Lipid composition of lens plasma membrane fractions enriched in fiber junctions

Little is known about the lipid environment of lens fiber junctions, the plasma membrane structure proposed to be responsible for passage of low molecular weight metabolites between adjacent lens fiber cells. Plasma membranes of the ocular lens are especially rich in fiber junctions. The resistance of junctional domains to disruption by detergent or alkali treatment provides the opportunity to isolate a lens plasma membrane fraction enriched in fiber junctions. When examined by electron microscopy, the fiber junction fraction prepared from bovine lenses was enriched with junctional structures by about twofold when compared to total plasma membrane. We compared the protein, phospholipid, and cholesterol concentration of total plasma membrane with fiber junctional membrane from rat and cow lens and from aged normal cataractous human lenses. The principal finding was that junctional membrane contained 20-40% more total lipid than that of the total plasma membrane. This was due to a proportionate increase in the relative content (mg/mg protein) of both phospholipid and cholesterol. Exclusive of one exception (nucleus of bovine lens), the cholesterol/phospholipid molar ratios of the two fractions were similar. In the bovine nucleus, the cholesterol/phospholipid molar ratio was substantially higher in the fiber junctional-enriched membrane fraction than in the total plasma membrane, suggesting a special association of cholesterol with bovine nuclear fiber junctions. The relative lipid compositions of the plasma membrane and fiber junction-enriched fractions from human normal and cataractous lenses were similar, suggesting that human senile cataractogenesis involves changes in the lens plasma membrane more subtle than would be reflected by gross changes in the membrane lipid composition.     

Immunocytochemical localization of the main intrinsic polypeptide (MIP) in ultrathin frozen sections of rat lens

The in situ distribution of the 26-kdalton Main Intrinsic Polypeptide (MIP or MP 26), a putative gap junction protein in ocular lens fibers, was defined at the electron microscope level using indirect immunoferritin labeling of ultrathin frozen sections of rat lens. MIP was found distributed throughout the plasma membrane of the lens fiber cell, with no apparent distinction between junctional and nonjunctional membrane. MIP was not detectable in the basal or lateral plasma membrane of the lens epithelial cell, including the interepithelial cell gap junctions; nor was MIP detectable in the plasma membrane or gap junctions of the hepatocyte. Previous reports have indicated that the protein composition of the lens fiber cell junction differs from that of the hepatocyte gap junction. The evidence presented here suggests that the composition of the fiber cell junction and plasma membrane is also immunocytochemically distinct from that of its progenitor, the lens epithelial cell.     

Calcium regulation by lens plasma membrane vesicles

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.     

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.     

Direct evidence for cholesterol crystalline domains in biological membranes: role in human pathobiology

This review will discuss the use of small-angle X-ray diffraction approaches to study the organization of lipids in plasma membranes derived from two distinct mammalian cell types: arterial smooth muscle cells and ocular lens fiber cells. These studies indicate that cholesterol at an elevated concentration can self-associate and form immiscible domains in the plasma membrane, a phenomenon that contributes to both physiologic and pathologic cellular processes, depending on tissue source. In plasma membrane samples isolated from atherosclerotic smooth muscle cells, the formation of sterol-rich domains is associated with loss of normal cell function, including ion transport activity and control of cell replication. Analysis of meridional diffraction patterns from intact and reconstituted plasma membrane samples indicates the presence of an immiscible cholesterol domain with a unit cell periodicity of 34 Å, consistent with a cholesterol monohydrate tail-to-tail bilayer, under disease conditions. These cholesterol domains were observed in smooth muscle cells enriched with cholesterol in vitro as well as from cells obtained ex vivo from an animal model of atherosclerosis. By contrast, well-defined cholesterol domains appear to be essential to the normal physiology of fiber cell plasma membranes of the human ocular lens. The organization of cholesterol into separate domains underlies the role of lens fiber cell plasma membranes in maintaining lens transparency. These domains may also interfere with cataractogenic aggregation of soluble lens proteins at the membrane surface. Taken together, these analyses provide examples of both physiologic and pathologic roles that sterol-rich domains may have in mammalian plasma membranes. These findings support a model of the membrane in which cholesterol aggregates into structurally distinct regions that regulate the function of the cell membrane.     

Direct evidence for cholesterol crystalline domains in biological membranes: role in human pathobiology

This review will discuss the use of small-angle X-ray diffraction approaches to study the organization of lipids in plasma membranes derived from two distinct mammalian cell types: arterial smooth muscle cells and ocular lens fiber cells. These studies indicate that cholesterol at an elevated concentration can self-associate and form immiscible domains in the plasma membrane, a phenomenon that contributes to both physiologic and pathologic cellular processes, depending on tissue source. In plasma membrane samples isolated from atherosclerotic smooth muscle cells, the formation of sterol-rich domains is associated with loss of normal cell function, including ion transport activity and control of cell replication. Analysis of meridional diffraction patterns from intact and reconstituted plasma membrane samples indicates the presence of an immiscible cholesterol domain with a unit cell periodicity of 34 A, consistent with a cholesterol monohydrate tail-to-tail bilayer, under disease conditions. These cholesterol domains were observed in smooth muscle cells enriched with cholesterol in vitro as well as from cells obtained ex vivo from an animal model of atherosclerosis. By contrast, well-defined cholesterol domains appear to be essential to the normal physiology of fiber cell plasma membranes of the human ocular lens. The organization of cholesterol into separate domains underlies the role of lens fiber cell plasma membranes in maintaining lens transparency. These domains may also interfere with cataractogenic aggregation of soluble lens proteins at the membrane surface. Taken together, these analyses provide examples of both physiologic and pathologic roles that sterol-rich domains may have in mammalian plasma membranes. These findings support a model of the membrane in which cholesterol aggregates into structurally distinct regions that regulate the function of the cell membrane.     

Evidence for distinct cholesterol domains in fiber cell membranes from cataractous human lenses

Previous studies in our laboratory have provided direct evidence for the existence of distinct cholesterol domains within the plasma membranes of human ocular lens fiber cells. The fiber cell plasma membrane is unique in that it contains unusually high concentrations of cholesterol, with cholesterol to phospholipid (C/P) mole ratios ranging from 1 to 4. Since membrane cholesterol content is disturbed in the development of cataracts, it was hypothesized that perturbation of cholesterol domain structure occurs in cataracts. In this study, fiber cell plasma membranes were isolated from both normal (control) and cataractous lenses and assayed for cholesterol and phospholipid. Control and cataractous whole lens membranes had C/P mole ratios of 3.1 and 1.7, respectively. Small angle x-ray diffraction approaches were used to directly examine the structural organization of the cataractous lens plasma membrane versus control. Both normal and cataractous oriented membranes yielded meridional diffraction peaks corresponding to a unit cell periodicity of 34.0 A, consistent with the presence of immiscible cholesterol domains. However, comparison of diffraction patterns indicated that cataractous lens membranes contained more pronounced and better defined cholesterol domains than controls, over a broad range of temperature (5-40 degrees C) and relative humidity (52-92%) levels. In addition, diffraction analyses of the sterol-poor regions of cataractous membranes indicated increased membrane rigidity as compared with control membranes. Modification of the membrane lipid environment, such as by oxidative insult, is believed to be one potential mechanism for the formation of highly resolved cholesterol domains despite significantly reduced cholesterol content. The results of this x-ray diffraction study provide evidence for fundamental changes in the lens fiber cell plasma membrane structure in cataracts, including the presence of more prominent and highly ordered, immiscible cholesterol domains.     

Is the cytoskeleton-plasma membrane complex involved in lens protein biosynthesis?

Calf lens fiber cells contain a population of polyribosomes that direct, at leastin vitro, the synthesis of a specific plasma membrane protein MP26. This protein may serve as a marker in terminal differentiation, since it is absent in the lens epithelium but appears in lens fiber plasma membranes. The MP26 manufacturing polyribosomes are found to be associated with a structural complex in which also the cytoskeleton and plasma membranes participate. They can be released from the complex by treatment with DNAse I. This result presumably reflects the involvement of actin in the linkage of the MP26 synthesizing polyribosomes to the cytoskeleton-membrane complex.     

MAINTENANCE OF IONS,PROTEINS AND WATER IN LENS FIBER CELLS BEFORE AND AFTER TREATMENT WITH NON-IONIC DETERGENTS

If the plasma membrane and its associated transport proteins are solely responsible for maintenance of the asymmetric solute distribution then disruption of the plasma membrane would quickly lead to the symmetric distribution of all unattached inorganic ions between the cell and the extracellular environment. To test this hypothesis fresh pig lenses were incubated in Hanks ’ balanced salt solution in either absence or presence of non-ionic detergents (0.2 % Triton X-100 or 0.2 % Brij 58). Both detergents caused permeabilization of every lens fiber cell as shown by electron microscopy. The flux kinetics of K⁺, Mg^{2 +}, Na⁺, Ca^{2 +}, water and protein out of and into the permeabilized lens fiber cells was measured. Triton X-100 caused a faster flux rate of all solutes than did Brij 58. The Triton X-100 induced flux of solutes and water was associated with a decrease in lens ATP. Incubation of untreated lenses in solutions of different osmotic pressures at 0 °C demonstrated that the major fraction of lens water was osmotically unresponsive. Thus the asymmetric distribution of solutes in lens fiber cells is dependent on an intact plasma membrane and on a co-operative ATP-dependent association between K⁺, Mg^{2 +}, water and cytomatrix proteins.     

Fatty acid acylation of lens fiber plasma membrane proteins. MP26 and alpha-crystallin are palmitoylated

We describe in this report the fatty acylation of some of the main polypeptides from the eye lens fibers. MP26, the major lens fiber plasma membrane protein, and probably MP22, its natural degradation product, are palmitoylated in a post-translational process. This is also the case for alpha-crystallin, a major cytoplasmic structural protein shown to interact directly with the plasma membrane. Furthermore, a 65 kDa non-identified polypeptide and a high molecular weight component are also modified in the same way.     

Paralemnin of the lens

Paralemmin was identified in the chicken lens as a protein with mol. wt 65 kDa and a splice variant of 60 kDa, both soluble in Triton X-100. Paralemmin is localized to the plasma membrane of fiber cells, and was not detected in the annular pad cells. Thus in the chick lens it is another feature of fiber cell differentiation. Its localization to the short side of the fiber cell and the sites of fiber cell interlocking suggests that paralemmin may play a role in the development of such interdigitating processes.     

BASP1 in the lens

BASP1 was detected in the embryonic and adult chicken lens, using immunological methods and by peptide sequence analysis. The protein was predominantly expressed in fiber cells and only faintly detected in annular pad cells. Localization of the protein was along the plasma membrane of fiber cells often in discrete areas. The role of BASP1 in the lens requires further study.     

Molecular organization of protein-lipid components of the crystalline lens

Lens transparency is primarily a physical phenomenon and is a manifestation of the lens structural organization. Traditionally the lens is considered as a "sac filled with proteins uniformly". Such studies have described overall average properties of the lens but have dealt with neither structural nor functional inhomogeneities in the lens tissue. All morphological, biochemical and physiological processes of the lens are aimed at the maintenance of transparency and refractive index. Minimizing of the lens light scatter is created in the lens by the processes that organize regularity at two structural levels: the fiber cytoplasmic matrix (cytoskeleton and soluble protein) and the fiber cell plasma membrane. Biochemical fractions of the lens are considered that are responsible for the physical basis of lens transparency.     

Characterization of alpha-crystallin-plasma membrane binding

Alpha-crystallin, a large lenticular protein complex made up of two related subunits (alphaA- and alphaB-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract. To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and recombinant human alphaA- and alphaB-crystallins conjugated to a small fluorescent tag (Alexa350). Both alphaA and alphaB homopolymer complexes, as well as a reconstituted 3:1 heteromeric complex, bind to lens membranes in a specific, saturable, and partially irreversible manner that is sensitive to both time and temperature. The amount of alpha-crystallin that binds to the membrane increases under acidic pH conditions and upon removal of exposed intrinsic membrane protein domains but is not affected at high ionic strength, suggesting that alpha-crystallin binds to the fiber cell plasma membranes mainly through hydrophobic interactions. The binding capacity and affinity for the reconstituted 3:1 heteromeric complex were measured to be 3. 45 +/- 0.11 ng/microg of membrane and 4.57 +/- 0.50 x 10(-4) microg(-1) of membrane, respectively. The present membrane binding data support the hypothesis that the physical properties of a mixed alpha-crystallin complex may hold particular relevance for the function of alpha-crystallin within the lens.     

Changes in fibronectin staining in the human lens during ageing and cataractogenesis

The content and localization of fibronectin, an extracellular glycoprotein, in the serial sections of lenses of normal human donors and cataractous patients of different ages were determined by the indirect immunoperoxidase staining technique. This was followed by the evaluation with quantitative morphometric analysis. It was shown that fibronectin was present in the area of cell contacts as single deposits of faint orange-brown stained material in the lens samples of young donors. The fibronectin level was raised in lens sections from aged donors. Its accumulation was detected mostly within the spaces of the lens fiber cells. At different stages of cataractogenesis a dramatic decrease of the fibronectin content was detected in the lens sections obtained from patients of different ages. A new linear spectrophotometric technique was developed for evaluation of the lens transparency, to correlate the lens opacity with corresponding histological data obtained from the immunostaining technique. Morphological studies performed further suggested that the lens fiber cell plasma membrane structures were deteriorated. This was observed as changes of fibronectin staining in the lens sections at different periods of human ageing and cataract development. It is concluded that a decrease of fibronectin staining in the human lens is an indication for the structural damage of the lens fiber cell plasma membranes during ageing and cataractogenesis.     

ERM proteins of the lens

Ezrin and radixin and protein 4.1 were detected in the lens of the eye. These proteins were mainly present in the young elongating cortical fiber cells and localized to the plasma membranes. Moesin was not detected. Ezrin, radixin, and protein 4.1 provide another means whereby actin is linked to the plasma membrane in addition to the known adherens junctions in the lens.     

In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane

Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro- synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl- [35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine- labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.     

Characterization of lens fiber cell triton insoluble fraction reveals ERM (ezrin, radixin, moesin) proteins as major cytoskeletal-associated proteins

To understand lens fiber cell elongation- and differentiation-associated cytoskeletal remodeling, here we identified and characterized the major protein components of lens fiber cell Triton X-100 insoluble fraction by mass spectrometry and immunoblot analysis. This analysis identified spectrin, filensin, vimentin, tubulin, phakinin, and β-actin as major cytoskeletal proteins in the lens fibers. Importantly, ezrin, radixin, and moesin (ERM), heat-shock cognate protein 70, and β/γ-crystallins were identified as major cytoskeletal-associated proteins. ERM proteins were confirmed to exist as active phosphorylated forms that exhibited intense distribution in the organelle free-zone fibers. Furthermore, ERM protein phosphorylation was found to be dramatically reduced in Rho GTPase-targeted transgenic mouse lenses. These data identify the ERM proteins, which cross-link the plasma membrane and actin, as major and stable cytoskeletal-associated proteins in lens fibers, and indicate a potential role(s) for the ERMs in fiber cell actin cytoskeletal and membrane organization.     

Preparation, characterization, and localization of antisera against bovine MP26, an integral protein from lens fiber plasma membrane

Polyclonal antisera were prepared in rabbits using both native and chymotrypsin-digested bovine lens fiber plasma membranes. MP26, the principal protein of lens fiber plasma membranes, and CT20, a chymotryptic fragment of MP26, were isolated electrophoretically and used to purify anti-MP26 and anti-CT20 activity from the respective antisera by affinity chromatography. These affinity-purified antisera were characterized by immunoreplica. Immunofluorescence microscopy localized MP26 on sections of methacrylate-embedded lenses in the lens fiber plasma membranes, but not the lens epithelium. Immunocytochemistry of isolated native or chymotrypsin-digested lens fiber plasma membranes localized both the MP26 and the CT20 only in the nonjunctional plasma membranes, with no detectable activity in the lens fiber junctions themselves. Electron microscopy revealed a second set of pentalaminar profiles, thinner by 4 nm than the lens fiber junctions, which contained demonstrable anti-MP26 and anti-CT20 activity following immunocytochemistry. These results indicate either that MP26 is not a component of the lens fiber junctions, or that significant conformational changes accompany assembly of MP26 into lens fiber junctions, resulting in the masking of MP26 antigenic determinants.