Fiber Cell Membranes Research Articles

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.

Chemical crosslinking analysis of β-dystroglycan in dystrophin-deficient skeletal muscle.

Background: In Duchenne muscular dystrophy, primary abnormalities in the membrane cytoskeletal protein dystrophin trigger the loss of sarcolemmal linkage between the extracellular matrix component laminin-211 and the intracellular cortical actin membrane cytoskeleton. The disintegration of the dystrophin-associated glycoprotein complex renders the plasma membrane of contractile fibres more susceptible to micro-rupturing, which is associated with abnormal calcium handling and impaired cellular signalling in dystrophinopathy. Methods: The oligomerisation pattern of β-dystroglycan, an integral membrane protein belonging to the core dystrophin complex, was studied using immunoprecipitation and chemical crosslinking analysis. A homo-bifunctional and non-cleavable agent with water-soluble and amine-reactive properties was employed to study protein oligomerisation in normal versus dystrophin-deficient skeletal muscles. Crosslinker-induced protein oligomerisation was determined by a combination of gel-shift analysis and immunoblotting. Results: Although proteomics was successfully applied for the identification of dystroglycan as a key component of the dystrophin-associated glycoprotein complex in the muscle membrane fraction, mass spectrometric analysis did not efficiently recognize this relatively low-abundance protein after immunoprecipitation or chemical crosslinking. As an alternative approach, comparative immunoblotting was used to evaluate the effects of chemical crosslinking. Antibody decoration of the crosslinked microsomal protein fraction from wild type versus the mdx-4cv mouse model of dystrophinopathy revealed oligomers that contain β-dystroglycan. The protein exhibited a comparable reduction in gel electrophoretic mobility in both normal and dystrophic samples. The membrane repair proteins dysferlin and myoferlin, which are essential components of fibre regeneration, as well as the caveolae-associated protein cavin-1, were also shown to exist in high-molecular mass complexes. Conclusions: The muscular dystrophy-related reduction in the concentration of β-dystroglycan, which forms in conjunction with its extracellular binding partner α-dystroglycan a critical plasmalemmal receptor for laminin-211, does not appear to alter its oligomeric status. Thus, independent of direct interactions with dystrophin, this sarcolemmal glycoprotein appears to exist in a supramolecular assembly in muscle.

Chemical crosslinking analysis of β-dystroglycan in dystrophin-deficient skeletal muscle

Background: In Duchenne muscular dystrophy, primary abnormalities in the membrane cytoskeletal protein dystrophin trigger the loss of sarcolemmal linkage between the extracellular matrix component laminin-211 and the intracellular cortical actin membrane cytoskeleton. The disintegration of the dystrophin-associated glycoprotein complex renders the plasma membrane of contractile fibres more susceptible to micro-rupturing, which is associated with abnormal calcium handling and impaired cellular signalling in dystrophinopathy. Methods: The oligomerisation pattern of β-dystroglycan, an integral membrane protein belonging to the core dystrophin complex, was studied using immunoprecipitation and chemical crosslinking analysis. A homo-bifunctional and non-cleavable agent with water-soluble and amine-reactive properties was employed to study protein oligomerisation in normal versus dystrophin-deficient skeletal muscles. Crosslinker-induced protein oligomerisation was determined by a combination of gel-shift analysis and immunoblotting. Results: Although proteomics was successfully applied for the identification of dystroglycan as a key component of the dystrophin-associated glycoprotein complex in the muscle membrane fraction, mass spectrometric analysis did not efficiently recognize this relatively low-abundance protein after immunoprecipitation or chemical crosslinking. As an alternative approach, comparative immunoblotting was used to evaluate the effects of chemical crosslinking. Antibody decoration of the crosslinked microsomal protein fraction from wild type versus the mdx-4cv mouse model of dystrophinopathy revealed oligomers that contain β-dystroglycan. The protein exhibited a comparable reduction in gel electrophoretic mobility in both normal and dystrophic samples. The membrane repair proteins dysferlin and myoferlin, which are essential components of fibre regeneration, as well as the caveolae-associated protein cavin-1, were also shown to exist in high-molecular mass complexes. Conclusions: The muscular dystrophy-related reduction in the concentration of β-dystroglycan, which forms in conjunction with its extracellular binding partner α-dystroglycan a critical plasmalemmal receptor for laminin-211, does not appear to alter its oligomeric status. Thus, independent of direct interactions with dystrophin, this sarcolemmal glycoprotein appears to exist in a supramolecular assembly in muscle.

Chemical crosslinking analysis of β-dystroglycan in dystrophin-deficient skeletal muscle

Background: In Duchenne muscular dystrophy, primary abnormalities in the membrane cytoskeletal protein dystrophin trigger the loss of sarcolemmal linkage between the extracellular matrix component laminin-211 and the intracellular cortical actin membrane cytoskeleton. The disintegration of the dystrophin-associated glycoprotein complex renders the plasma membrane of contractile fibres more susceptible to micro-rupturing, which is associated with abnormal calcium handling and impaired cellular signalling in dystrophinopathy. Methods: The oligomerisation pattern of β-dystroglycan, an integral membrane protein belonging to the core dystrophin complex, was studied using immunoprecipitation and chemical crosslinking analysis. A homo-bifunctional and non-cleavable agent with water-soluble and amine-reactive properties was employed to study protein oligomerisation in normal versus dystrophin-deficient skeletal muscles. Crosslinker-induced protein oligomerisation was determined by a combination of gel-shift analysis and immunoblotting. Results: Although proteomics was successfully applied for the identification of dystroglycan as a key component of the dystrophin-associated glycoprotein complex in the muscle membrane fraction, mass spectrometric analysis did not efficiently recognize this relatively low-abundance protein after immunoprecipitation or chemical crosslinking. As an alternative approach, comparative immunoblotting was used to evaluate the effects of chemical crosslinking. Antibody decoration of the crosslinked microsomal protein fraction from wild type versus the mdx-4cv mouse model of dystrophinopathy revealed oligomers that contain β-dystroglycan. The protein exhibited a comparable reduction in gel electrophoretic mobility in both normal and dystrophic samples. The membrane repair proteins dysferlin and myoferlin, which are essential components of fibre regeneration and counteract the dystrophic phenotype, were also shown to exist in high-molecular mass complexes. Conclusions: The muscular dystrophy-related reduction in the concentration of β-dystroglycan, which forms in conjunction with its extracellular binding partner α-dystroglycan a critical plasmalemmal receptor for laminin-211, does not appear to alter its oligomeric status. Thus, independent of direct interactions with dystrophin, this sarcolemmal glycoprotein appears to exist in a supramolecular assembly in muscle.

Characterization of an N-terminal mutant of αA-crystallin αA–R21Q associated with congenital cataract

Several mutations associated with congenital cataracts in human beings target conserved arginine residues in αA-crystallin. The N-terminal region of αA-crystallin is a “mutational hotspot,” with multiple cataract-related mutations reported in this region. Two mutations at arginine 21 in the N-terminal domain of αA-crystallin — αA–R21L and αA–R21W have been associated with congenital cataract. A third mutant of R21, αA–R21Q, was recently identified to be associated with congenital cataract in a South Australian family. The point mutation was reported to compromise the quaternary structure of αA-crystallin by preventing its assembly into higher ordered oligomers. To assess the effect of the αA–R21Q mutation on αA-crystallin function, recombinant αA–R21Q was expressed, purified and characterized in vitro. Compared to wild-type αA-crystallin, the recombinant αA–R21Q exhibits enhanced chaperone-like activity, increased surface hydrophobicity, lesser stability in urea and increased susceptibility to digestion by trypsin. αA–R21Q demonstrated increased binding affinity towards unfolding ADH and bovine lens fiber cell membranes. αA–R21Q homo-oligomers and hetero-oligomers also prevented H2O2–induced apoptosis in ARPE–19 cells. Taken together, αA–R21Q exhibited a gain of function despite subtle structural differences as compared to wild-type αA-crystallin. This study further validates the involvement of arginine 21 in regulating αA-crystallin structure and function.

High-Yield Purification, Preservation, and Serial Transplantation of Human Satellite Cells.

SummaryInvestigation of human muscle regeneration requires robust methods to purify and transplant muscle stem and progenitor cells that collectively constitute the human satellite cell (HuSC) pool. Existing approaches have yet to make HuSCs widely accessible for researchers, and as a result human muscle stem cell research has advanced slowly. Here, we describe a robust and predictable HuSC purification process that is effective for each human skeletal muscle tested and the development of storage protocols and transplantation models in dystrophin-deficient and wild-type recipients. Enzymatic digestion, magnetic column depletion, and 6-marker flow-cytometric purification enable separation of 104 highly enriched HuSCs per gram of muscle. Cryostorage of HuSCs preserves viability, phenotype, and transplantation potential. Development of enhanced and species-specific transplantation protocols enabled serial HuSC xenotransplantation and recovery. These protocols and models provide an accessible system for basic and translational investigation and clinical development of HuSCs.

Aquaporin 0 Modulates Lens Gap Junctions in the Presence of Lens-Specific Beaded Filament Proteins.

PurposeThe objective of this study was to understand the molecular and physiologic mechanisms behind the lens cataract differences in Aquaporin 0-knockout-Heterozygous (AQP0-Htz) mice developed in C57 and FVB (lacks beaded filaments [BFs]) strains.MethodsLens transparency was studied using dark field light microscopy. Water permeability (Pf) was measured in fiber cell membrane vesicles. Western blotting/immunostaining was performed to verify expression of BF proteins and connexins. Microelectrode-based intact lens intracellular impedance was measured to determine gap junction (GJ) coupling resistance. Lens intracellular hydrostatic pressure (HP) was determined using a microelectrode/manometer system.ResultsLens opacity and spherical aberration were more distinct in AQP0-Htz lenses from FVB than C57 strains. In either background, compared to wild type (WT), AQP0-Htz lenses showed decreased Pf (approximately 50%), which was restored by transgenic expression of AQP1 (TgAQP1/AQP0-Htz), but the opacities and differences between FVB and C57 persisted. Western blotting revealed no change in connexin expression levels. However, in C57 AQP0-Htz and TgAQP1/AQP0-Htz lenses, GJ coupling resistance decreased approximately 2.8-fold and the HP gradient decreased approximately 1.9-fold. Increased Pf in TgAQP1/AQP0-Htz did not alter GJ coupling resistance or HP.ConclusionsIn C57 AQP0-Htz lenses, GJ coupling resistance decreased. HP reduction was smaller than the coupling resistance reduction, a reflection of an increase in fluid circulation, which is one reason for the less severe cataract in C57 than FVB. Overall, our results suggest that AQP0 modulates GJs in the presence of BF proteins to maintain lens transparency and homeostasis.

Cholesterol Bilayer Domains in the Eye Lens Health: A Review

The most unique biochemical characteristic of the eye lens fiber cell plasma membrane is its extremely high cholesterol content, the need for which is still unclear. It is evident, however, that the disturbance of Chol homeostasis may result in damages associated with cataracts. Electron paramagnetic resonance methods allow discrimination of two types of lipid domains in model membranes overloaded with Chol, namely, phospholipid-cholesterol domains and pure Chol bilayer domains. These domains are also detected in human lens lipid membranes prepared from the total lipids extracted from lens cortices and nuclei of donors from different age groups. Independent of the age-related changes in phospholipid composition, the physical properties of phospholipid-Chol domains remain the same for all age groups and are practically identical for cortical and nuclear membranes. The presence of Chol bilayer domains in these membranes provides a buffering capacity for cholesterol concentration in the surrounding phospholipid-Chol domains, keeping it at a constant saturating level and thus keeping the physical properties of the membrane consistent with and independent of changes in phospholipid composition. It seems that the presence of Chol bilayer domains plays an integral role in the regulation of cholesterol-dependent processes in fiber cell plasm membranes and in the maintenance of fiber cell membrane homeostasis.

Lipid-Protein Interactions in Fiber Cell Plasma Membrane Isolated From Human and Porcine Eye Lenses

The protein content in human lens fiber cell plasma membranes is extremely high, increases with age, and is higher in the nucleus than in cortex. This strongly affects the organization and physical properties of the lipid bilayer portion of intact fiber cell plasma membranes including the alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter. Investigated membranes were isolated from the eye lenses of pigs and human donors. They were studied using electron paramagnetic resonance spin-labeling method that provides an opportunity to discriminate coexisting lipid domains and to evaluate the relative amounts of phospholipids and cholesterol in them. Fiber-cell membranes were found to contain three distinct lipid environments: bulk, boundary and trapped domains that appear due to the presence of membrane proteins. In nuclear membranes the amount of boundary and trapped phospholipids as well as the amount of cholesterol in trapped lipid domains was greater than that in cortical membranes. Cholesterol was to a large degree excluded from trapped lipid domains in cortical membranes. The difference between the amounts of lipids in domains uniquely formed due to the presence of membrane proteins increased with the donors‘ age. The rigidity of nuclear membranes was greater than that of cortical membranes for all age groups. The nuclear fiber cell plasma membranes were less permeable to oxygen than cortical membranes and both of them become less permeable to oxygen with age. In nuclear porcine membranes the amounts of lipids in domains created due to the presence of membrane proteins were greater than those in cortical membranes and the differences were larger than the differences observed for human membranes. Lipids in porcine nuclear fiber cell membranes were more rigid and less permeable to oxygen than in human nuclear membranes.

A novel missense mutation of Mip causes semi-dominant cataracts in the Nat mouse.

Major intrinsic protein of lens fiber (MIP) is one of the proteins essential for maintaining lens transparency while also contributing to dominant cataracts in humans. The Nodai cataract (Nat) mice harbor a spontaneous mutation in Mip and develop early-onset nuclear cataracts. The Nat mutation is a c.631G>A mutation (MipNat), resulting in a glycine-to-arginine substitution (p.Gly211Arg) in the sixth transmembrane domain. The MipNat/Nat homozygotes exhibit congenital cataracts caused by the degeneration of lens fiber cells. MIP normally localizes to the lens fiber cell membranes. However, the MipNat/Nat mice were found to lack an organelle-free zone, and the MIP was mislocalized to the nuclear membrane and perinuclear region. Furthermore, the MipNat/+ mice exhibited milder cataracts than MipNat/Nat mice due to the slight degeneration of the lens fiber cells. Although there were no differences in the localization of MIP to the membranes of lens fiber cells in MipNat/+ mice compared to that in wild-type mice, the protein levels of MIP were significantly reduced in the eyes. These findings suggest that cataractogenesis in MipNat mutants are caused by defects in MIP expression. Overall, the MipNat mice offer a novel model to better understand the phenotypes and mechanisms for the development of cataracts in patients that carry missense mutations in MIP.

Calmodulin Gates Aquaporin 0 Permeability through a Positively Charged Cytoplasmic Loop

Aquaporin 0 (AQP0), the major intrinsic protein of the eye lens, plays a vital role in maintaining lens clarity by facilitating the transport of water across lens fiber cell membranes. AQP0 reduces its osmotic water permeability constant (Pf) in response to increases in the external calcium concentration, an effect that is mediated by an interaction with the calcium-binding messenger protein, calmodulin (CaM), and phosphorylation of the CaM-binding site abolishes calcium sensitivity. Despite recent structural characterization of the AQP0-CaM complex, the mechanism by which CaM modulates AQP0 remains poorly understood. By combining atomistic molecular dynamics simulations and oocyte permeability assays, we conclude that serine phosphorylation of AQP0 does not inhibit CaM binding to the whole AQP0 protein. Instead, AQP0 phosphorylation alters calcium sensitivity by modifying the AQP0-CaM interaction interface, particularly at an arginine-rich loop that connects the fourth and fifth transmembrane helices. This previously unexplored loop, which sits outside of the canonical CaM-binding site on the AQP0 cytosolic face, mechanically couples CaM to the pore-gating residues of the second constriction site. We show that this allosteric loop is vital for CaM regulation of the channels, facilitating cooperativity between adjacent subunits and regulating factors such as serine phosphorylation. Similar allosteric interactions may also mediate CaM modulation of the properties of other CaM-regulated proteins.

Changes in the Properties and Organization of Human Lens Lipid Membranes Occurring with Age

ABSTRACTPurpose: This research was undertaken to document the changes in the organization and properties of human lens lipid membranes that occur with age.Methods: Human lens lipid membranes prepared from the total lipids extracted from clear lens cortices and nuclei of donors from age groups 0–20 and 21–40 years were investigated. An electron paramagnetic resonance technique and nitroxide spin labels (analogues of phospholipids and cholesterol) were used.Results: Two distinct lipid domains, the phospholipid–cholesterol domain (PCD) and the pure cholesterol bilayer domain (CBD), were detected in all investigated membranes. Profiles of the acyl chain order, fluidity, hydrophobicity, and oxygen transport parameter across discriminated coexisting lipid domains were assessed. Independent of the age-related changes in phospholipid composition, the physical properties of the PCD remained the same for all age groups and were practically identical for cortical and nuclear membranes. However, the properties of pure CBDs changed significantly with the age of the donor and were related to the size of the CBD, which increased with the age of the donor and was greater in nuclear than in cortical membranes. A more detailed analysis revealed that the size of the CBD was determined mainly by the cholesterol content in the membrane.Conclusions: This paper presents data from four age groups: 0–20, 21–40, 41–60, and 61–70 years. Data from age groups 41–60 and 61–70 years were published previously. Combining the previously published data with those data obtained in the present work allowed us to show the changes in the organization of cortical and nuclear lens lipid membranes as functions of age and cholesterol. It seems that the balance between age-related changes in membrane phospholipid composition and cholesterol content plays an integral role in the regulation of cholesterol-dependent processes in fiber cell membranes and in the maintenance of fiber cell membrane homeostasis.

Identification and Functional Assessment of Age-Dependent Truncations to Cx46 and Cx50 in the Human Lens.

PurposeMany proteins in the lens undergo extensive posttranslational modifications (PTMs) with age, leading to alterations in their function. The extent to which lens gap junction proteins, Cx46 and Cx50, accumulate PTMs with aging is not known. In this study, we identified truncations in Cx46 and Cx50 in the human lens using mass spectrometry. We also examined the effect of truncations on channel function using electrophysiological measurements.MethodsHuman lenses were dissected into cortex, outer nucleus, and nucleus regions, and fiber cell membranes were subjected to trypsin digestion. Tryptic peptides were analyzed by liquid chromatography (LC)–electrospray tandem mass spectrometry (ESI/MS/MS). Effects of truncations on channel conductance, permeability, and gating were assessed in transfected cells.ResultsCleavage sites were identified in the C-terminus, the cytoplasmic loop, and the N-terminus of Cx46 and Cx50. Levels of C-terminal truncations, which were found at residues 238 to 251 in Cx46 and at residues 238 to 253 and 274 to 284 in Cx50, were similar in different lens regions. In contrast, levels of truncations in cytoplasmic loop and N-terminal domains of Cx46 and Cx50 increased dramatically from outer cortex to nucleus. Most of the C-terminally truncated proteins were functional, whereas truncations in the cytoplasmic loop did not result in the formation of functional channels.ConclusionsAccumulation of cytoplasmic loop and N-terminal truncations in the core might lead to decreases in coupling with age. This reduction is expected to lead to an increase in intracellular calcium and a decrease in levels of glutathione in the nucleus. These changes may ultimately lead to age-related nuclear cataracts.

Connexin23 deletion does not affect lens transparency

While connexin46 (Cx46) and connexin50 (Cx50) are crucial for maintaining lens transparency and growth, the contributions of a more recently identified lens fiber connexin, Cx23, are poorly understood. Therefore, we studied the consequences of absence of Cx23 in mouse lenses. Cx23-null mice were generated by homologous Cre recombination. Cx23 mRNA was abundantly expressed in wild type lenses, but not in Cx23-null lenses. The transparency and refractive properties of Cx23-null lenses were similar to wild type lenses when examined by darkfield microscopy. Neither the focusing ability nor the light scattering was altered in the Cx23-null lenses. While both Cx46 and Cx50 localized to appositional fiber cell membranes (as in wild type lenses), their levels were consistently (but not significantly) decreased in homozygous Cx23-null lenses. These results suggest that although Cx23 expression can influence the abundance of the co-expressed lens fiber connexins, heterozygous or homozygous expression of a Cx23-null allele does not alter lens transparency.

Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells.

Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.

Exertion‐induced rhabdomyolysis of the long head of the triceps

Exertion‐induced rhabdomyolysis of the long head of the triceps

αB‐Crystallin Phosphorylation as a Precursor to Cataractogenesis

PurposeThe molecular mechanism behind age‐related cataract formation is yet to be elucidated, although phosphorylation induced hyperactivity of αB‐crystallin (αB) and its subsequent association with fibre cell membranes has been implicated. This study evaluates the effect phosphorylation of αB at three well characterised sites (S19, S45 and S59) has on its chaperone activity and aims to characterise αB's membrane association with a focus on its role in cataractogenesis.MethodsThe chaperone activity of αB phosphomimics (S→D) were measured via aggregation assays using creatine phosphokinase (CPK) as a substrate. In vivo association of αB phosphoisotypes with two abundant membrane proteins, connexin 46 (cx46) and aquaporin 0 (AQP0) in aged human lenses was visualised using Duolink proximity assays and confocal microscopy.ResultsThe chaperone activity of αB increased with an increase in modification sites. Modification at the S19 and S59 residues activated αB to a greater extent than at S45. In the lens, phosphorylated αB was found to interact with Cx46 and AQP0 at cell membranes.ConclusionsThis study confirms that multiple phosphorylation events of αB cause a cumulative increase in activity, and that membrane association, at least in part, is mediated by interactions with membrane pore proteins. The proposed mechanism of cataractogenesis is that phosphorylation induced increases in substrate affinity of αB may lead to increased association with membrane pore proteins, leading to the obstruction of the diffusion of small molecules into inner regions of the lens, contributing to cataract formation.

Amounts of phospholipids and cholesterol in lipid domains formed in intact lens membranes: Methodology development and its application to studies of porcine lens membranes

An electron paramagnetic resonance spin-labeling method has been developed that allows quantitative evaluation of the amounts of phospholipids and cholesterol in lipid domains of intact fiber-cell plasma membranes isolated from cortical and nuclear regions of eye lenses. The long term goal of this research is the assessment of organizational changes in human lens fiber cell membranes that occur with age and during cataract development. The measurements needed to be performed on lens membranes prepared from eyes of single donors and from single eyes. For these types of studies it is necessary to separate the age/cataract related changes from preparation/technique related changes. Human lenses differ not only because of age, but also because of the varying health histories of the donors. To solve these problems the sample-to-sample preparation/technique related changes were evaluated for cortical and nuclear lens membranes prepared from single porcine eyes. It was assumed that the differences due to the age (animals were two year old) and environmental conditions for raising these animals were minimal. Mean values and standard deviations from preparation/technique changes for measured amounts of lipids in membrane domains were calculated. Statistical analysis (Student's t-test) of the data also allowed determining the differences of mean values which were statistically significant with P ≤ 0.05. These differences defined for porcine lenses will be used for comparison of amounts of lipids in domains in human lens membranes prepared from eyes of single donors and from single eyes. Greater separations will indicate that differences were statistically significant with (P ≤ 0.05) and that they came from different than preparation/technique sources. Results confirmed that in nuclear porcine membranes the amounts of lipids in domains created due to the presence of membrane proteins were greater than those in cortical membranes and the differences were larger than the differences observed for human intact fiber cell membranes [Raguz, M. Mainali, L., O'Brien, W.J., and Subczynski, W.K. (2015) Exp. Eye Res.]. Lipids in porcine nuclear fiber cell plasma membranes were more rigid and less permeable to oxygen than in human nuclear membranes. Most likely the significant differences in the lipid composition were responsible for the observed differences.

Fragile X Syndrome FMRP Co-localizes with Regulatory Targets PSD-95, GABA Receptors, CaMKIIα, and mGluR5 at Fiber Cell Membranes in the Eye Lens.

Fmr1 and FMRP underlie Fragile X Syndrome (FXS) and are linked with related autism spectrum disorders (ASD). Fmr1 also has an essential role in eye and lens development. Lenses express FMRP along with γ-aminobutyric acid (GABA) receptors (GABARs), post-synaptic density protein 95 (PSD-95), Tyr-phosphatase STEP, CaMKIIα and Alzheimer's disease Aβ precursor protein, which are verified targets of FMRP regulation in neurons and outline major topics in FXS/ASD research. PSD-95 as well as CaMKIIα transcripts undergo polypryimidine tract binding protein dependent alternative splicing in lens, consistent with PSD-95 translation in lens. At least 13 GABAR subunits and GAD25/65/67 GABA metabolism enzymes are expressed in lenses beginning in embryonic development, matching neural development. Interestingly, GABAergic drugs (e.g. baclofen) studied as FXS/ASD therapeutics are shown to resolve developmental vision defects in experimental myopia. Here, we demonstrated that FMRP co-localizes at fiber cell membranes with PSD-95, GABAAδ, GABAAβ3, GABBR1, STEP, CaMKIIα, and mGluR5 in young adult lenses. GAD65 and GABA detection was greatest at the peri-nuclear lens region where fiber cell terminal differentiation occurs. These findings add to an extensive list of detailed parallels between fiber cell and neuron morphology and their lateral membrane spine/protrusions, also reflected in the shared expression of genes involved in the morphogenesis and function of these membrane structures, and shared use of associated regulatory mechanisms first described as distinguishing the neuronal phenotype. Future studies can determine if GABA levels currently studied as a FXS/ASD biomarker in the brain, and generated by GAD25/65/67 in a comparable cell environment in the lens, may be similarly responsive to Fmr1 mutation in lens. The present demonstration of FMRP and key regulatory targets in the lens identifies a potential for the lens to provide a new research venue, in the same individual, to inform about Fmr1/FMRP pathobiology in brain as well as lens.

Role of Aquaporin 0 in lens biomechanics

Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent ex vivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5−/−), AQP0 KO (heterozygous KO: AQP0+/−; homozygous KO: AQP0−/−; all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0+/− lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.