The structural organization and protein composition of lens fiber junctions

The structural organization and protein composition of lens fiber junctions isolated from adult bovine and calf lenses were studied using combined electron microscopy, immunolocalization with monoclonal and polyclonal anti-MIP and anti-MP70 (two putative gap junction-forming proteins), and freeze-fracture and label-fracture methods. The major intrinsic protein of lens plasma membranes (MIP) was localized in single membranes and in an extensive network of junctions having flat and undulating surface topologies. In wavy junctions, polyclonal and monoclonal anti-MIPs labeled only the cytoplasmic surface of the convex membrane of the junction. Label-fracture experiments demonstrated that the convex membrane contained MIP arranged in tetragonal arrays 6-7 nm in unit cell dimension. The apposing concave membrane of the junction displayed fracture faces without intramembrane particles or pits. Therefore, wavy junctions are asymmetric structures composed of MIP crystals abutted against particle-free membranes. In thin junctions, anti-MIP labeled the cytoplasmic surfaces of both apposing membranes with varying degrees of asymmetry. In thin junctions, MIP was found organized in both small clusters and single membranes. These small clusters also abut against particle-free apposing membranes, probably in a staggered or checkerboard pattern. Thus, the structure of thin and wavy junctions differed only in the extent of crystallization of MIP, a property that can explain why this protein can produce two different antibody-labeling patterns. A conclusion of this study is that wavy and thin junctions do not contain coaxially aligned channels, and, in these junctions, MIP is unlikely to form gap junction-like channels. We suggest MIP may behave as an intercellular adhesion protein which can also act as a volume-regulating channel to collapse the lens extracellular space. Junctions constructed of MP70 have a wider overall thickness (18-20 nm) and are abundant in the cortical regions of the lens. A monoclonal antibody raised against this protein labeled these thicker junctions on the cytoplasmic surfaces of both apposing membranes. Thick junctions also contained isolated clusters of MIP inside the plaques of MP70. The role of thick junctions in lens physiology remains to be determined.     

Biochemical and genetic investigations on gap junctions from mammalian cells

Gap junction protein (26K) in mouse or rat liver has been studied using a rabbit antiserum directed against the sodium dodecylsulfate denatured 26K protein from mouse liver. The liver 26K protein has been localized in gap junction plaques of hepatic plasma membranes by immuno electron microscopy. Affinity purified anti-26K antiserum showed weak cross reactivity with mouse or bovine lens gap junction protein (MIP26). This result suggests some structural homology between the different gap junction proteins in liver and lens. After partial hepatectomy of young rats the liver 26K protein appears to be degraded and later resynthesized. A variant of established Chinese hamster fibroblastoid cells has been isolated and shown to be defective in metabolic cooperation via gap junctions.     

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.     

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.     

Biochemical and genetic investigations on gap junctions from mammalian cells

Gap junction protein (26K) in mouse or rat liver has been studied using a rabbit antiserum directed against the sodium dodecylsulfate denatured 26K protein from mouse liver. The liver 26K protein has been localized in gap junction plaques of hepatic plasma membranes by immuno electron microscopy. Affinity purified anti-26K antiserum showed weak cross reactivity with mouse or bovine lens gap junction protein (MIP26). This result suggests some structural homology between the different gap junction proteins in liver and lens. After partial hepatectomy of young rats the liver 26K protein appears to be degraded and later resynthesized. A variant of established Chinese hamster fibroblastoid cells has been isolated and shown to be defective in metabolic cooperation via gap junctions.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

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.     

Comparative analysis of the major polypeptides from liver gap junctions and lens fiber junctions

Gap junctions from rat liver and fiber junctions from bovine lens have similar septilaminar profiles when examined by thin-section electron microscopy and differ only slightly with respect to the packing of intramembrane particles in freeze-fracture images. These similarities have often led to lens fiber junctions being referred to as gap junctions. Junctions from both sources were isolated as enriched subcellular fractions and their major polypeptide components compared biochemically and immunochemically. The major liver gap junction polypeptide has an apparent molecular weight of 27,000, while a 25,000-dalton polypeptide is the major component of lens fiber junctions. The two polypeptides are not homologous when compared by partial peptide mapping in SDS. In addition, there is not detectable antigenic similarity between the two polypeptides by immunochemical criteria using antibodies to the 25,000-dalton lens fiber junction polypeptide. Thus, in spite of the ultrastructural similarities, the gap junction and the lens fiber junction are comprised of distinctly different polypeptides, suggesting that the lens fiber junction contains a unique gene product and potentially different physiological properties.     

The arthropod gap junction and pseudo-gap junction: Isolation and preliminary biochemical analysis

Summary The hepatopancreas of the crayfish, Procambarus clarkii, contains an unusual abundance of gap junctions, suggesting that this tissue might provide an ideal source from which to isolate the arthropod-type of gap junction. A membrane fraction obtained by subcellular fractionation of this organ contained smooth septate junctions, zonulae adhaerentes, gap junctions and pentalaminar membrane structures (pseudo-gap junctions) as determined by electron microscopy. A further enrichment of plasma membranes and gap junctions was achieved by the use of linear sucrose gradients and extraction with 5 mM NaOH. The enrichment of gap junctions correlated with the enrichment of a 31 Kd protein band on polyacrylamide gels. Extraction with 20 mM NaOH or 0.5% (w/v) Sarkosyl NL97 resulted in the disruption and/or solubilization of gap junctions. Negative staining revealed a uniform population of 9.6 nm diameter subunits within the gap junctions with an apparent sixfold symmetry. Using antisera to the major gap junctional protein of rat liver (32 Kd) and to the lens membrane protein (MP 26), we failed to detect any homologous antigenic components in the arthropod material by immunoblotting-enriched gap junction fractions or by immunofluorescence on tissue sections. The enrichment of another membrane structure (pseudo-gap junctions), closely resembling a gap junction, correlated with the enrichment of two protein bands, 17 and 16Kd, on polyacrylamide gels. These structures appeared to have originated from intracellular myelin-like figures in phagolysosomal structures. They could be distinguished from gap junctions on the basis of their thickness, detergent-alkali insolubility, and lack of association with other plasma membrane structures, such as the septate junction. Pseudo-gap junctions may be related to a class of pentalaminar contacts among membranes involved in intracellular fusion in many eukaryotic cell types. We conclude that pseudo-gap junctions and gap junctions are different cellular structures, and that gap junctions from this arthropod tissue are uniquely different from mammalian gap junctions of rat liver in their detergentalkali solubility, equilibrium density on sucrose gradients, and protein content (antigenic properties).     

The Role of MIP in Lens Fiber Cell Membrane Transport

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in Cat ^Fr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (p _H2O ) on the order of 1 μm/sec whereas normal fiber cell membrane p _H2O was 17 μm/sec frog, 32 μm/sec rabbit and 43 μm/sec mouse. Cat ^Fr mouse lens fiber cell p _H2O was reduced by 13 μm/sec for heterozygous and 30 μm/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the p _H2O conferred by MIP is not sensitive to Hg²⁺ whereas that of CHIP28 (AQP1) is blocked by Hg²⁺. The fiber cell membrane p _H2O was also not sensitive to Hg²⁺ whereas lens epithelial cell p _H2O (136 μm/sec in rabbit) was blocked by Hg²⁺. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous Cat ^Fr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses. Received: 17 February 1999/Revised: 16 April 1999     

The major polypeptide (MIP) of lens fiber junctions and its synthesis in cultured differentiating lens epithelial cells

Lens and liver contain many gap junctions, which for a long time have been considered to be very similar. Recent results, however, point to differences on morphological and biochemical levels, especially when the liver gap junction polypeptide (26,000 Daltons) is compared with the main intrinsic polypeptide (MIP) from lens junctions. The lens fiber specific MIP, which represents a marker molecule for lens cell differentiation could be detected by indirect immunofluorescence as well as by immunodiffusion in lens epithelial cells, which differentiated in vitro under distinct culture conditions. The fine structure of these differentiated cells is presented.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Structure and distribution of gap junctions in lens epithelium and fiber cells

Summary We report a comparative study of gap junctions in lens epithelia of frog, rabbit, rat and human, using a double mounting method for freeze-fracture electron microscopy. The gap junctions on the narrow sides of hexagonal cortical fiber cells of various species were also studied with the same technique. Gap junctions were commonly present between epithelial cells of the entire undifferentiated epithelium, between fiber cells on both wide and narrow sides, and between epithelial cells and fiber cells. Structural diversity of gap junctions, based on connexon arrangements, was evident in lens epithelia among the four species studied. Gap junctions with random arrays of connexons were found predominantly in frog lens epithelium, while the crystalline and striated configurations were mainly observed in the epithelia of human and rat, and of rabbit, respectively. On the other hand, there was no structural variation of gap junctions observed on either wide or narrow sides of lens fiber cells from any species studied. Only the random-type gap junction was found. However, the distribution of gap junctions was unique on the narrow sides. There was a single row of junctional plaques along the middle of the narrow sides, whereas the wide sides showed an uneven distribution pattern. The gap junctions between epithelial cells and fiber cells had a random packing of connexons.     

A lens intercellular junction protein, MP26, is a phosphoprotein

The major protein present in the plasma membrane of the bovine lens fiber cell (MP26), thought to be a component of intercellular junctions, was phosphorylated in an in vivo labeling procedure. After fragments of decapsulated fetal bovine lenses were incubated with [32P]orthophosphate, membranes were isolated and analyzed by SDS PAGE and autoradiography. A number of lens membrane proteins were routinely phosphorylated under these conditions. These proteins included species at Mr 17,000 and 26,000 as well as a series at both 34,000 and 55,000. The label at Mr 26,000 appeared to be associated with MP26, since (a) boiling the membrane sample in SDS led to both an aggregation of MP26 and a loss of label at Mr 26,000, (b) the label at 26,000 was resistant to both urea and nonionic detergents, and (c) two-dimensional gels showed that a phosphorylated Mr 24,000 fragment was derived from MP26 with V8 protease. Studies with proteases also provided for a localization of most label within approximately 20 to 40 residues from the COOH-terminus of MP26. Published work indicates that the phosphorylated portion of MP26 resides on the cytoplasmic side of the membrane, and that this region of MP26 contains a number of serine residues. The same region of MP26 was labeled when isolated lens membranes were reacted with a cAMP-dependent protein kinase prepared from the bovine lens. After the in vivo labeling of lens fragments, phosphoamino acid analysis of MP26 demonstrated primarily labeled serines, with 5-10% threonines and no tyrosines. Treatments that lowered the intracellular calcium levels in the in vivo system led to a selective reduction of MP26 phosphorylation. In addition, forskolin and cAMP stimulated the phosphorylation of MP26 and other proteins in concentrated lens homogenates. These findings are of interest because MP26 appears to serve as a protein of cell-to-cell channels in the lens, perhaps as a lens gap junction protein.     

Limited proteolysis of gap junction protein is intrinsic in mammalian 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.     

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.     

Primary Cultures of Embryonic Chick Lens Cells as a Model System to Study Lens Gap Junctions and Fiber Cell Differentiation

A major limitation in lens gap junction research has been the lack of experimentally tractable ex vivo systems to study the formation and regulation of fiber-type gap junctions. Although immortalized lens-derived cell lines are amenable to both gene transfection and siRNA-mediated knockdown, to our knowledge none are capable of undergoing appreciable epithelial-to-fiber differentiation. Lens central epithelial explants have the converse limitation. A key advance in the field was the development of a primary embryonic chick lens cell culture system by Drs. Sue Menko and Ross Johnson. Unlike central epithelial explants, these cultures also include cells from the peripheral (preequatorial and equatorial) epithelium, which is the most physiologically relevant population for the study of fiber-type gap junction formation. We have modified the Menko/Johnson system and refer to our cultures as dissociated cell-derived monolayer cultures (DCDMLs). We culture DCDMLs without serum to mimic the avascular lens environment and on laminin, the major matrix component of the lens capsule. Here, I review the features of the DCDML system and how we have used it to study lens gap junctions and fiber cell differentiation. Our results demonstrate the power of DCDMLs to generate new findings germane to the mammalian lens and how these cultures can be exploited to conduct experiments that would be impossible, prohibitively expensive and/or difficult to interpret using transgenic animals in vivo.     

NCAM in the differentiation of embryonic lens tissue

The role of the neural cell adhesion molecule (NCAM)2 in ocular lens differentiation was investigated in chicken embryos. Changes in expression of NCAM were documented by immunohistology of frozen sections. This analysis revealed that NCAM diminished during lens fiber differentiation, in contrast to the gap junction-associated protein MP26 which became more abundant. The form of NCAM expressed was determined by Western blot analysis of proteins extracted from the different regions of the Embryonic Day 6 lenses. All regions expressed NCAM with an apparent molecular weight of 140 kDa and relatively low levels of polysialylation. The function of NCAM in lens differentiation was investigated using antibodies that inhibit NCAM-mediated adhesion. Two parameters that change during maturation of the lens epithelial cells were monitored: the thickness of the tissue, indicating the length of lens cells, and the particle arrangement of gap junctions, reflecting the state of junctional differentiation. When epithelial cell explants of Embryonic Day 6 lenses were cultured for 5 days, the cells elongated and displayed an increase in the loose, random intramembranous particle arrangements characteristic of maturing lens fiber gap junctions. When the explants were cultured in the presence of anti-NCAM Fabs, the epithelia were thinner than in matched controls and had particle arrangements characteristic of a less mature state. The expression of NCAM during lens differentiation and the effects of attenuating NCAM function suggest that adhesion mediated by NCAM is an essential event in lens cell differentiation.     

On the structural organization of isolated bovine lens fiber junctions

Junctions between fiber cells of bovine lenses have been isolated in milligram quantities, without using detergents or proteases. The structure of the isolated junctions has been studied by thin-section, negative-stain, and freeze-fracture electron microscopy and by x-ray diffraction. The junctions are large and most often have an undulating surface topology as determined by thin sectioning and freeze-fracture. These undulations resemble the tongue-and-groove interdigitations between lens fiber cells previously seen by others (D. H. Dickson and G. W. Crock, 1972, Invest. Ophthalmol. 11:809-815). In sections, the isolated junctions display a pentalamellar structure approximately 13- 14 nm in overall thickness, which is significantly thinner than liver gap junctions. Each junctional membrane contains in the plane of the lipid bilayers distinct units arranged in a square lattice with a center-to-center spacing of 6.6 nm. Freeze-fracture replicas of the junctions fractured transversely show that the repeating units extend across the entire thickness of each membrane. Each unit is probably constructed from four identical subunits, with each subunit containing a protein of an apparent molecular weight of 27,000. We conclude that the lens junctions are structurally and chemically, different from gap junctions and could represent a new kind of intercellular contact, not simply another crystalline state of the gap junction protein.     

家兔晶状体纤维的超微结构:纤维细胞的间隙连接

本文报道晶状体纤维细胞间间隙连接的形态结构。我们利用冰冻断裂技术,在不同部位的球-和-凹连结的头部以及在纤维细胞和纤维细胞之间都观察到间隙连接的存在。通过极其丰富的上述连接,可实现细胞间代谢物和离子的传递。作者认为:对正常晶状体纤维细胞之间的间隙连接的深入了解,将会为晶状体发病机制的研究提供新的线索。     

MP26 in the bovine lens: a post-embedding immunocytochemical study

Gold immunolabeling of bovine lens tissue embedded in Lowicryl K4M, using a polyclonal antibody specific for a major component of lens fiber plasma membrane of 26 K molecular weight, shows that this constituent is absent from the epithelial cell plasma membrane and associated only with the junctional and non-junctional domains of the lens fiber plasma membrane.     

Reconstitution of native-type noncrystalline lens fiber gap junctions from isolated hemichannels

Gap junctions contain numerous channels that are clustered in apposed membrane patches of adjacent cells. These cell-to-cell channels are formed by pairing of two hemichannels or connexons, and are also referred to as connexon pairs. We have investigated various detergents for their ability to separately solubilize hemichannels or connexon pairs from isolated ovine lens fiber membranes. The solubilized preparations were reconstituted with lipids with the aim to reassemble native-type gap junctions and to provide a model system for the characterization of the molecular interactions involved in this process. While small gap junction structures were obtained under a variety of conditions, large native-type gap junctions were assembled using a novel two-step procedure: in the first step, hemichannels that had been solubilized with octylpolyoxyethylene formed connexon pairs by dialysis against n-decyl-beta-D-maltopyranoside. In the second step, connexon pairs were reconstituted with phosphatidylcholines by dialysis against buffer containing Mg2+. This way, double-layered gap junctions with diameter < or = 300 nm were obtained. Up to several hundred channels were packed in a noncrystalline arrangement, giving these reconstituted gap junctions an appearance that was indistinguishable from that of the gap junctions in the lens fiber membranes.