Lens Membrane Research Articles

Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts

Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein–protein contacts but also due to increased lipid–protein complementarity. Since each tetramer interacts with four such ‘glue’ cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.

Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts.

Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.

Aquaporin-0-protein interactions elucidated by crosslinking mass spectrometry

Aquaporin-0 (AQP0) constitutes 50 % of the lens membrane proteome and plays important roles in lens fiber cell adhesion, water permeability, and lens transparency. Previous work has shown that specific proteins, such as calmodulin (CaM), interact with AQP0 to modulate its water permeability; however, these studies often used AQP0 peptides, rather than full-length protein, to probe these interactions. Furthermore, the specific regions of interaction of several known AQP0 interacting partners, i.e. αA and αB-crystallins, and phakinin (CP49) remain unknown. The purpose of this study was to use crosslinking mass spectrometry (XL-MS) to identify interacting proteins with full-length AQP0 in crude lens cortical membrane fractions and to determine the specific protein regions of interaction. Our results demonstrate, for the first time, that the AQP0 N-terminus can engage in protein interactions. Specific regions of interaction are elucidated for several AQP0 interacting partners including phakinin, α-crystallin, connexin-46, and connexin-50. In addition, two new interacting partners, vimentin and connexin-46, were identified.

Abnormal function of EPHA2/p.R957P mutant in congenital cataract.

To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity. A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited. Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals. Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery. The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software. Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2 (EPHA2). Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins. The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay, respectively. The cell migration was analyzed by wound healing assay. Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo. A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif (SAM) domain of EPHA2. Functional studies demonstrated the variant's impact: reduced EPHA2 protein expression, altered subcellular localization, and disrupted interactions with other lens membrane proteins. This mutant notably enhanced human lens epithelial cell migration, and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast (DIC) optics. Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.

Neurotrophic Keratopathy after wide retinal endolaser and postoperative Ketorolac eye drops: A case series.

We report a series of 5 cases, happened in a period of 5 months, who developed neurotrophic keratopathy (NK) following pars plana vitrectomy (PPV) and retinal endolaser for rhegmatogenous retinal detachment (RRD). In our several decennary experience of surgical center predominantly based on vitreoretinal surgery, we had rare cases of postoperative NK. These recent cases of post-surgical NK happened contextually to our change of postoperative non-steroidal anti-inflammatory drugs (NSAIDs) drops, based on Ketorolac Tromethamine 0.5% eye drops. Five patients with a mean age of 61 ± 7.3 years were treated with one or more PPV with intraoperative peripheral endolaser for RRD. Nobody had previous herpetic keratitis, systemic disease like diabetes mellitus or other predisposing factors for NK. In the postoperative period, all patients received Ketorolac Tromethamine 0.5% eye drops for a mean period of 54 ± 25 days. During follow-up visits they developed NK and they were successfully treated with suspension of Ketorolac eye drops, application of therapeutic contact lens or amniotic membrane patch and topical lubricant therapy. Postoperative Ketorolac eye drops, in patients who underwent PPV with endolaser, may reduce the corneal sensitivity, predispose to epithelial disruption and NK development. Studies are needed to explore the effect of NSAIDs on corneal sensitivity reduction in patient who will undergo PPV and extensive endolaser.

Interaction of βL- and γ-Crystallin with Phospholipid Membrane Using Atomic Force Microscopy.

Highly concentrated lens proteins, mostly β- and γ-crystallin, are responsible for maintaining the structure and refractivity of the eye lens. However, with aging and cataract formation, β- and γ-crystallin are associated with the lens membrane or other lens proteins forming high-molecular-weight proteins, which further associate with the lens membrane, leading to light scattering and cataract development. The mechanism by which β- and γ-crystallin are associated with the lens membrane is unknown. This work aims to study the interaction of β- and γ-crystallin with the phospholipid membrane with and without cholesterol (Chol) with the overall goal of understanding the role of phospholipid and Chol in β- and γ-crystallin association with the membrane. Small unilamellar vesicles made of Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Chol/POPC) membranes with varying Chol content were prepared using the rapid solvent exchange method followed by probe tip sonication and then dispensed on freshly cleaved mica disk to prepare a supported lipid membrane. The βL- and γ-crystallin from the cortex of the bovine lens was used to investigate the time-dependent association of βL- and γ-crystallin with the membrane by obtaining the topographical images using atomic force microscopy. Our study showed that βL-crystallin formed semi-transmembrane defects, whereas γ-crystallin formed transmembrane defects on the phospholipid membrane. The size of semi-transmembrane defects increases significantly with incubation time when βL-crystallin interacts with the membrane. In contrast, no significant increase in transmembrane defect size was observed in the case of γ-crystallin. Our result shows that Chol inhibits the formation of membrane defects when βL- and γ-crystallin interact with the Chol/POPC membrane, where the degree of inhibition depends upon the amount of Chol content in the membrane. At a Chol/POPC mixing ratio of 0.3, membrane defects were observed when both βL- and γ-crystallin interacted with the membrane. However, at a Chol/POPC mixing ratio of 1, no association of γ-crystallin with the membrane was observed, which resulted in a defect-free membrane, and the severity of the membrane defect was decreased when βL-crystallin interacted with the membrane. The semi-transmembrane or transmembrane defects formed by the interaction of βL- and γ-crystallin on phospholipid membrane might be responsible for light scattering and cataract formation. However, Chol suppressed the formation of such defects in the membrane, likely maintaining lens membrane homeostasis and protecting against cataract formation.

Protecting the Eye Lens from Oxidative Stress through Oxygen Regulation.

Molecular oxygen is a primary oxidant that is involved in the formation of active oxygen species and in the oxidation of lipids and proteins. Thus, controlling oxygen partial pressure (concentration) in the human organism, tissues, and organs can be the first step in protecting them against oxidative stress. However, it is not an easy task because oxygen is necessary for ATP synthesis by mitochondria and in many biochemical reactions taking place in all cells in the human body. Moreover, the blood circulatory system delivers oxygen to all parts of the body. The eye lens seems to be the only organ that is protected from the oxidative stress through the regulation of oxygen partial pressure. The basic mechanism that developed during evolution to protect the eye lens against oxidative damage is based on the maintenance of a very low concentration of oxygen within the lens. This antioxidant mechanism is supported by the resistance of both the lipid components of the lens membrane and cytosolic proteins to oxidation. Any disturbance, continuous or acute, in the working of this mechanism increases the oxygen concentration, in effect causing cataract development. Here, we describe the biophysical basis of the mechanism and its correlation with lens transparency.

Binding of βL-Crystallin with Models of Animal and Human Eye Lens-Lipid Membrane.

Several discoveries show that with age and cataract formation, β-crystallin binds with the lens membrane or associates with other lens proteins, which bind with the fiber cell plasma membrane, accompanied by light scattering and cataract formation. However, how lipids (phospholipids and sphingolipids) and cholesterol (Chol) influence β-crystallin binding to the membrane is unclear. This research aims to elucidate the role of lipids and Chol in the binding of β-crystallin to the membrane and the membrane's physical properties (mobility, order, and hydrophobicity) with β-crystallin binding. We used electron paramagnetic resonance (EPR) spin-labeling methods to investigate the binding of βL-crystallin with a model of porcine lens-lipid (MPLL), model of mouse lens-lipid (MMLL), and model of human lens-lipid (MHLL) membrane with and without Chol. Our results show that βL-crystallin binds with all of the investigated membranes in a saturation manner, and the maximum parentage of the membrane surface occupied (MMSO) by βL-crystallin and the binding affinity (Ka) of βL-crystallin to the membranes followed trends: MMSO (MPLL) > MMSO (MMLL) > MMSO (MHLL) and Ka (MHLL) > Ka (MMLL) ≈ Ka (MPLL), respectively, in which the presence of Chol reduces the MMSO and Ka for all membranes. The mobility near the headgroup regions of the membranes decreases with an increase in the binding of βL-crystallin; however, the decrease is more pronounced in the MPLL and MMLL membranes than the MHLL membrane. In the MPLL and MMLL membranes, the membranes become slightly ordered near the headgroup with an increase in βL-crystallin binding compared to the MHLL membrane. The hydrophobicity near the headgroup region of the membrane increases with βL-crystallin binding; however, the increase is more pronounced in the MPLL and MMLL membranes than the MHLL membrane, indicating that βL-crystallin binding creates a hydrophobic barrier for the passage of polar molecules, which supports the barrier hypothesis in cataract formation. However, in the presence of Chol, there is no significant increase in hydrophobicity with βL-crystallin binding, suggesting that Chol prevents the formation of a hydrophobic barrier, possibly protecting against cataract formation.

Comparison between Placement of an Amniotic Membrane Contact Lens and Temporary Amniotic Membrane Transplantation

Purpose: We compared the clinical utilities of an amniotic contact lens and temporary amniotic membrane transplantation in patients with persistent corneal epithelial defects.Methods: Patients diagnosed with persistent corneal epithelial defects from November 2019 to July 2022 were randomly assigned to either lens placement for 2 weeks or temporary transplantation for 1 week, and the outcomes were retrospectively analyzed. The best-corrected visual acuity, corneal sensation, tear film break-up time, and corneal opacity were measured before and after the interventions. The treatment success rates, recurrences, and other complications were analyzed during the follow-up period.Results: We placed lenses in 32 patients (32 eyes) and 20 patients (20 eyes) underwent transplantation. In both groups, significant differences in best-corrected visual acuity, corneal sensation, tear film break-up time, and corneal opacity were apparent after intervention vs. before. None of the parameters showed any significant differences in terms of their rates of change.Conclusions: In patients with persistent corneal epithelial defects, amniotic contact lens placement is an effective outpatient procedure and is not inferior to temporary amniotic membrane transplantation.

Interactions of beta-crystallin with a model of human eye lens-lipid membranes.

β-Crystallin helps maintain the eye lens's structure and refractivity. With age and cataract formation, α-, β-, and γ-crystallin may undergo various mutations and post-translational modifications, and consequently form higher molecular weight complexes, some of which bind to lens membranes. However, the binding of β-crystallin alone to the lens membrane is still unclear. This study aims to investigate the binding of β-crystallin to the model of the human lens-lipid (MHLL) membrane and the physical properties of the MHLL membrane after β-crystallin binding. Small unilamellar vesicles of MHLL membranes, resembling the human lens-lipid composition, were prepared using the rapid solvent exchange method and probe-tip sonication. The 1 mol% cholesterol analogue spin label (CSL) integrated into the membrane, which resides near the lens-lipid headgroup region, probes β-crystallin binding to the membrane using the electron paramagnetic resonance (EPR) spin-labeling method. Our results showed that β-crystallin binds to the MHLL membrane with a binding affinity (Ka) of 0.082 ± 0.006 μM−1 and saturates around 5% of the membrane surface occupied. Furthermore, β-crystallin binding to the MHLL membrane decreased the mobility parameter, making the headgroups of the membrane less mobile. However, β-crystallin binding to the MHLL membrane showed no significant change in maximum splitting, meaning it does not affect the order near the headgroup regions of the MHLL membrane. Moreover, the hydrophobicity of the MHLL membrane also did not change significantly with the binding of β-crystallin, meaning hydrophobicity near the headgroup regions does not significantly change with β-crystallin binding. Our results showed that β-crystallin binding with MHLL membranes changes the physical property of membranes, implying that β-crystallin binding to the lens membrane may change the integrity of the membrane and has the potential to promote cataract formation.

Binding of Alpha-Crystallin to Cortical and Nuclear Lens Lipid Membranes Derived from a Single Lens.

Several studies reported that α-crystallin concentrations in the eye lens cytoplasm decrease with a corresponding increase in membrane-bound α-crystallin with age and cataracts. The influence of the lipid and cholesterol composition difference between cortical membrane (CM) and nuclear membrane (NM) on α-crystallin binding to membranes is still unclear. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the α-crystallin binding to bovine CM and NM derived from the total lipids extracted from a single lens. Compared to CMs, NMs have a higher percentage of membrane surface occupied by α-crystallin and binding affinity, correlating with less mobility and more order below and on the surface of NMs. α-Crystallin binding to CM and NM decreases mobility with no significant change in order and hydrophobicity below and on the surface of membranes. Our results suggest that α-crystallin mainly binds on the surface of bovine CM and NM and such surface binding of α-crystallin to membranes in clear and young lenses may play a beneficial role in membrane stability. However, with decreased cholesterol content within the CM, which mimics the decreased cholesterol content in the cataractous lens membrane, α-crystallin binding increases the hydrophobicity below the membrane surface, indicating that α-crystallin binding forms a hydrophobic barrier for the passage of polar molecules, supporting the barrier hypothesis in developing cataracts.

Molecular dynamics simulations of the human ocular lens with age and cataract

The human ocular lens consists primarily of elongated, static fibers characterized by high stability and low turnover, which differ dramatically in their composition and properties from other biological membranes. Cholesterol (Chol) and sphingolipids (SL) are present at high concentrations, including saturated SLs, such as dihyrosphingomyelin (DHSM). Past molecular dynamics simulations demonstrated that the presence of DHSM and high Chol concentration contributes to higher order in lipid membranes. This current study simulated more complex models of human lens membranes. Models were developed representing physiological compositions in cataractous lenses aged 74 ± 6 years and in healthy lenses aged 22 ± 4, 41 ± 6, and 69 ± 3 years. With older age, Chol and ceramide concentrations increase and glycerophospholipid concentration decreases. With cataract, ceramide concentration increases and Chol and glycerophospholipid concentrations decrease. Surface area per lipid, deuterium order parameters (SCD), sterol tilt angle, electron density profiles, bilayer thickness, chain interdigitation, two-dimensional radial distribution functions (2D-RDF), lipid clustering, and hydrogen bonding were calculated for all simulations. All systems exhibited low surface area per lipid and high bilayer thickness, indicative of strong vertical packing. SCD parameters suggest similarly, with saturated tails in the hydrophobic core of the membrane having elevated order. Vertical packing and acyl tail order increased with both age and cataract condition. Lateral diffusion decreased with age and cataracts, with the older and cataractous models demonstrating increased long-range structure by the 2D-RDF analysis. In future work examining the membrane proteins of the lens, these models can serve as a physiologically accurate representation of the lens lipidome.

Efficacy of N-Acetylcysteine (NAC) Against Malondialdehyde (MDA) Levels in Diabetic Cataracts: In vivo Study

Background: Diabetes mellitus is a systemic condition that affects various organs, including the eyes. N-acetylcysteine ​​(NAC) functions as an antioxidant because it belongs to a thiol group synthesis glutathione. However, the availability of cysteine ​​in the body is only 15%, so cysteine ​​supplementation can help with oxidative stress in diabetic cataracts. That it will prevent the reaction of lipid peroxidase and the formation of PUFA in the lens membrane, which causes damage to lens cells and is characterized by an increase in malondialdehyde. This study aims to determine the comparison of malondialdehyde levels in the lenses of diabetic cataract rats given or without topical NAC.
 Methods: 36 rats were divided into the control group (received streptozotocin) and treatment group (received streptozotocin and topical NAC. Intraperitoneal injection of streptozotocin was given 90 mg/kg BW and performed once a week for 3 weeks. GDP examination was performed the next day after the rats fasted for 16 hours from the vein. In the tail, GDP > 110 mg/dl categorized as diabetes. Topical NAC was given 4 times per day for 3 weeks. At the end of the study, the lens was extracted for the measurement of malondialdehyde.
 Results: The mean MDA levels in the control group were higher (2.90±0.71nmol/ml) than in the treatment group (2.33±0.38nmol/ml), p<0.05.
 Conclusion: NAC was effective in reducing oxidative stress levels in diabetic cataract lenses by lowering MDA levels better than the group that did not receive MDA in vivo.

Membrane elasticity modulated by cholesterol in model of porcine eye lens-lipid membrane

Experimental evidence shows that the eye lens loses its elasticity dramatically with age. It has also been reported that the cholesterol (Chol) content in the eye lens fiber cell plasma membrane increases significantly with age. High Chol content leads to the formation of cholesterol bilayer domains (CBDs) in the lens membrane. The role of high Chol associated with lens elasticity is unclear. The purpose of this research is to investigate the membrane elasticity of the model of porcine lens-lipid (MPLL) membrane with increasing Chol content to elucidate the role of high Chol in lens membrane elasticity. In this study, we used atomic force microscopy (AFM) to study the mechanical properties (breakthrough force and area compressibility modulus (KA)) of the MPLL membrane with increasing Chol content where KA is the measure of membrane elasticity. We varied Chol concentration in Chol/MPLL membrane from 0 to ∼71 mol%. Supported Chol/MPLL membranes were prepared by fusion of small unilamellar vesicles (SUVs) on top of a flat mica surface. SUVs of the Chol/MPLL lipid mixture were prepared with the rapid solvent exchange method followed by probe-tip sonication. For the Chol/MPLL mixing ratio of 0, AFM image showed the formation of two distinct phases of the membrane, i.e., liquid-disordered phase (ld) and solid-ordered phase (so) membrane. However, with Chol/MPLL mixing ratio of 0.5 and above, only liquid-ordered phase (lo) membrane was formed. Also, two distinct breakthrough forces corresponding to ld and so were observed for Chol/MPLL mixing ratio of 0, whereas only one breakthrough force was observed for membranes with Chol/MPLL mixing ratio of 0.5 and above. No significant difference in the membrane surface roughness was measured with increasing Chol content for these membranes; however, breakthrough force and KA for lo membrane increased when Chol/MPLL mixing ratio was increased from 0.5 to 1. Interestingly above the Chol/MPLL mixing ratio of 1, both breakthrough force and KA decreased, indicating the formation of CBDs. Furthermore, these results showed that membrane elasticity increases at high Chol content, suggesting that high Chol content in lens membrane might be responsible for maintaining lens membrane elasticity.

Optimization of Giant Unilamellar Vesicle Electroformation for Phosphatidylcholine/Sphingomyelin/Cholesterol Ternary Mixtures.

Artificial vesicles are important tools in membrane research because they enable studying membrane properties in controlled conditions. Giant unilamellar vesicles (GUVs) are specially interesting due to their similarity in size to eukaryotic cells. We focus on optimization of GUV production from phosphatidylcholine/sphingomyelin/cholesterol mixtures using the electroformation method. This mixture has been extensively researched lately due to its relevance for the formation of lipid rafts. We measured the effect of voltage, frequency, lipid film thickness, and cholesterol (Chol) concentration on electroformation successfulness using spin-coating for reproducible lipid film deposition. Special attention is given to the effect of Chol concentrations above the phospholipid bilayer saturation threshold. Such high concentrations are of interest to groups studying the role of Chol in the fiber cell plasma membranes of the eye lens or development of atherosclerosis. Utilizing atomic force and fluorescence microscopy, we found the optimal lipid film thickness to be around 30 nm, and the best frequency–voltage combinations in the range of 2–6 V and 10–100 Hz. Increasing the Chol content, we observed a decrease in GUV yield and size. However, the effect was much less pronounced when the optimal lipid film thickness was used. The results underline the need for simultaneous optimization of both electrical parameters and thickness in order to produce high-quality GUVs for experimental research.

An AFM Approach Applied in a Study of α-Crystallin Membrane Association: New Insights into Lens Hardening and Presbyopia Development.

The lens of the eye loses elasticity with age, while α-crystallin association with the lens membrane increases with age. It is unclear whether there is any correlation between α-crystallin association with the lens membrane and loss in lens elasticity. This research investigated α-crystallin membrane association using atomic force microscopy (AFM) for the first time to study topographical images and mechanical properties (breakthrough force and membrane area compressibility modulus (KA), as measures of elasticity) of the membrane. α-Crystallin extracted from the bovine lens cortex was incubated with a supported lipid membrane (SLM) prepared on a flat mica surface. The AFM images showed the time-dependent interaction of α-crystallin with the SLM. Force spectroscopy revealed the presence of breakthrough events in the force curves obtained in the membrane regions where no α-crystallin was associated, which suggests that the membrane’s elasticity was maintained. The force curves in the α-crystallin submerged region and the close vicinity of the α-crystallin associated region in the membrane showed no breakthrough event within the defined peak force threshold, indicating loss of membrane elasticity. Our results showed that the association of α-crystallin with the membrane deteriorates membrane elasticity, providing new insights into understanding the molecular basis of lens hardening and presbyopia.

Alpha-Crystallin Association with the Model of Human and Animal Eye Lens-Lipid Membranes is Modulated by Surface Hydrophobicity of Membranes

Purpose This research aims to probe the interaction of α-crystallin with a model of human, porcine, and mouse lens-lipid membranes. Methods Cholesterol/model of human lens-lipid (Chol/MHLL), cholesterol/model of porcine lens-lipid (Chol/MPLL), and cholesterol/model of mouse lens-lipid (Chol/MMLL) membranes with 0–60 mol% Chol were prepared using the rapid solvent exchange method and probe-tip sonication. The hydrophobicity near the surface of model lens-lipid membranes and α-crystallin association with these membranes were investigated using the electron paramagnetic resonance spin-labeling approach. Results With increased Chol content, the hydrophobicity near the surface of Chol/MHLL, Chol/MPLL, and Chol/MMLL membranes, the maximum percentage of membrane surface occupied (MMSO) by α-crystallin, and the association constant (K a) decreased, showing that surface hydrophobicity of model lens-lipid membranes modulated the α-crystallin association with these membranes. The different MMSO and K a for different model lens-lipid membranes with different rates of decrease of MMSO and K a with increased Chol content and decreased hydrophobicity near the surface of these membranes suggested that the lipid composition also modulates α-crystallin association with membranes. Despite different lipid compositions, complete inhibition of α-crystallin association with model lens-lipid membranes was observed at saturating Chol content forming cholesterol bilayer domains (CBDs) with the lowest hydrophobicity near the surface of these membranes. The decreased mobility parameter with increased α-crystallin concentration suggested that membranes near the surface became less mobile due to α-crystallin association. The decreased mobility parameter and increased maximum splitting with increased Chol content suggested that membranes became less mobile and more ordered near the surface with increased Chol content. Conclusions This study suggested that the interaction of α-crystallin with model lens-lipid membranes is hydrophobic. Furthermore, our data indicated that Chol and CBDs reduce α-crystallin association with lens membrane, likely increase α-crystallin concentration in lens cytoplasm, and possibly favor the chaperone-like activity of α-crystallin maintaining lens cytoplasm homeostasis.

Alpha-crystallin-membrane interactions using atomic force microscopy

Several studies suggest that the lens loses its elasticity with age. It has also been reported that α-crystallin binding to the lens membrane increases with age. It is unclear whether there is any correlation between α-crystallin binding to lens membrane and loss in lens elasticity with age. The purpose of this research is to investigate the membrane elasticity with α-crystallin binding. We have used atomic force microscopy (AFM) to study the mechanical properties (breakthrough force and membrane's area compressibility modulus (KA)) of the membrane with α-crystallin binding, where KA is the measure of membrane elasticity.

α-Crystallin interacts with the model of human eye lens-lipid membrane via hydrophobic interaction

α-Crystallin, a significant protein of the mammalian eye lens, binds to the lens membrane progressively with age and cataract formation with the corresponding decline in the level of α-crystallin in the lens cytoplasm. Despite various studies of α-crystallin binding to the lens membranes and phospholipid vesicles, the nature of such binding is still unclear. This study investigated a cholesterol/model of human lens-lipid (Chol/MHLL) membrane with Chol/MHLL mixing ratios varying from 0 to 60 mol% Chol.

Visualising UV-A light-induced damage to plasma membranes of eye lens.

An eye lens is constantly exposed to the solar UV radiation, which is considered the most important external source of age-related changes to eye lens constituents. The accumulation of modifications of proteins and lipids with age can eventually lead to the development of progressive lens opacifications, such as cataracts. Though the impact of solar UV radiation on the structure and function of proteins is actively studied, little is known about the effect of photodamage on plasma membranes of lens cells. In this work we exploit Fluorescence Lifetime Imaging Microscopy (FLIM), together with viscosity-sensitive fluorophores termed molecular rotors, to study the changes in viscosity of plasma membranes of porcine eye lens resulting from two different types of photodamage: Type I (electron transfer) and Type II (singlet oxygen) reactions. We demonstrate that these two types of photodamage result in clearly distinct changes in viscosity - a decrease in the case of Type I damage and an increase in the case of Type II processes. Finally, to simulate age-related changes that occur in vivo, we expose an intact eye lens to UV-A light under anaerobic conditions. The observed decrease in viscosity within plasma membranes is consistent with the ability of eye lens constituents to sensitize Type I photodamage under natural irradiation conditions. These changes are likely to alter the transport of metabolites and predispose the whole tissue to the development of pathological processes such as cataracts.