Cholesterol Content Regulates the Interaction of αA-, αB-, and α-Crystallin with the Model of Human Lens-Lipid Membranes.

α-Crystallin (αABc) is a major protein comprised of αA-crystallin (αAc) and αB-crystallin (αBc) that is found in the human eye lens and works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress. However, with age and cataract formation, the concentration of αABc in the eye lens cytoplasm decreases, with a corresponding increase in the membrane-bound αABc. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the role of cholesterol (Chol) and Chol bilayer domains (CBDs) in the binding of αAc, αBc, and αABc to the Chol/model of human lens-lipid (Chol/MHLL) membranes. The maximum percentage of membrane surface occupied (MMSO) by αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trends: MMSO (αAc) > MMSO (αBc) ≈ MMSO (αABc), indicating that a higher amount of αAc binds to these membranes compared to αBc and αABc. However, with an increase in the Chol concentration in the Chol/MHLL membranes, the MMSO by αAc, αBc, and αABc decreases until it is completely diminished at a mixing ratio of 1.5. The Ka of αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trend: Ka (αBc) ≈ Ka (αABc) > Ka (αAc), but it was close to zero with the diminished binding at a Chol/MHLL mixing ratio of 1.5. The mobility near the membrane headgroup regions decreased with αAc, αBc, and αABc binding, and the Chol antagonized the capacity of the αAc, αBc, and αABc to decrease mobility near the headgroup regions. No significant change in membrane order near the headgroup regions was observed, with an increase in αAc, αBc, and αABc concentrations. Our results show that αAc, αBc, and αABc bind differently with Chol/MHLL membranes at mixing ratios of 0 and 0.5, decreasing the mobility and increasing hydrophobicity near the membrane headgroup region, likely forming the hydrophobic barrier for the passage of polar and ionic molecules, including antioxidants (glutathione), creating an oxidative environment inside the lens, leading to the development of cataracts. However, all binding was completely diminished at a mixing ratio of 1.5, indicating that high Chol and CBDs inhibit the binding of αAc, αBc, and αABc to membranes, preventing the formation of hydrophobic barriers and likely protecting against cataract formation.

Association of Alpha-Crystallin with Human Cortical and Nuclear Lens Lipid Membrane Increases with the Grade of Cortical and Nuclear Cataract.

Eye lens α-crystallin has been shown to become increasingly membrane-bound with age and cataract formation; however, to our knowledge, no studies have investigated the membrane interactions of α-crystallin throughout the development of cataracts in separated cortical membrane (CM) and nuclear membrane (NM) from single human lenses. In this study, four pairs of human lenses from age-matched male and female donors and one pair of male lenses ranging in age from 64 to 73 years old (yo) were obtained to investigate the interactions of α-crystallin with the NM and CM throughout the progression of cortical cataract (CC) and nuclear cataract (NC) using the electron paramagnetic resonance spin-labeling method. Donor health history information (diabetes, smoker, hypertension, radiation treatment), sex, and race were included in the data analysis. The right eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 1, NC: 2), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Similarly, left eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 2, NC: 3), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Analysis of α-crystallin binding to male and female eye lens CM and NM revealed that the percentage of membrane surface occupied (MSO) by α-crystallin increases with increasing grade of CC and NC. The binding of α-crystallin resulted in decreased mobility, increased order, and increased hydrophobicity on the membrane surface in male and female eye lens CM and NM. CM mobility decreased with an increase in cataracts for both males and females, whereas the male lens NM mobility showed no significant change, while female lens NM showed increased mobility with an increase in cataract grade. Our data shows that a 68 yo female donor (long-term smoker, pre-diabetic, and hypertension; grade 3 CC) showed the largest MSO by α-crystallin in CM from both the left and right lens and had the most pronounced mobility changes relative to all other analyzed samples. The variation in cholesterol (Chol) content, size and amount of cholesterol bilayer domains (CBDs), and lipid composition in the CM and NM with age and cataract might result in a variation of membrane surface mobility, membrane surface hydrophobicity, and the interactions of α-crystallin at the surface of each CM and NM. These findings provide insight into the effect of decreased Chol content and the reduced size and amount of CBDs in the cataractous CM and NM with an increased binding of α-crystallin with increased CC and NC grade, which suggests that Chol and CBDs might be a key component in maintaining lens transparency.

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

Highly concentrated lens proteins, mostly ß- and γ-crystallin, are responsible for maintaining the structure and refractivity of the eye lens. However, with aging and cataract formation, ß- 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 ß- and γ-crystallin are associated with the lens membrane is unknown. This work aims to study the interaction of ß- 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 ß- 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 ßL- and γ-crystallin from the cortex of the bovine lens was used to investigate the time-dependent association of ßL- and γ-crystallin with the membrane by obtaining the topographical images using atomic force microscopy. Our study showed that ßL-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 ßL-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 ßL- 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 ßL- 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 ßL-crystallin interacted with the membrane. The semi-transmembrane or transmembrane defects formed by the interaction of ßL- 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.

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

Several discoveries show that with age and cataract formation, ß-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 ß-crystallin binding to the membrane is unclear. This research aims to elucidate the role of lipids and Chol in the binding of ß-crystallin to the membrane and the membrane's physical properties (mobility, order, and hydrophobicity) with ß-crystallin binding. We used electron paramagnetic resonance (EPR) spin-labeling methods to investigate the binding of ßL-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 ßL-crystallin binds with all of the investigated membranes in a saturation manner, and the maximum parentage of the membrane surface occupied (MMSO) by ßL-crystallin and the binding affinity (Ka) of ßL-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 ßL-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 ßL-crystallin binding compared to the MHLL membrane. The hydrophobicity near the headgroup region of the membrane increases with ßL-crystallin binding; however, the increase is more pronounced in the MPLL and MMLL membranes than the MHLL membrane, indicating that ßL-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 ßL-crystallin binding, suggesting that Chol prevents the formation of a hydrophobic barrier, possibly protecting against cataract formation.

Quantification of Age-Related Changes in the Lateral Organization of the Lipid Portion of the Intact Membranes Isolated from the Left and Right Eye Lenses of the Same Human Donor.

The continuous wave EPR spin-labeling method was used to evaluate age-related changes in the amounts of phospholipids (PLs) and cholesterol (Chol) in domains present in intact, cortical, and nuclear fiber cell plasma membranes isolated separately from the left and right eye lenses of the same human donor. The relative amounts of boundary plus trapped PLs were evaluated with the PL analog 12-doxylstearic acid spin label (12-SASL) and the relative amounts of trapped Chol with the Chol analog androstane spin label (ASL). The donors ranged in age from 15 to 70 years. Both the left and right eye lenses from donors aged 60, 65, and 70 years had nuclear cataracts; additionally, the right eye lens only of the 60-year-old donor had a cortical cataract. In transparent lenses, the relative amounts of boundary plus trapped PLs increase monotonously with donor age, and, at all ages, this amount was greater in nuclear compared with cortical membranes. Moreover, in transparent lenses, the relative amount of trapped Chol increases with age in nuclear membranes. However, the EPR spectrum of ASL from cortical membranes of 15- to 60-year-old donors shows only the weakly immobilized component assigned to ASL in the bulk plus Chol bilayer domain. Only the cortical membranes of 61- to 70-year-old donors contain both weakly and strongly immobilized components. The strongly immobilized component is assigned to ASL in trapped lipids. We speculate that the age of 60 years may be considered as a "threshold" for appearance of trapped lipids in cortical membranes. The relative amounts of boundary plus trapped PLs in lenses with nuclear cataracts is lower than that predicted from the tendency of the age-dependent increase observed for transparent lenses. The differences in amounts of lipids in the indicated left and right eye domains of each donor are smaller than the differences in single donors of a similar age.

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid.

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young's modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition. Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

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.

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.

Alpha-Crystallin-Membrane Association Modulated by Phospholipid Acyl Chain Length and Degree of Unsaturation.

α-crystallin-membrane association increases with age and cataracts, with the primary association site of α-crystallin being phospholipids. However, it is unclear if phospholipids' acyl chain length and degree of unsaturation influence α-crystallin association. We used the electron paramagnetic resonance approach to investigate the association of α-crystallin with phosphatidylcholine (PC) membranes of different acyl chain lengths and degrees of unsaturation and with and without cholesterol (Chol). The association constant (Ka) of α-crystallin follows the trends, i.e., Ka (14:0−14:0 PC) > Ka (18:0−18:1 PC) > Ka (18:1−18:1 PC) ≈ Ka (16:0−20:4 PC) where the presence of Chol decreases Ka for all membranes. With an increase in α-crystallin concentration, the saturated and monounsaturated membranes rapidly become more immobilized near the headgroup regions than the polyunsaturated membranes. Our results directly correlate the mobility and order near the headgroup regions of the membrane with the Ka, with the less mobile and more ordered membrane having substantially higher Ka. Furthermore, our results show that the hydrophobicity near the headgroup regions of the membrane increases with the α-crystallin association, indicating that the α-crystallin-membrane association forms the hydrophobic barrier to the transport of polar and ionic molecules, supporting the barrier hypothesis in cataract development.

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 (Ka) decreased, showing that surface hydrophobicity of model lens-lipid membranes modulated the α-crystallin association with these membranes. The different MMSO and Ka for different model lens-lipid membranes with different rates of decrease of MMSO and Ka 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.

Association of Alpha-Crystallin with Fiber Cell Plasma Membrane of the Eye Lens Accompanied by Light Scattering and Cataract Formation.

α-crystallin is a major protein found in the mammalian eye lens that works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress in the eye lens. These functions of α-crystallin are significant for maintaining lens transparency. However, with age and cataract formation, the concentration of α-crystallin in the eye lens cytoplasm decreases with a corresponding increase in the membrane-bound α-crystallin, accompanied by increased light scattering. The purpose of this review is to summarize previous and recent findings of the role of the: (1) lens membrane components, i.e., the major phospholipids (PLs) and sphingolipids, cholesterol (Chol), cholesterol bilayer domains (CBDs), and the integral membrane proteins aquaporin-0 (AQP0; formally MIP26) and connexins, and (2) α-crystallin mutations and post-translational modifications (PTMs) in the association of α-crystallin to the eye lens's fiber cell plasma membrane, providing thorough insights into a molecular basis of such an association. Furthermore, this review highlights the current knowledge and need for further studies to understand the fundamental molecular processes involved in the association of α-crystallin to the lens membrane, potentially leading to new avenues for preventing cataract formation and progression.

Mechanical properties of the high cholesterol-containing membrane: An AFM study.

Cholesterol (Chol) content in most cellular membranes does not exceed 50 mol%, only in the eye lens's fiber cell plasma membrane, its content surpasses 50 mol%. At this high concentration, Chol induces the formation of pure cholesterol bilayer domains (CBDs), which coexist with the surrounding phospholipid-cholesterol domain (PCD). Here, we applied atomic force microscopy to study the mechanical properties of Chol/phosphatidylcholine membranes where the Chol content was increased from 0 to 75 mol%, relevant to eye lens membranes. The surface roughness of the membrane decreases with an increase of Chol content until it reaches 60 mol%, and roughness increases with a further increment in Chol content. We propose that the increased roughness at higher Chol content results from the formation of CBDs. Force spectroscopy on the membrane with Chol content of 50 mol% or lesser exhibited single breakthrough events, whereas two distinct puncture events were observed for membranes with the Chol content greater than 50 mol%. We propose that the first puncture force corresponds to the membranes containing coexisting PCD and CBDs. In contrast, the second puncture force corresponds to the "CBD water pocket" formed due to coexisting CBDs and PCD. Membrane area compressibility modulus (KA) increases with an increase in Chol content until it reaches 60 mol%, and with further increment in Chol content, CBDs are formed, and KA starts to decrease. Our results report the increase in membrane roughness and decrease KA at very high Chol content (>60 mol%) relevant to the eye lens membrane.

Cholesterol and cholesterol bilayer domains inhibit binding of alpha-crystallin to the membranes made of the major phospholipids of eye lens fiber cell plasma membranes.

The concentration of α-crystallin decreases in the eye lens cytoplasm, with a corresponding increase in membrane-bound α-crystallin during cataract formation. The eye lens's fiber cell plasma membrane consists of extremely high cholesterol (Chol) content, forming cholesterol bilayer domains (CBDs) within the membrane. The role of high Chol content in the lens membrane is unclear. Here, we applied the continuous-wave electron paramagnetic resonance spin-labeling method to probe the role of Chol and CBDs on α-crystallin binding to membranes made of four major phospholipids (PLs) of the eye lens, i.e., phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Small unilamellar vesicles (SUVs) of PC, SM*, and PS with 0, 23, 33, 50, and 60 mol% Chol and PE* with 0, 9, and 33 mol% Chol were prepared using the rapid solvent exchange method followed by probe-tip sonication. The 1 mol% CSL spin-labels used during SUVs preparation distribute uniformly within the Chol/PL membrane, enabling the investigation of Chol and CBDs' role on α-crystallin binding to the membrane. For PC, SM*, and PS membranes, the binding affinity (Ka) and the maximum percentage of membrane surface occupied (MMSO) by α-crystallin decreased with an increase in Chol concentration. The Ka and MMSO became zero at 50 mol% Chol for PC and 60 mol% Chol for SM* membranes, representing that complete inhibition of α-crystallin binding was possible before the formation of CBDs within the PC membrane but only after the formation of CBDs within the SM* membrane. The Ka and MMSO did not reach zero even at 60 mol% Chol in the PS membrane, representing CBDs at this Chol concentration were not sufficient for complete inhibition of α-crystallin binding to the PS membrane. Both the Ka and MMSO were zero at 0, 9, and 33 mol% Chol in the PE* membrane, representing no binding of α-crystallin to the PE* membrane with and without Chol. The mobility parameter profiles decreased with an increase in α-crystallin binding to the membranes; however, the decrease was more pronounced for the membrane with lower Chol concentration. These results imply that the membranes become more immobilized near the headgroup regions with an increase in α-crystallin binding; however, the Chol antagonizes the capacity of α-crystallin to decrease the mobility near the headgroup regions of the membranes. The maximum splitting profiles remained the same with an increase in α-crystallin concentration, but there was an increase in the maximum splitting with an increase in the Chol concentration in the membranes. It implies that membrane order near the headgroup regions does not change with an increase in α-crystallin concentration but increases with an increase in Chol concentration in the membrane. Based on our data, we hypothesize that the Chol and CBDs decrease hydrophobicity (increase polarity) near the membrane surface, inhibiting the hydrophobic binding of α-crystallin to the membranes. Thus, our data suggest that Chol and CBDs play a positive physiological role by preventing α-crystallin binding to lens membranes and possibly protecting against cataract formation and progression.

Interaction of Alpha-Crystallin with Phospholipid Membranes.

Purpose/Aim: The amount of membrane-bound α-crystallin increases significantly with age and cataract formation, accompanied by a corresponding decline in the level of α-crystallin in the lens cytoplasm. The purpose of this research is to evaluate the binding affinity of α-crystallin to the phospholipid membranes as well as the physical properties of the membranes after α-crystallin binding. Materials and Methods: The continuous wave and saturation recovery electron paramagnetic resonance (EPR) methods were used to obtain the information about the binding affinity and the physical properties of the membrane. In this approach, the cholesterol analog spin label CSL was incorporated in the membrane and the binding of α-crystallin to the membrane was monitored by this spin label. Small uni-lamellar vesicles were prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with 1% of CSL. The measured membrane properties included the mobility parameter, fluidity, and the oxygen transport parameter. Results: The binding affinity (Ka ) of α-crystallin with the POPC membrane was estimated to be 4.9 ± 2.4 µM-1. The profiles of mobility parameter showed that mobility parameter decreased with an increase in the binding of α-crystallin. The profiles of spin-lattice relaxation rate showed that the spin-lattice relaxation rate decreased with an increase in binding. These results show that the binding of α-crystallin makes the membrane more immobilized near the head group region of the phospholipids. Furthermore, the profiles of the oxygen transport parameter indicated that the oxygen transport parameter decreased with an increase of binding, indicating the binding of α-crystallin forms a barrier for the passage of non-polar molecules which supports the barrier hypothesis. Conclusions: The binding of α-crystallin to the membrane alters the physical properties of the membranes, and this plays a significant role in modulating the integrity of the membranes. EPR techniques are useful in studying α-crystallin membrane interactions.

Interaction of alpha-crystallin with four major phospholipids of eye lens membranes.

It is well-studied that the significant factor in cataract formation is the association of α-crystallin, a major eye lens protein, with the fiber cell plasma membrane of the eye lens. The fiber cell plasma membrane of the eye lens consists of four major phospholipids (PLs), i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). Despite several attempts to study the interaction of α-crystallin with PLs of the eye lens membrane, the role of individual PL for the binding with α-crystallin is still unclear. We recently developed the electron paramagnetic resonance (EPR) spin-labeling method to study the binding of α-crystallin to the PC membrane (Mainali et al., 2020a). Here, we use the recently developed EPR method to explicitly measure the binding affinity (Ka) of α-crystallin to the individual (PE*, PS, and SM) and two-component mixtures (SM/PE, SM/PS, and SM/PC in 70:30 and 50:50 mol%) of PL membranes as well as the physical properties (mobility parameter and maximum splitting) of these membranes upon binding with α-crystallin. One of the key findings of this study was that the Ka of α-crystallin binding to individual PL membranes followed the trends: Ka(PC) > Ka(SM) > Ka(PS) > Ka(PE*), indicating PE* inhibits binding the most whereas PC inhibits binding the least. Also, the Ka of α-crystallin binding to two-component mixtures of PL membranes followed the trends: Ka(SM/PE) > Ka(SM/PS) > Ka(SM/PC), indicating SM/PC inhibits binding the most whereas SM/PE inhibits binding the least. Except for the PE* membrane, for which there was no binding of α-crystallin, the mobility parameter for all other membranes decreased with an increase in α-crystallin concentration. It represents that the membranes become more immobilized near the headgroup regions of the PLs when more and more α-crystallin binds to them. The maximum splitting increased only for the SM and the SM/PE (70:30 mol%) membranes, with an increase in the binding of α-crystallin. It represents that the PL headgroup regions of these membranes become more ordered after binding of α-crystallin to these membranes. Our results showed that α-crystallin binds to PL membranes in a saturable manner. Also, our data suggest that the binding of α-crystallin to PL membranes likely occurs through hydrophobic interaction between α-crystallin and the hydrophobic fatty acid core of the membranes, and such interaction is modulated by the PL headgroup's size and charge, hydrogen bonding between headgroups, and PL curvature. Thus, this study provides an in-depth understanding of α-crystallin interaction with the PL membranes made of individual and two-component mixtures of the four major PLs of the eye lens membranes.

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.

Purpose/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.

Confocal Microscopy Confirmed that in Phosphatidylcholine Giant Unilamellar Vesicles with very High Cholesterol Content Pure Cholesterol Bilayer Domains Form.

The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. This high Chol content forms pure Chol bilayer domains (CBDs) and Chol crystals in model membranes and membranes formed from the total lipid extracts from human lenses. CBDs have been detected using electron paramagnetic resonance (EPR) spin-labeling approaches. Here, we confirm the presence of CBDs in giant unilamellar vesicles prepared using the electroformation method from Chol/1-palmitoyl-2-oleoylphosphocholine and Chol/distearoylphosphatidylcholine mixtures. Confocal microscopy experiments using phospholipid (PL) analog (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'-disulfonic acid) and cholesterol analog fluorescent probes (23-(dipyrrometheneboron difluoride)-24-norcholesterol) were performed, allowing us to make three major conclusions: (1) In all membranes with a Chol/PL mixing ratio (expressed as a molar ratio) >2, pure CBDs were formed within the bulk PL bilayer saturated with Chol. (2) CBDs were present as the pure Chol bilayer and not as separate patches of Chol monolayers in each leaflet of the PL bilayer. (3) CBDs, presented as single large domains, were always located at the top of giant unilamellar vesicles, independent of the change in sample orientation (right-side-up/upside-down). Results obtained with confocal microscopy and fluorescent Chol and PL analogs, combined with those obtained using EPR and spin-labeled Chol and PL analogs, contribute to the understanding of the organization of lipids in the fiber cell plasma membranes of the human eye lens.

Characterization of the distribution of spin-lattice relaxation rates of lipid spin labels in fiber cell plasma membranes of eye lenses with a stretched-exponential function.

The stretched exponential function (SEF) was used to analyze and interpret saturation recovery (SR) electron paramagnetic resonance (EPR) data obtained from spin-labeled porcine eye-lens membranes. This function has two fitting parameters: the characteristic spin-lattice relaxation rate (T 1str -1) and the stretching parameter (ß), which ranges between zero and one. When ß = 1, the function is a single exponential. It is assumed that the SEF arises from a distribution of single exponential functions, each described by a T 1 value. Because T 1 -1s are determined primarily by the rotational diffusion of spin labels, they are a measure of membrane fluidity. Since ß describes the distribution of T 1 -1s, it can be interpreted as a measure of membrane heterogeneity. The SEF was used to analyze SR data obtained from intact cortical and nuclear fiber cell plasma membranes extracted from the eye lenses of two-year old animals and spinlabeled with phospholipid- and cholesterol-analogs. The lipid environment sensed by these probe molecules was found to be less fluid and more heterogeneous in nuclear membranes than in cortical membranes. Parameters T 1str -1 and ß were also used for a multivariate K-means cluster analysis of stretched-exponential data. This analysis indicates that SEF data can be assigned accurately to clusters in nuclear or cortical membranes. In future work, the SEF will be applied to analyze data from human eye lenses of donors with differing health histories.

Detection of cholesterol bilayer domains in intact biological membranes: Methodology development and its application to studies of eye lens fiber cell plasma membranes.

Four purported lipid domains are expected in plasma membranes of the eye lens fiber cells. Three of these domains, namely, bulk, boundary, and trapped lipids, have been detected. The cholesterol bilayer domain (CBD), which has been detected in lens lipid membranes prepared from the total lipids extracted from fiber cell plasma membranes, has not yet been detected in intact fiber cell plasma membranes. Here, a saturation-recovery electron paramagnetic resonance spin-labeling method has been developed that allows identification of CBDs in intact fiber cell plasma membranes of eye lenses. This method is based on saturation-recovery signal measurements of the cholesterol-analog spin label located in the lipid bilayer portion of intact fiber cell membranes as a function of the partial pressure of molecular oxygen with which the samples are equilibrated. The capabilities and limitations of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses where CBDs were detected in porcine nuclear intact membranes for which CBDs were also detected in lens lipid membranes. CBDs were not detected in porcine cortical intact and lens lipid membranes. CBDs were detected in intact membranes isolated from both cortical and nuclear fiber cells of lenses obtained from human donors. The cholesterol content in fiber cell membranes of these donors was always high enough to induce the formation of CBDs in cortical as well as nuclear lens lipid membranes. The results obtained for intact membranes, when combined with those obtained for lens lipid membranes, advance our understanding of the role of high cholesterol content and CBDs in lens biology, aging, and/or cataract formation.