 0-induced fiber cell to fiber cell adhesion in the eye lens

Cell-to-cell adhesion (CTCA), which is key for establishing lens transparency, is a critical function of Aquaporin 0 (AQP0). The aim of this investigation was to find out the possible mechanism by which AQP0 exerts CTCA between fiber cells, since there are two proposals currently, either an AQP0-AQP0 interaction or an AQP0-lipid interaction. We studied the mechanism of AQP0-induced CTCA in intact AQP0 and C-terminally cleaved AQP0 (CTC-AQP0). Assays showed CTCA between L-cells transfected with intact AQP0 or CTC-AQP0 and parental L-cells indicating AQP0-membrane interaction. Both forms of AQP0 significantly (P < 0.001) promoted adhesion to negatively charged l-α-phosphatidylserine lipid vesicles signifying AQP0-lipid interaction. AQP0-expressing L-cells also promoted adhesion of WT and AQP0-KO mouse lens fiber cell membrane vesicles (FCMVs) significantly (P < 0.001). However, when FCMVs of WT or AQP0-KO were plated over parental L-cells, only WT vesicles adhered significantly, corroborating AQP0-membrane interaction. After incubating with extracellular domain-specific AQP0 antibody, L-cells expressing intact AQP0 or CTC-AQP0 showed a significant reduction (P < 0.001) in the adhesion of AQP0-KO FCMVs indicating extracellular loop involvement in CTCA. WT FCMVs from outer cortex and inner cortex promoted adhesion to parental L-cells, without any statistically significant difference in adhesion efficiency (P > 0.05). Ultrastructure studies of WT, AQP0-KO and transgenic lenses showed AQP0 is critical for fiber CTCA and compaction. The data collected clearly demonstrate that the positively charged amino acids in the AQP0 extracellular loop domains interact with the negatively charged lipids in the plasma membrane to promote CTCA for compaction of fiber cells.