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.

Properties of membranes derived from the total lipids extracted from clear and cataractous lenses of 61-70-year-old human donors.

Human lens-lipid membranes prepared from the total lipids extracted from clear and cataractous lens cortexes and nuclei of 61-70-year-old donors by use of a rapid solvent-exchange method were investigated. The measured cholesterol-to-phospholipid (Chol/PL) molar ratio in these membranes was 1.8 and 4.4 for cortex and nucleus of clear lenses, respectively, and 1.14 and 1.45 for cataractous lenses. Properties and organization of the lipid bilayer were investigated by use of electron paramagnetic resonance spin-labeling methods. Formation of Chol crystals was confirmed by use of differential scanning calorimetry. Pure cholesterol bilayer domains (CBDs) were formed in all the membranes investigated. It was shown that in clear lens membranes of the nucleus, Chol exists in three different environments: (1) dispersed in phospholipid bilayers (PCDs), (2) in CBDs, and (3) in Chol crystals. In clear lens membranes of the cortex, and in cortical and nuclear cataractous lens membranes, Chol crystals were not detected, because of the lower Chol content. Profiles of membrane properties (alkyl-chain order, fluidity, oxygen transport, and hydrophobicity) across the PCD were very similar for clear and cataractous membranes. Profiles of the oxygen transport parameter across the CBD were, however, different for cortical clear and cataractous membranes-the amount and size of CBDs was less in cataractous membranes. These results suggest that high Chol content, formation of CBDs, and formation of Chol crystals should not be regarded as major predispositions for the development of age-related cataracts.

Properties of membranes derived from the total lipids extracted from the human lens cortex and nucleus.

Human lens lipid membranes prepared using a rapid solvent exchange method from the total lipids extracted from the clear lens cortex and nucleus of 41- to 60-year-old donors were investigated using electron paramagnetic resonance spin-labeling. Profiles of the phospholipid alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity were assessed across coexisting membrane domains. Membranes prepared from the lens cortex and nucleus were found to contain two distinct lipid environments, the bulk phospholipid-cholesterol domain and the cholesterol bilayer domain (CBD). The alkyl chains of phospholipids were strongly ordered at all depths, indicating that the amplitude of the wobbling motion of alkyl chains was small. However, profiles of the membrane fluidity, which explicitly contain time (expressed as the spin-lattice relaxation rate) and depend on the rotational motion of spin labels, show relatively high fluidity of alkyl chains close to the membrane center. Profiles of the oxygen transport parameter and hydrophobicity have a rectangular shape and also indicate a high fluidity and hydrophobicity of the membrane center. The amount of CBD was greater in nuclear membranes than in cortical membranes. The presence of the CBD in lens lipid membranes, which at 37°C showed a permeability coefficient for oxygen about 60% smaller than across a water layer of the same thickness, would be expected to raise the barrier for oxygen transport across the fiber cell membrane. Properties of human membranes are compared with those obtained for membranes made of lipids extracted from cortex and nucleus of porcine and bovine eye lenses.

Properties of fiber cell plasma membranes isolated from the cortex and nucleus of the porcine eye lens.

The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eye lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes, because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes-namely, the domain formed by boundary lipids and the domain formed by trapped lipids-were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region.

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies.

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 degrees C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.

Resistance of alpha-crystallin quaternary structure to UV irradiation.

The damaging effect of UV radiation (lambda > 260 nm) on bovine alpha-crystallin in solution was studied by small-angle X-ray scattering, gel permeation chromatography, electrophoresis, absorption and fluorescence spectroscopy, and differential scanning calorimetry. The results obtained show that damage to even a large number of subunits within an alpha-crystallin oligomer does not cause significant rearrangement of its quaternary structure, aggregation of oligomers, or the loss of their solubility. Due to the high resistance of its quaternary structure, alpha-crystallin is able to prevent aggregation of destabilized proteins (especially of gamma- and beta-crystallins) and so to maintain lens transparency throughout the life of an animal (the chaperone-like function of alpha-crystallin).

Oxindolealanine in age-related human cataracts.

The present study was performed in order to obtain structural and quantitative information regarding the modifications that take place in the human lens as a result of tryptophan oxidation. In particular, the early tryptophan oxidation product, oxindolealanine (OIA) has been detected in lyophilized and hydrolyzed cataractous lenses by mass spectrometry. OIA was confirmed in human cataract samples by observing its ion (m/z 221), fragmentation pattern and absorption spectrum. Quantitative results indicate that there are differences in the amounts of OIA in the nucleus versus the cortex in human cataractous lenses. Expressed as a ratio to the level of phenylalanine (Phe), the nucleus has more than one and a half times greater levels of OIA as compared to the cortex [nucleus=(3.7+/-0.7)x10(-2) versus cortex=(2.3+/-0.3)x10(-2)]. Furthermore, the average value for the OIA/Phe ratio in the calf lens (controls) was (0.8+/-0.2)x10(-2) as compared to (3.7+/-0.7)x10(-2) in human cataractous lens nucleus (p<0.05). The quantitative results correspond to a 4.6-fold increase of OIA in human cataractous lenses. In a separate series of experiments using HPLC with photodiode array (PDA) detection only, the differences in OIA levels in cataract nucleus versus cortex and cataracts versus controls closely matched the LC/MS data. The results suggest that OIA levels are elevated in human cataractous lenses thus providing further evidence to implicate tryptophan oxidation in this process.

Status of caveolin-1 in various membrane domains of the bovine lens.

Recent studies of the distribution and relative concentration of caveolin-1 in fractions of bovine lens epithelial and fiber cells have led to the novel concept that caveolin-1 may largely exist as a peripheral membrane protein in some cells. Caveolin-1 is typically viewed as a scaffolding protein for caveolae in plasma membrane. In this study, membrane from cultured bovine lens epithelial cells and bovine lens fiber cells were divided into urea soluble and insoluble fractions. Cytosolic lipid vesicles were also recovered from the lens epithelial cells. Lipid-raft domains were recovered from fiber cells following treatment with detergents and examined for caveolin and lipid content. Aliquots of all fractions were Western blotted for caveolin-1. Fluorescence microscopy and double immunofluorescence labeling were used to examine the distribution of caveolin-1 in cultured epithelial cells. Electron micrographs revealed an abundance of caveolae in plasma membrane of cultured lens epithelial cells. About 60% of the caveolin-1 in the epithelial-crude membrane was soluble in urea, a characteristic of peripheral membrane proteins. About 30% of the total was urea-insoluble membrane protein that likely supports the structure of caveolae. The remaining caveolin was part of cytosolic lipid vesicles. By contrast, most caveolin in the bovine lens fiber cell membrane was identified as intrinsic protein, being present at relatively low concentrations in caveolae-free lipid raft domains enriched in cholesterol and sphingomyelin. We estimate that these domains occupied 25-30% of the fiber cell membrane surface. Thus, the status of caveolin-1 in lens epithelial cells appears markedly different from that in fiber cells.

Protein-bound UV filters in normal human lenses: the concentration of bound UV filters equals that of free UV filters in the center of older lenses.

PURPOSE: To survey the levels of protein-bound UV filters in the cortices and nuclei of normal human lenses as a function of age and to relate this to the concentration of free UV filters. METHODS: Levels of each of the three kynurenine (Kyn) UV filters, 3-hydroxykynurenine glucoside (3OHKG), Kyn, and 3-hydroxykynurenine (3OHKyn), covalently attached to proteins, were determined by using a newly developed method of reductive capture, after base treatment of the intact lens proteins. RESULTS: The data show that, in the normal lens, each of the three UV filters became bound to proteins to a significant extent only after age 50 and, further, that the levels in the nucleus were much higher than in the cortex. These findings are consistent with the lens barrier that forms in middle age. 3OHKG was present at the highest levels followed by Kyn, with 3OHKyn being attached in the lowest amount. The ratio was 145:4:1 (3OHKG-Kyn-3OHKyn), with a total protein-bound UV filter concentration in the lens nucleus after age 50 of approximately 1300 picomoles/mg protein. This ratio is in agreement with 3OHKG being the most abundant free UV filter in the human lens and 3OHKyn being present in the lowest concentration with free Kyn present in intermediate amounts. CONCLUSIONS: The three Kyn UV filters are bound to the nuclear proteins of all normal lenses over the age of 50. Indeed in the center of older normal lenses, the concentration of UV filters bound to proteins is approximately equal to that of the free filters. Since bound UV filters promote oxidation of proteins after exposure to wavelengths of light that penetrate the cornea, lenses in middle-aged and older individuals may be more prone to photooxidation than those of young people.

Molecular identification and characterisation of the glycine transporter (GLYT1) and the glutamine/glutamate transporter (ASCT2) in the rat lens.

Glutathione (GSH) is an essential antioxidant required for the maintenance of lens transparency. In the lens, GSH is maintained at unusually high concentrations as a result of direct GSH uptake and/or intracellular de novo synthesis from its precursor amino acids; cysteine, glycine and glutamine/glutamate. With increasing age, the levels of GSH, particularly in the core of the lens, are significantly reduced. It has been proposed that alterations in the transport of GSH and/or its precursor amino acids may contribute to the changes in GSH levels in older lenses. As considerable uncertainty exists about the molecular identity of GSH transporters in the lens, we have focused on identifying transporters involved in the uptake of the precursor amino acids required for GSH synthesis. Previously, we identified an uptake system for cyst(e)ine mediated by the Xc(-) exchanger and the Excitatory Amino Acid Transporters (EAATs) in the rat lens. In this current study, we have identified and localised additional uptake systems that contribute to GSH synthesis. Transcripts for GLYT1 (glycine transporter) and ASCT2 (glutamine/glutamate transporter) were detected in rat lens fiber cells using the reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis confirmed the expression of both GLYT1 and ASCT2 at the protein level. Immunocytochemistry revealed GLYT1 expression to be restricted to cortical regions of the lens. Labelling was predominantly cytoplasmic with some labelling of the membrane. In contrast, ASCT2 was expressed throughout the lens extending from the outer cortex through to the core. In the outer cortex, ASCT2 expression was predominantly cytoplasmic. However, with deeper distance into the lens, labelling became more membraneous indicating insertion of ASCT2 into the membranes of mature fiber cells of the lens core. The molecular identification and localisation of GLYT1 and ASCT2 in the lens suggests that these transporters may be responsible for the uptake of the precursor amino acids, glycine and glutamine, which are involved in GSH synthesis. Moreover, the presence of ASCT2 in the centre of the lens raises the possibility that ASCT2 may work with the Xc(-) exchanger to accumulate cysteine where it can potentially act as a low molecular mass antioxidant.

Connexin 46 and connexin 50 in selenite cataract.

The purpose of this work was to determine if the lens gap junction proteins connexin 46 (Cx46) and connexin 50 (Cx50) were altered with the development of selenite-induced cataract. Cataracts were induced in young Sprague-Dawley rats with a single subcutaneous injection of sodium selenite; age-matched uninjected rats served as controls. Membrane fractions were isolated from homogenates of cortex and nucleus of normal and cataractous lenses by differential and discontinuous sucrose gradient centrifugation. Aliquots of urea-insoluble protein from membrane fractions were analyzed by quantitative densitometry of Western blots probed with antibodies to Cx46 and Cx50. A significant decrease in the more slowly migrating Cx46-reactive band, which represents phosphorylated Cx46, was found in the major membrane fraction of the cortex of cataractous lenses. There was no significant difference in the amounts of either Cx46 or Cx50 associated with selenite cataract in any of the membrane fractions examined. These results suggest that alteration of gap junction function (as evidenced by the change in phosphorylation of Cx46) may be associated with the development of the selenite cataract, but that neither Cx46 nor Cx50 is subject to the well-characterized proteolysis associated with the selenite cataract model.

Oxidative stress-induced up-regulation of the chloride channel and Na+/Ca2+ exchanger during cataractogenesis in diabetic rats.

We have determined the abundance of the chloride channel, ClC-3, and Na(+)/Ca(2+) exchanger proteins in isolated rat lens cortex fiber cells by immunofluorescence method using polyclonal anti-ClC-3 antibodies and monoclonal antibodies against the canine cardiac Na(+)/Ca(2+) exchanger protein. These proteins were also quantified in the lens cortex of streptozotocin-injected rats by Western blots. Also, mRNA for ClC-3 was determined by Northern blot analysis. The isolated rat lens cortical fibers expressed basal levels of ClC-3 and Na(+)/Ca(2+) exchanger proteins. As compared to controls, the ClC-3 protein in the lens cortex of diabetic rats (blood glucose>400 mg%) increased by 2.5-fold in 7 days and 4.5-fold in 14 days. However, the ClC-3 protein decreased to near-normal values in 40 days. The changes in ClC-3 mRNA closely followed the protein levels. Similarly, as compared to controls, on Day 7, the Na(+)/Ca(2+) exchanger protein in the diabetic rat lens cortex increased by 3.5-fold and on Day14 by 5.5-fold. Subsequently, it decreased to control levels on Day 40. Treatment with the antioxidant, Trolox (2 mg/kg body weight), prevented the initial increase in ClC-3 and Na(+)/Ca(2+) exchanger proteins. The up-regulation of ClC-3 and Na(+)/Ca(2+) exchanger proteins during the early stages of diabetes and its prevention by antioxidants suggests that the proteins regulating ion transport may have a pathophysiological role in the development of diabetic cataracts.

Microtubule configuration and membranous vesicle transport in elongating fiber cells of the rat lens.

This study examines the microtubule configuration and its close association with the Golgi complex and Golgi-derived membranous vesicles in elongating fiber cells of the rat lens. Since fiber cells elongate tremendously during lens differentiation, we hypothesize that a microtubule-based motor system exists in the elongating fiber cells for transporting important membrane proteins and organelles to the target regions for cell growth. The newly synthesized membrane proteins are known to be transported from the trans-Golgi network in the form of vesicles to the target plasma membrane. By thin-section TEM, we observed a large number of vesicles of various sizes and shapes randomly distributed throughout the cytoplasm of elongating fiber cells. Both Golgi complex and vesicles exhibited characteristic normal structural features seen in other cell types and thus represented real vesicular organelles in the fiber cells. A large number of microtubules were regularly arranged into bundles parallel to the long axis of fiber cells as examined in both longitudinal and cross-section views. Many of these microtubules were closely associated or in intimate contact with the Golgi complex and vesicles in elongating fiber cells. The microtubule polarity assay revealed that microtubules exhibited a unidirectional polarity for the entire length of fiber cells as examined in both anterior and posterior cortical fiber segments. Namely, the minus end of microtubules was towards the anterior lens pole while the plus end was headed towards the posterior pole. This suggests that multiple molecular motors such as kinesin and dynein are needed for carrying the vesicles to both lens poles, since conventional kinesin is known to transport vesicular organelles towards the plus end whereas cytoplasmic dynein carries them towards the minus end of microtubules. By immunoblot analysis, we indeed detected the presence of both kinesin (120 kD) and dynein (70 kD) in homogenate prepared from lens cortical fibers. Moreover, immunogold TEM demonstrated that the aquaporin 0 (formally MIP26) antibody was localized on the membranous vesicles as well as plasma membranes of the cortical fiber cells. This study suggests that a microtubule-based motor system exists in the lens and plays an important role in transporting membrane proteins such as aquaporin 0 in the vesicles during fiber cell differentiation and elongation.

Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers.

PURPOSE: Human lens membranes contain unusually high levels of cholesterol and sphingolipids, lipids known to segregate into liquid-ordered domains. The current study was conducted to pursue the determination and characterization of these domains in membranes of clear and cataractous human lenses. METHODS: Cortical and nuclear regions of aged clear and cataractous lenses were obtained. After lysis with Triton X-100 at 4 degrees C and sucrose linear-density centrifugation, sedimenting and nonsedimenting fractions (when present) were collected. Phospholipids were analyzed by (31)P-nuclear magnetic resonance (NMR) and mass spectrometry. Caveolae and raft markers were tested by Western blot analysis. RESULTS: Only samples from clear lenses exhibited a nonsedimenting band. Phospholipid contents were comparable for sedimenting fractions of clear and cataractous membranes. Cholesterol to phospholipid molar ratios in light-density bands were nearly 7, three times greater than in sedimenting fractions. The portion of total cholesterol present in nonsedimenting fractions increased from 5.5% in the cortex to 14% in the nucleus. Two lysophospholipids comprising approximately 10% of all phospholipids in total membranes were undetectable in nonsedimenting fractions. Caveolin-1 was enriched in these fractions. CONCLUSIONS: Phospholipid compositional differences between lighter and heavier fractions from clear lenses were relatively minor and could not, alone, account for the substantial enrichment of cholesterol in the lighter fractions. Specific proteins, such as caveolin-1, must recruit cholesterol and induce clustering. Undetectable amounts of light-density domains in cataractous membranes suggest either disruption of these aggregates and thus the function of proteins within them, possibly relevant to lens transparency, and/or greater density of these clusters due to stronger binding of insoluble crystallins to membranes.

Analysis of alpha-crystallin chaperone function using restriction enzymes and citrate synthase.

PURPOSE: To compare the abilities of [alpha]A-crystallin, [alpha]B-crystallin, and mini-[alpha]A-crystallin (a synthetic peptide chaperone representing the functional unit of [alpha]A-crystallin) to protect against heat-induced inactivation of citrate synthase (CS) and restriction enzymes, SmaI and NdeI. METHODS: Restriction enzymes, SmaI and NdeI were heated at different temperatures in the presence of various amounts of molecular chaperones and tested for their ability to cleave plasmid DNA. The aggregation of CS was measured at 43 degrees C while the loss in activity was monitored at 37 degrees C in the presence of various crystallins. RESULTS: Restriction enzyme activities were protected by the crystallin subunits up to 37 degrees C for SmaI and 43 degrees C for NdeI. However, the mini-[alpha]A-crystallin was unable to protect endonuclease activity. The crystallin subunits and the peptide chaperone were able to suppress thermal aggregation of CS at 43 degrees C, but failed to stabilize its activity at 37 degrees C. CONCLUSIONS: The ability of [alpha]-crystallin subunits to stabilize denaturing proteins varies from enzyme to enzyme as evidenced by the inactivation of CS and protection of SmaI and NdeI activity in the presence of [alpha]-crystallin subunits. Additionally, our results show that there could be more than one site in [alpha]A-crystallin responsible for its chaperone-like action. By addition of crystallin subunits to restriction enzymes prior to or during storage, transport, or assay would maintain or improve their activity thereby decreasing their cost.

Surface tongue-and-groove contours on lens MIP facilitate cell-to-cell adherence.

The lens major intrinsic protein (MIP, AQP0) is known to function as a water and solute channel. However, MIP has also been reported to occur in close membrane contacts between lens fiber cells, indicating that it has adhesive properties in addition to its channel function. Using atomic force and cryo-electron microscopy we document that crystalline sheets reconstituted from purified ovine lens MIP mostly consisted of two layers. MIP lattices in the apposing membranes were in precise register, and determination of the membrane sidedness demonstrated that MIP molecules bound to each other via their extracellular surfaces. The surface structure of the latter was resolved to 0.61 nm and revealed two protruding domains providing a tight "tongue-and-groove" fit between apposing MIP molecules. Cryo-electron crystallography produced a projection map at 0.69 nm resolution with a mirror symmetry axis at 45 degrees to the lattice which was consistent with the double-layered nature of the reconstituted sheets. These data strongly suggest an adhesive function of MIP, and strengthen the view that MIP serves dual roles in the lens.

Galectin-3 is associated with the plasma membrane of lens fiber cells.

PURPOSE: To discover proteins that have the potential to contribute to the tight packing of fiber cells in the lens. METHODS: Crude fiber cell membranes were isolated from ovine lens cortex. Proteins were separated by two-dimensional gel electrophoresis, and selected protein spots identified by micro-sequencing. The identification of galectin-3 was confirmed by immunoblotting with a specific antibody. The association of galectin-3 with the fiber cell plasma membrane was investigated using immunofluorescence microscopy, solubilization trials with selected reagents, and immunoprecipitation to identify candidate ligands. RESULTS: A cluster of three protein spots with an apparent molecular weight of 31,000 and isoelectric points ranging between 7 and 8.5 were resolved and identified as galectin-3. This protein was associated peripherally with the fiber cell plasma membrane and interacted with MP20, an abundant intrinsic membrane protein that had been identified previously as a component of membrane junctions between fiber cells. CONCLUSIONS: The detection of galectin-3 in the lens is a novel result and adds to the growing list of lens proteins with adhesive properties. Its location at the fiber cell membrane and its association with the junction-forming MP20 is consistent with a potential role in the development or maintenance of the tightly packed lens tissue architecture.

Direct evidence for immiscible cholesterol domains in human ocular lens fiber cell plasma membranes.

The molecular structure of human ocular lens fiber cell plasma membranes was examined directly using small angle x-ray diffraction approaches. A distinct biochemical feature of these membranes is their high relative levels of free cholesterol; the mole ratio of cholesterol to phospholipid (C/P) measured in these membranes ranges from 1 to 4. The organization of cholesterol in this membrane system is not well understood, however. In this study, the structure of plasma membrane samples isolated from nuclear (3.3 C/P) and cortical (2.4 C/P) regions of human lenses was evaluated with x-ray diffraction approaches. Meridional diffraction patterns obtained from the oriented membrane samples demonstrated the presence of an immiscible cholesterol domain with a unit cell periodicity of 34.0 A, consistent with a cholesterol monohydrate bilayer. The dimensions of the sterol-rich domains remained constant over a broad range of temperatures (5-20 degrees C) and relative humidity levels (31-97%). In contrast, dimensions of the surrounding sterol-poor phase were significantly affected by experimental conditions. Similar structural features were observed in membranes reconstituted from fiber cell plasma membrane lipid extracts. The results of this study indicate that the lens fiber cell plasma membrane is a complex structure consisting of separate sterol-rich and -poor domains. Maintenance of these separate domains may be required for the normal function of lens fiber cell plasma membrane and may interfere with the cataractogenic aggregation of soluble lens proteins at the membrane surface.

Analysis of small GTP-binding proteins of the lens by GTP overlay assay reveals the presence of unique GTP-binding proteins associated with fiber cells.

Low molecular weight GTP-binding proteins are molecular switches which are thought to play pivotal roles in cell growth, differentiation, cytoskeletal organization and vesicular trafficking. In this study, members of this family of proteins have been identified and characterized in the eye lens, for the first time. [alpha 33P]GTP blot overlay assays of monkey and human lens water soluble and membranous insoluble fractions revealed the presence of specific GTP-binding proteins in the range of 20-30 kDa (small GTPases) in both fractions, with much higher amounts in the membranous insoluble fraction. In the insoluble fraction, in addition to 20-30 kDa GTPases, there are three distinct GTP-binding proteins, ranging from 33-45 kDa. The small GTPases (20-30 kDa) were present throughout the lens in epithelium, cortex and nucleus, while the 33-45 kDa GTP-binding protein bands were exclusively associated with the cortex and nucleus (fiber cells). Analysis of lens fractions by two-dimensional electrophoresis, immunoprecipitation using monoclonal and sequence specific polyclonal antibodies and C3 exoenzyme mediated ADP-ribosylation demonstrated the presence of Ras, Rap, Rho, Rac, Rab and several other small GTPases. The 33-45 kDa GTP-binding proteins that are associated with lens fiber cells appear to be distinct from the small GTPases and from heterotrimeric GTPases, and were not detected in brain or heart tissue. The presence of different complements of GTP-binding proteins in lens fibers and epithelial, cells suggests their involvement in important regulatory functions, possibly related to cell growth, differentiation and organization of the cytoskeleton.

Actin filament bundles in cortical fiber cells of the rat lens.

The distribution and organization of actin filament bundles were studied in cortical fiber cells of rat lenses at various ages (3 days to 2.5 months old), using thin-section electron microscopy, immunocytochemistry and immunoblotting. Electron microscopy showed that actin bundles were regularly found along cortical fiber cell membranes of the lens at all ages studied. The actin bundles were commonly arranged in three distinct units, one bundle in each fiber cell, located at the intersections where three hexagonal fiber cells meet as seen in cross sections. These actin bundles were approximately 150 nm in diameter and were composed of 7-nm small filaments. They were aligned parallel to the long axis of fiber cells as judged by both cross and longitudinal sections. The outside border of each bundle was always surrounded by a zone of 10-nm intermediate filaments which have the same orientation as that of the actin bundles. In longitudinal sections, elongated actin bundles were always parallel to the cell membranes. A number of individual actin bundles sometimes were found to form a chain with periodic short intervals. In addition, actin bundles were frequently associated with adherens junctions near the intersections and other regions of fiber cell membranes. By immunoelectron microscopy, we demonstrated that these filament bundles indeed contained actins. By rhodamine-phalloidin labelling, we found that labeled actin bundles appeared as large, distinct dots at the corners of hexagonal fiber cells in all ages studied. In addition, non-bundle F-actins were labeled preferentially along the cell membranes of the short sides of hexagonal fiber cells. This resulted in a unique zigzag pattern of actin labeling commonly seen in the cortical fiber cells of a mature rat lens. Finally, we showed that alpha-actinin was associated with the actin bundles in the fiber cells by immunofluorescent double labeling and immunoblotting. It is suggested that this unique arrangement of actin bundles in fiber cells may provide a stabilizing structure for forming a sharp angle at each corner of fiber cells, thereby the hexagonal shape of the cells can be maintained.