Fiber Cell Plasma Membranes Research Articles

Characterization of the ovine-lens plasma-membrane protein-kinase substrates.

The cAMP-dependent protein-kinase-catalyzed phosphorylation of the two major intrinsic lens fiber cell plasma membrane proteins, MP20 and MP26, is likely restricted to the inner cortical and nuclear regions of the lens in vivo. The ovine-lens-specific connexin, MP70, that has been identified as Cx50 in mice and Cx45.6 in the chick, is also a protein kinase substrate although it does not appear to be phosphorylated by a number of protein kinases including cAMP-dependent protein kinase, calmodulin-dependent protein kinase or protein kinase C. Rather, an extrinsic lens membrane fraction was isolated which contained protein kinase activity that catalyzed the phosphorylation of MP70; this protein kinase activity was cAMP-independent, Ca(2+)-independent, Mg(2+)-dependent, phosphorylated MP70 on a serine residue(s) and migrated with a molecular mass of 35 kDa on a gel filtration column. Both MP70 phosphorylation and the endogenous protein kinase activity were restricted to the lens outer cortical region. This membrane-associated protein kinase activity represents the first reported partial characterization of an endogenous lens fiber cell protein kinase activity that catalyzes the phosphorylation of a lens connexin protein. The phosphatase-induced shift in the electrophoretic mobility of MP70 is not reversed by this protein kinase, indicating that MP70 is likely phosphorylated on different residues by two or more protein kinases.

Protein associated with human lens ‘native’ membrane during aging and cataract formation

Plasma membrane contains extrinsic as well as intrinsic proteins. Changes in the extrinsic proteins of lens membrane during human aging and cataract formation have not been investigated in detail. Unlike previous studies which examined lens membrane after being stripped of extrinsic proteins by treatment with chaotropic agents, we have isolated whole or 'native' lens membrane on a sucrose gradient by ultracentrifugation of the total water-insoluble protein. Essentially all of the water-insoluble protein from young to aged to cataractous human lens appeared membrane associated. In young lens (20-37 years old), most of the membrane banded at the 25/45% sucrose interface fraction. This fraction contained relatively little urea-soluble protein and likely represents fiber-cell plasma membrane with its physiologically associated extrinsic and intrinsic proteins. With aging (62-80 years old), about one-third of the membrane, as judged by the distribution of cholesterol, banded at a much higher density (50/58% sucrose fraction). The higher density was due to a great increase in the membrane's relative protein content (protein/cholesterol). Although this extra protein was composed of both urea-insoluble and -soluble fractions, the urea-soluble protein predominated in all lenses. Cataractous lens differed from aged-clear lens in that much more of the total membrane (70-75%) had shifted to the high density and participated in this massive binding of cytosolic proteins. Although alpha-crystallin was the principal extrinsic-membrane protein in young lens, high molecular weight aggregate of modified (acidic) crystallins accounted for the increased extrinsic protein in aging. The extrinsic proteins bound to both clear-aged and cataractous lens membrane were aggregated. In conclusion, examination of human lens native membrane fractions revealed that the association of crystallins with membrane in both aging and cataracts was much greater than previously recognized and most of this increased protein was non-covalently bound to the membrane. Much more of the lens total membrane from cataractous than clear-aged lens was involved in this massive protein association and the protein bound to cataract membrane appeared more highly aggregated.

Identification and characterization of tropomodulin and tropomyosin in the adult rat lens.

The lens fiber cells express all the major components of the erythrocyte membrane skeleton including spectrin, protein 4.1 and ankyrin. We have used immunoblot and immunoprecipitation analyses, as well as immunofluorescence localization to identify and characterize two additional components of the membrane skeleton in the rat lens: tropomyosin and the tropomyosin-binding protein tropomodulin. In the erythrocyte, tropomyosin and tropomodulin are proposed to stabilize and limit the lengths of the short actin filaments of the spectrin-actin network, thus influencing the organization and mechanical properties of the erythrocyte membrane skeleton. Antibodies directed against erythrocyte tropomodulin specifically recognize a M(r) 43,000 polypeptide from rat lens that comigrates with erythrocyte tropomodulin on SDS-gels. A non-muscle isoform of tropomyosin is also present in the lens. This tropomyosin isoform migrates on SDS-gels with a M(r) of approximately 28,000 and is distinct from the two erythrocyte isoforms of tropomyosin (M(r) 27,000 and 29,000). Indirect immunofluorescence staining of 5 microns cryosections of adult rat lens reveals that both tropomodulin and tropomyosin colocalize with rhodamine phalloidin staining for actin filaments on fiber cell plasma membranes. Lens tropomodulin exhibits many characteristics that are similar to its erythrocyte counterpart. For example, lens tropomodulin binds tropomyosin in a solid-phase blot binding assay, and extraction experiments with Triton X-100, urea and NaOH show that the membrane-bound tropomodulin in the lens is a tightly associated peripheral membrane protein that is a component of the Triton-insoluble cytoskeleton. However, unlike the erythrocyte, there are approximately 2000 actin monomers per tropomodulin in the lens.(ABSTRACT TRUNCATED AT 250 WORDS)

A 28 kDa sarcolemmal antigen in kidney principal cell basolateral membranes: relationship to orthogonal arrays and MIP26.

Two recently cloned water channels, CHIP28 and WCH-CD, are homologous to MIP26, an integral membrane channel-forming protein found in lens fiber plasma membranes. CHIP28 is found in basolateral and apical plasma membranes of kidney proximal tubules and thin descending limbs of Henle, whereas WCH-CD is apically located in collecting duct principal cells. So far, the putative water channel that may be responsible for the high constitutive permeability of principal cell basolateral membranes has not been identified. Interestingly, freeze-fracture electron microscopy has shown that characteristic orthogonal arrays of intramembrane particles (OAPs) are found on the basolateral plasma membranes of collecting duct principal cells, and that morphologically identical OAPs present in lens fiber cell plasma membranes contain the protein MIP26. Similar OAPs have also been detected on plasma membranes of other cell types including gastric parietal cells, astroglial cells and skeletal muscle fibers. By indirect immunofluorescence, western blotting and northern blotting, MIP26 was found only in lens fibers. In addition, functional studies on reconstituted and oocyte-expressed MIP26 excluded the possibility that MIP26 might be a basolateral water channel in the kidney. However, a polyclonal antibody raised against skeletal muscle sarcolemmal vesicles, which are enriched in OAPs, produced an intense staining of principal cell basolateral plasma membranes in kidney collecting duct and immunoprecipitated a 28 kDa protein from kidney papilla. The immunoprecipitated protein from papilla was not recognized by anti-CHIP28 or anti-MIP26 antibodies, indicating that principal cell basolateral membranes contain a novel member of the CHIP/MIP family. Because this antibody also stained brain astrocyte end feet, which are enriched in OAPs, it is possible that the 28 kDa protein is related to these structures. We conclude that OAPs probably contain related but distinct proteins that may have different membrane channel functions in different cell types.

Isolation of a Non-sedimenting Membrane Fraction from the Water Soluble Fraction of Bovine Lens

A membrane fraction was isolated from the water soluble fraction of bovine lens by increasing the density of the water soluble fraction with KBr and subjecting it to overnight centrifugation at 100 000 g. We have called this fraction, which floats to the top of the mixture upon centrifugation, the non-sedimenting membrane fraction (NSMF). Electron microscopy of the NSMF revealed that it is composed of the expected membrane structures of unit membrane, fiber junction and cytoskeleton. Significantly less of the total membrane of the NSMF was devoted to fiber junction (22·8%) than in the sedimenting membrane fraction (SMF) (41·1%) prepared by sucrose density centrifugation of the water insoluble fraction. The NSMF accounted for about 7-12% of the total bovine lens membrane, and preliminary experiments demonstrated that a similar fraction could be isolated from the water soluble fraction of lenses from rats, rabbits, chickens and humans. The NSMF contained about 0·9 mg total lipid per mg total membrane protein, which was significantly greater than the value obtained for the SMF (0·5 mg total lipid per mg total membrane protein). The greater relative amount of total lipid in the NSMF was due to a significantly greater relative amount of phospholipid in the NSMF which was further reflected by the observation that the cholesterol:phospholipid molar ratio of the NSMF (0·58) was significantly less than that of the SMF (0·88). Thus the relative lipid composition of the NSMF was significantly different than that of the SMF. Although the phospholipid content of the NSMF was greater than that of the SMF, the compositions of the phospholipids in the two membrane fractions were similar. The NSMF possessed essentially the same polypeptides (both extrinsic and intrinsic) which were found in the SMF. The NSMF was found to be distributed throughout the lens in a proportionate manner. We conclude that the NSMF may account for most of the lipid which remains in the water soluble fraction of the normal bovine lens after sedimentation of the water insoluble fraction. This membrane fraction substantially differs from the SMF in terms of structure and relative lipid composition. We speculate that the NSMF may represent a specialized domain of the fiber cell plasma membrane which has been previously unrecognized.

ESR spin label and ultrastructural monotoring of protein-lipid interactions in the lends fiber-cell plasma membranes in relation to human ageing and cataractogenesis

The Electron Spin Resonance (ESR) technique and the protein spin labels 2,2,3,4,9-pentamethyl-1,2,3,4-tetrahydro-γ-carboline-3-oxyl and 4-( N-maleimido)-2,2,6,6,-tetramethylpiperidine-1-oxyl were used in this study to probe the fluidity and binding ability to protein functional groups in human lens membranes. The image and the stage of cataract as well as the ultrastructural characteristics of the lens fiber cell membranes were evaluated in parallel studies. ESR measurements in membrane structures of the transparent lenses of different ages have shown that the sorbtion parameter of the carboline label on the surface of protein-lipid components was usually weakly expressed but increased with aging and the extent of immobilization of the bound label was not significant. At different stages of the lens opacification the carboline analogue spin label increasingly bound to the lens membranous structures. A spin label signal can be enhanced by addition to the samples of the paramagnetic probe K 3Fe(CN) 6. The maleimide spin label bound to the protein SH-groups in the cataractous lens membranes at a slow rate, however in transparent lenses a gradual increase of the rapidly binding phase of this label could be detected. The results show an appearance of at least two types of the reactive SH-groups in membranes of human transparent lenses. The electron microscopic studies suggested that the age-related and cataractogenic changes in the lens matter are accompanied by deterioration of the lenticular fiber plasma membranes and formation of the coalescing globules with a diameter of 220–500 nm. At the stage of cataract with advanced opacities, which is biochemically characterized by the increase in number of the carboline label binding sites with the surface proteins and annular lipids of membranes, a mass of amorphous aggregates filled with the electron-grey debris is formed contributing to significant scatter of light. It appears, therefore, that the suggested ESR spin label technique can be effectively used to monitor the aggregation process of protein membrane components which takes place during human cataractogenesis.

Selective association of crystallins with lens 'native' membrane during dynamic cataractogenesis

Plasma membrane with its associated extrinsic proteins was isolated from normal and cataractous rat lenses by centrifugation of the total water insoluble fraction from homogenized lenses on a discontinuous sucrose gradient. Membrane, which we call "native" membrane, was recovered mainly from the 25/45% sucrose interface. Development of the experimental U18666A cataract resulted in plasma membrane shifting to higher density (the 50/55% sucrose fraction) and great increases in the urea soluble protein content of the lens. At early stages of cataract development, most of the increased urea soluble protein was membrane associated, presumably as extrinsic protein. With advancing cataract, most of the urea soluble protein appeared in an essentially membrane-free pellet fraction. The urea soluble protein associated with the cataract membrane was shown by combined IEF, SDS-PAGE, Western blotting, amino acid compositional analysis and protein sequence determinations to be mainly composed of modified alpha- and beta-crystallins. Alpha A-crystallin truncated by not more than 27 residues from the carboxyl terminus plus beta b1 crystallin truncated by 49 residues from the amino terminus were conclusively identified. In addition to beta b1, a population of six alpha-crystallin derived polypeptides were specifically enriched in the cataract membrane fraction. Four of these six alpha-crystallins appear to be truncated from their carboxyl terminus, a modification which should have increased their hydrophobicity. The pellet fraction, which accumulated in the lens nucleus as the cataract advanced, was enriched in urea soluble gamma-crystallin derived polypeptides. We suggest that protein insolubilization in this experimental cataract involves the selective and tight association of principally modified alpha-crystallins to the fiber cell plasma membrane.

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 caractous 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 capacity 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 of 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.

Ankyrin of the Ocular Lens

Ankyrin was identified in the human, bovine and chicken lens as a protein of molecular weight 216 kilodaltons. It is specifically extracted from association with the fiber cell plasma membranes by high-ionic-strength salt solution and is predominantly found in young fiber cells. A protein which is unrelated to plakoglobin, protein 4.1 or ankyrin is also specifically extracted by high-salt solution.

Structural Organization of the Lens Fiber Cell Plasma Membrane Protein MP18

The 18,000-dalton bovine lens fiber cell intrinsic membrane protein MP18 was phosphorylated on a serine residue by both cAMP-dependent protein kinase and protein kinase C. In addition, this protein bound calmodulin and was recognized by a monoclonal antibody (2D10). These different regions were localized using enzymatic and chemical fragmentation of electrophoretically purified MP18 that had been phosphorylated with either cAMP-dependent protein kinase or protein kinase C. Partial digestion of 32P-labeled MP18 with protease V8 resulted in a Mr = 17,000 peptide that bound calmodulin, but neither contained 32P or was recognized by the monoclonal antibody 2D10. Furthermore, the 17-kDa peptide had the same N-terminal amino acid sequence as MP18. Thus, the monoclonal antibody 2D10 recognition site and the protein kinase phosphorylation site(s) are close together and confined to a small region in the C terminus of MP18. This conclusion was confirmed in experiments where MP18 was fragmented with trypsin, endoproteinase Lys-C, or CNBr. The location of the phosphorylation site was confirmed by sequencing the small 32P-labeled, C-terminal peptide that resulted from protease V8 digestion of 32P-labeled MP18. This peptide contained a consensus sequence for cAMP-dependent protein kinase.

Fluorescence studies on the age related changes in bovine and human lens membrane structure.

Bovine and human lens fiber cell plasma membranes were isolated as the urea-insoluble fraction for a study of age related changes. The changes in fluorescence intensity, both intrinsic (tryptophan) and extrinsic probes ANS (1-anilinonaphthalene-8-sulfonic acid) and DPH (1,6-diphenyl 1,3,5-hexatriene), as well as DPH anisotropy and lifetime were measured. The results of tryptophan fluorescence indicate that tryptophan residues in membrane proteins are in a very hydrophobic environment and do not show a change with aging. ANS reacts with surface protein in the polar-apolar interface, while DPH penetrates into the interior of membranes. Both probes show a decrease in fluorescence intensity in the old membranes. The decrease in ANS fluorescence may result from the conversion of MP26 to MP22, while the decrease of DPH fluorescence intensity may indicate a decrease in accessibility of lipid to DPH. To further delineate the change, fluorescence anisotropy and lifetime data of the lipid probe DPH were obtained. While lifetimes do not change with age, anisotropy shows a definite age-dependent increase. Anisotropy is related to the degree of lipid structural order. Greater anisotropy values were found for older membrane samples, indicating an increased rigidity with age, which may be partially caused by the increased cholesterol/phospholipid (C/P) ratio as reported in the literature.

Immunochemical characterization of a Mr 115 lens fiber cell-specific extrinsic membrane protein.

Monoclonal and polyclonal antibodies have been produced against a lens fiber cell extrinsic membrane protein, with a relative molecular weight of approximately 115 kd. Enzyme Linked Immunosorbent Assays (ELISA) of retina, ciliary body-iris, liver, and skeletal muscle, utilizing these antibodies, suggest that the antigen is unique to the lens. Immunocytochemistry indicates that the antigen is present only in the differentiated fiber cell, and is absent from the lens epithelium. Further, immunocytochemical reactivity is predominantly associated with the fiber cell plasma membrane. However, sequential extraction of fiber cell homogenate, followed by quantitative, competitive ELISA analysis, indicates that most of the antigen is recovered in the neutral buffer extract. ELISA analysis using monoclonal antibodies indicates that an analogous antigen is present in human and rabbit lenses. On the basis of these results we characterize this antigen as a conserved extrinsic membrane protein, which is unique to the differentiated lens fiber cell. The relationship of this antigen to a previously described Mr 95 beaded filament-associated protein is discussed.

Limited proteolysis of gap junction protein is intrinsic in mammlian lens fiber-cell plasma membranes

We demonstrate that the limited proteolysis of the lens fiber-cell gap junction protein, MP26, is intrinsic in mammalian lens fiber plasma membranes. Incubations of isolated intact bovine lens fiber plasma membranes in buffer alone did not elicit proteolysis of MP26. Incubations in the buffer with detergent, however, resulted in the limited proteolysis of MP26 which was totally inhibited by calcium chelators, thiol-alkylating agents, and protease inhibitors. As the limited proteolysis required the presence of detergent, it must depend on an enzymatic activity intrinsic in the lens fiber plasma membranes or in MP26 itself.

Structural studies of lens fiber junction protein MP26 by cyanogen bromide cleavage

The lens fiber-cell plasma membrane MP26 from chick, bovine, and human lenses yielded identical cyanogen bromide peptide maps, confirming the essential conservation of structure in the junction protein of vertebrate lens fiber cells. Immunoblot analyses of the cyanogen bromide peptide maps of human lens MP26 and of its age-dependent proteolytic product MP22 confirmed that MP22 is a derivative of MP26. The findings in this study are the first consistent with the positioning of the methionine residues in lens MP26 as predicted by its cDNA-derived sequence.

The distribution of the main intrinsic membrane polypeptide in ocular lens.

The Main Intrinsic Polypeptide (MIP) of the ocular lens fiber cell plasma membrane was immunocytochemically localized at the ultrastructural level on ultrathin frozen sections of rat lens, and on extracted, gradient-purified bovine lens membranes. The results indicate that both the junctional and non-junctional membrane domains of the cortical lens fiber cell are MIP immunoreactive. Frozen thin section immunocytochemistry of the lens epithelium and hepatocytes, also using anti-MIP antibodies, revealed that these cells, and their intercellular junctions, are not MIP-immunoreactive. From these findings we conclude that 1) MIP, a putative fiber cell junctional protein, is present throughout the plasma membrane of the lens fiber cell, and is not confined to the fiber cell junctional domain, 2) MIP is not a detectable component of the lens epithelial cell membrane, or its intercellular junctions, 3) MIP is not detectable in gap junctions of hepatocytes.

Total poly(A+)-messenger RNA from bovine lens cofractionates with sucrose purified fiber cell plasma membrane

Poly(A+)-messenger RNA was isolated from bovine lens as well as poly(A+)-mRNA from crude and sucrose-purified plasma membrane. Bovine lens MP26 antisera was also propagated in rabbits, from which anti-MP26 IgG was partially purified and used to assay for the specific synthesis of MP26 during in vitro translation of the isolated poly(A+)-mRNAs. We found that membrane-associated poly(A+)-mRNA supported the synthesis of proteins which were identical to those translated from total lens fiber cell poly(A+)-mRNA. These proteins included MP26, as assayed by immunoprecipitation of [ 35S]-labeled MP26.

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.

δ-Crystallin is a chick lens fiber cell membrane extrinsic protein

In an attempt to resolve the present controversy concerning the reported presence of δ-crystallin in preparations of chick lens plasma membranes, a study was conducted on the extractability of chick lens fiber cell plasma membranes in the presence or absence of calcium ions. One lens from each animal was homogenized in calcium-containing buffer and the other in (calcium) chelating buffer. Each resulting water insoluble crude cell membrane fraction was evenly divided and membranes isolated from each portion by treatment with urea in either calcium-containing buffer or chelating buffer. The isolated membranes were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Gels of membranes isolated in the presence of calcium showed very prominent δ-crystallin bands while gels of membranes isolated under chelating conditions showed a marked depletion of δ-crystallin. The treatment sequence employed ruled out the possibility that the observed binding of δ-crystallin to the membranes was a result of a spurious association caused by calcium because excess soluble δ-crystallin was removed in discarded chelating buffer washes prior to the isolation of the membranes with urea in chelating buffer. The results of the study showed that δ-crystallin is an extrinsic protein of the chick lens fiber cell plasma membranes, and that its linkage to the membranes is calcium-dependent.

Immunocytochemical localization of the lens main intrinsic polypeptide (MIP26) in communicating junctions.

Plasma membranes of vertebrate lens fiber cells contain a major intrinsic polypeptide with an apparent molecular weight of 26,000 (MIP26). These plasma membranes are extremely rich in communicating junctions, and it has been suggested that MIP26 is a component of them. MIP26 was purified from cow lenses using preparative SDS gel electrophoresis followed by hydroxylapatite column chromatography. From gel electrophoresis patterns and aggregational properties it was concluded that the MIP26 preparation was homogeneous. The purified MIP26 was used to produce monospecific antibodies in rabbits as assessed by double immunodiffusion and crossed immunoelectrophoresis of purified MIP26 and solubilized lens plasma membranes against the antiserum. Indirect immunocytochemical studies were performed on open and closed lens plasma membrane vesicles by incubation in anti-MIP antiserum followed by ferritin-conjugated goat antirabbit IgG. The conjugate bound unequivocally to lens communicating junctions, indicating that MIP26 is a component of these structures.

Biochemical and structural features of chick lens gap junctions

Deoxycholate treatment of isolated chick lens fiber cell plasma membranes yields a fraction rich in gap junctions. Analysis by SDS-PAGE revealed the preponderance of a 26k dalton (MP 26k) component and a minor 43k dalton (MP 43k) component in the membranes with further specific enrichment of the MP 26k component in the gap junction rich fraction. Analysis of the isolated chick lens gap junctions by freeze-fracture and negative staining revealed aggregates of 9·0 nm intramembrane particles (connexons) arranged in a non-crystalline pleomorphic pattern. The results imply that MP 26k is the principal component of lens fiber cell gap junction connexons.