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.     

Plakoglobin is a component of the filamentous subplasmalemmal coat of lens cells

The plasma membranes of the cells of the superficial layer of the eye lens and the lens fibres are in close intercellular contact, leaving an intermembrane space of approximately 20 nm or less throughout their entire length. This plasma membrane is underlaid by a filamentous, cytoplasmic web containing actin, proteins of the spectrin and band 4.1 families, alpha-actinin and vinculin. Using immunofluorescence microscopy and immunoblotting of gel electrophoretically separated proteins, we show that plakoglobin, the plaque protein common to desmosomal and nondesmosomal adhering junctions, is present in lens cells and is also a component of the subplasmalemmal coat of these cells. Plakoglobin also exists in the extended regions of intercellular contacts between cultured lenticular cells where it often colocalizes with vinculin but does not occur in other vinculin-rich plasma membrane regions such as the focal adhesions at the ventral cell surface. Plakoglobin associated with plasma membrane regions can also be identified in various other adhesive cultured cells, but it is not detected in cells and tissues that do not establish firm intercellular junctions such as erythrocytes, platelets, cultured myeloma cells and smooth muscle tissue. We conclude that plakoglobin occurs, at least in lens cells, throughout the entire subplasmalemmal coat, coexisting in this situation not only with vinculin but also with spectrin and 4.1 protein(s). This colocalization infers the presence of a distinct, complex type of membrane-skeleton assembly involving the actin filament-associated junctional plaque elements plakoglobin and vinculin together with actin-associated proteins of the spectrin and band 4.1 protein families.     

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.     

Square arrays and their role in ridge formation in human lens fibers

Square arrays in human lens fibers were studied with freeze-fracture and thin-section TEM. In superficial fibers a number of patches of square array particles in the P face and pits in the E face are found in the smooth membrane. In the deeper cortex and the nucleus, fiber cells have undulating membranes and many ridges. Numerous patches of the particles (P face) are distributed in the concave regions, and the pits (E face) in the convex areas of the bumpy membrane. In most ridges, patches of the particles occur at regular intervals in the "valley" portion, while the pits are on the "crest" portion of ridges. Also, continuous square arrays having the same "valley" location as the regularly arranged patches are found in areas with extensive ridge patterns. The overlapping of the outer portions of two adjacent square arrays is found on the sides between the "crest" and the "valley" of the ridges. Structurally, square arrays are located in a nonjunctional part of the membrane; in an orthogonal crystalline arrangement; and with a particle size of about 6 nm and center-center spacing about 6.4 nm. They are structurally different from gap junctions found in the lens fibers. Thin-section studies reveal two types of cellular contacts: thin pentalamellar structures (about 12-13 nm in overall thickness) associated with the ridge patterns are believed to be square arrays; thick heptalamellar structures (about 16-17 nm in overall thickness) with a narrow gap in between the two central laminae are believed to be gap junctions. This study strongly suggests that square arrays are specifically involved in ridge formation in human lens fibers.     

Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers

The purified major intrinsic protein of the lens fiber plasma membrane (MP26) reconstituted into liposomes favored membrane-to-membrane close contacts as visualized by freeze fracture and immunoelectron microscopy. Reconstituted apposed unilamellar vesicles formed pentalaminar profiles, and multilamellar liposomes showed regions of stacked bilayers. Immunogold labeling, using antibody directed against MP26, demonstrated that this polypeptide is present in regions of membrane-to-membrane close interaction. Fracture faces displayed both randomly distributed clusters of 8-nm polygonal intramembrane particles and membrane domains where a bidimensional lattice of repeating subunits was present. The structural pleomorphism which characterized the MP26-reconstituted proteoliposomes seems quite comparable to that visualized in natural fiber plasma membrane domains.     

Basement Membrane Formation in Transfilter Tooth Culture and Its Relation to Odontoblast Differentiation

The mesenchymal cells of the developing tooth differentiate into odontoblasts as a result of an epithelio-mesenchymal interaction. Odontoblast differentiation was studied in vitro by cultivating dental mesenchyme and epithelium with interposed filters. Separation of the two components by enzyme treatment resulted in removal of the basement membrane. When the epithelium was grown alone, or transfilter from killed lens capsule, the basement membrane was not restored. Transfilter cultivation with dental mesenchyme resulted in basement membrane formation, but only if the filter pores allowed penetration of cytoplasmic processes. Hence, a close association between the epithelial and the mesenchymal cells seems to be a prerequisite for the restoration of the basement membrane. Differentiation of odontoblasts took place only in explants in which a basement membrane was formed. Differentiation did not occur when contact of the mesenchymal cells with the basement membrane was prevented by small pore size filters. Further experiments demonstrating an intact basement membrane suggested that membrane contacts between the epithelial and the mesenchymal cells are not needed for odontoblast differentiation. Hence, we suggest that differentiation of odontoblasts is triggered via contact of the mesenchymal cells with the basement membrane.     

Proteome Remodeling of the Eye Lens at 50 Years Identified With Data-Independent Acquisition

The eye lens is responsible for focusing and transmitting light to the retina. The lens does this in the absence of organelles, yet maintains transparency for at least 5 decades before onset of age-related nuclear cataract (ARNC). It is hypothesized that oxidative stress contributes significantly to ARNC formation. It is in addition hypothesized that transparency is maintained by a microcirculation system that delivers antioxidants to the lens nucleus and exports small molecule waste. Common data-dependent acquisition methods are hindered by dynamic range of lens protein expression and provide limited context to age-related changes in the lens. In this study, we utilized data-independent acquisition mass spectrometry to analyze the urea-insoluble membrane protein fractions of 16 human lenses subdivided into three spatially distinct lens regions to characterize age-related changes, particularly concerning the lens microcirculation system and oxidative stress response. In this pilot cohort, we measured 4788 distinct protein groups, 46,681 peptides, and 7592 deamidated sequences, more than in any previous human lens data-dependent acquisition approach. Principally, we demonstrate that a significant proteome remodeling event occurs at approximately 50 years of age, resulting in metabolic preference for anaerobic glycolysis established with organelle degradation, decreased abundance of protein networks involved in calcium-dependent cell–cell contacts while retaining networks related to oxidative stress response. Furthermore, we identified multiple antioxidant transporter proteins not previously detected in the human lens and describe their spatiotemporal and age-related abundance changes. Finally, we demonstrate that aquaporin-5, among other proteins, is modified with age by post-translational modifications including deamidation and truncation. We suggest that the continued accumulation of each of these age-related outcomes in proteome remodeling contribute to decreased fiber cell permeability and result in ARNC formation.     

Effect of plasma membrane contacts between cells on uridine incorporation and RNA polymerase activity

The effect of lateral plasma membrane contacts between neighbouring cells on uridine incorporation and endogenous RNA polymerase activity has been studied in kidney epithelial cells growing on a solid substrate in culture. A gradual increase of intercellular membrane contacts was parallelled by a gradual decrease in the rate of uridine incorporation. In contrast, the endogenous RNA polymerase activity remained unaltered when cells established membrane contacts between each other. The results are interpreted as reflecting a decreased uptake of uridine through the plasma membrane rather than a decline in RNA synthesis in cells with intercellular membrane contacts.     

Caspase remodeling of the spectrin membrane skeleton during lens development and aging

Terminal differentiation of lens fiber cells resembles the apoptotic process in that organelles are lost, DNA is fragmented, and changes in membrane morphology occur. However, unlike classically apoptotic cells, which are disintegrated by membrane blebbing and vesiculation, aging lens fiber cells are compressed into the center of the lens, where they undergo cell-cell fusion and the formation of specialized membrane interdigitations. In classically apoptotic cells, caspase cleavage of the cytoskeletal protein alpha-spectrin to approximately 150-kDa fragments is believed to be important for membrane blebbing. We report that caspase(s) cleave alpha-spectrin to approximately 150-kDa fragments and beta-spectrin to approximately 120- and approximately 80-kDa fragments during late embryonic chick lens development. These fragments continue to accumulate with age so that in the oldest fiber cells of the adult lens, most, if not all, of the spectrin is cleaved to discrete fragments. Thus, unlike classical apoptosis, where caspase-cleaved spectrin is short lived, lens fiber cells contain spectrin fragments that appear to be stable for the lifetime of the organism. Moreover, fragmentation of spectrin results in reduced membrane association and thus may lead to permanent remodeling of the membrane skeleton. Partial and specific proteolysis of membrane skeleton components by caspases may be important for age-related membrane changes in the lens.     

Co-axial association of recombinant eye lens aquaporin-0 observed in loosely packed 3D crystals

Aquaporin-0 (AQP0) is the major membrane protein in vertebrate eye lenses. It has been proposed that AQP0 tetramers mediate contact between membranes of adjacent lens fiber cells, which would be consistent with the extraordinarily narrow inter-cellular spacing. We have obtained 3D crystals of recombinant bovine AQP0 that diffract to 7.0 A resolution. The crystal packing was determined by molecular replacement and shows that, within the cubic lattice, AQP0 tetramers are associated head-to-head along their 4-fold axes. Oligomeric states larger than the tetramer were also observed in solution by native gel electrophoresis and analytical ultracentrifugation methods. In the crystals, there are no direct contacts between octamers, and it can thus be inferred that crystalline order is mediated solely by the detergent belts surrounding the membrane protein. Across the tetramer-tetramer interface, extracellular loops A and C interdigitate at the center and the perimeter of the octamer, respectively. The octamer structure is compared with that of the recently determined structure of truncated ovine AQP0 derived from electron diffraction of 2D crystals. Intriguingly, also in these crystals, octamers are observed, but with significantly different relative tetramer-tetramer orientations. The interactions observed in the loosely packed 3D crystals reported here may in fact represent an in vivo association mode between AQP0 tetramers from juxtaposed membranes in the eye lens.     

A 135-kd membrane protein of intercellular adherens junctions.

We report here on a new 135-kd membrane protein which is specifically associated with intercellular adherens-type junctions. This surface component was identified by a monoclonal antibody, ID-7.2.3, raised against detergent-extracted components of membranes of chicken cardiac muscle rich in intercalated discs. The antibodies stain extensively adherens junctions in intact cardiac muscle and in lens, as well as in cultured cells derived from these tissues. In living cultured cells only very little immunolabelling was obtained with ID-7.2.3 antibodies, probably due to the limited accessibility of the antibodies to the intercellular gap. However, upon the removal of extracellular Ca2+ ions a dissociation of the junction occurred, leading to the rapid exposure of the 135-kd protein. Immunoelectron microscopic labelling of EGTA-treated, or detergent-permeabilized cells indicated that the antigen is found along the plasma membrane and highly enriched in contact areas. Double immunolabelling for both the 135-kd protein and vinculin pointed to the close association of the two in intercellular junctions and to the apparent absence of the former protein from the vinculin-rich focal contacts of cultured cells and from dense plaque of smooth muscle. Immunoblotting indicated that the 135-kd protein is present in many tissues but is particularly enriched in heart, lens and brain.     

Endoplasmic reticulum–plasma membrane contacts: Principals of phosphoinositide and calcium signaling

The endoplasmic reticulum (ER) forms an extensive network of membrane contact sites with intra-cellular organelles and the plasma membrane (PM). Interorganelle contacts have vital roles in membrane lipid and ion dynamics. In particular, ER–PM contacts are integral to numerous inter-cellular and intra-cellular signaling pathways including phosphoinositide lipid and calcium signaling, mechanotransduction, metabolic regulation, and cell stress responses. Accordingly, ER–PM contacts serve important signaling functions in excitable cells including neurons and muscle and endocrine cells. This review highlights recent advances in our understanding of the vital roles for ER–PM contacts in phosphoinositide and calcium signaling and how signaling pathways in turn regulate proteins that form and function at ER–PM contacts.     

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.     

Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells

Surface protrusions at the leading edge of a moving cell that make contact with the surrounding extracellular matrix (ECM) are its main motor for locomotion and invasion. Chicken embryonic fibroblasts transformed by Rous sarcoma virus (RSV-CEF) form specialized membrane rosette-shaped contact sites on planar substrata as shown by interference reflection microscopy (IRM). Such activity is lacking in normal cells. These rosette contacts are more labile than other adhesion sites, such as focal and close contacts. Ultrastructural studies demonstrate that rosettes are sites at which membrane protrusions from the ventral cell surface contact the substratum. These protrusions are filled with meshworks of microfilaments and contain the pp60src oncogene product, actin, vinculin, and alpha-actinin. However, unlike focal contacts, at the rosettes these proteins interact to extend a highly motile membrane. Rosettes have the biological activity of degrading ECM components, as demonstrated by (1) local degradation of fibronectin substrata at sites of rosette contacts, but not focal and close contacts; (2) localization of putative antiprotease antibody at sites of rosette contacts, but not at focal an close contacts; and (3) local disruption of fibronectin matrix at sites of protrusive activity seen by transmission electron microscopy (TEM). In addition, formation of the rosette contact is insensitive to the ionophore monensin, and to inhibitors of proteolytic enzymes, while local fibronectin degradation at rosette contacts is inhibited by inhibitors of metalloproteases, 1,10-phenanthroline and NP-20. I consider these membrane protrusions of the rosette contacts in RSV-transformed cells specialized structural entities--invadopodia--that are involved in the local degradation of the ECM.     

Are gap junction membrane plaques implicated in intercellular vesicle transfer?

Gap junction channels are concentrated in specialised plaques of plasma membrane where cells are in close apposition. In this communication evidence is provided showing that these specialised regions of membrane also provide a site for vesicular transfer between cells. Vesicle distribution in eye lenses was found to generally reflect the reported distribution of gap junction membrane plaques. In certain areas of the lens gap junction membrane plaques and vesicles could be seen to form combined, complex structures. Ultrastructure of the vesicle and gap junction membrane plaque complexes was consistent with the vesicles moving through membrane plaques from one lens fibre cell to the next. To investigate whether transport of substances was consistent with intercellular vesicle transfer, transport of various markers was investigated. Time course experiments showing the rate of uptake of various markers into the lens did not show dramatic differences for molecules smaller or larger then gap junction pores formed by connexons. While considered as a primary intercellular transport mechanism in the lens, connexon pores were not the sole agent mediating the observed transport. Other reported mechanisms of intercellular transport in the lens can only account for the movement of relatively small molecules. Vesicular transport may therefore be a major form of transport into the outer lens layers for larger molecules. Implicit in these observations is a new hypothesis for intercellular vesicle movement via gap junction membrane plaques. Intercellular vesicle movement could possibly provide a path for large molecules associated with intact vesicles to be transported into the eye lens tissue.     

Molecular heterogeneity of adherens junctions

We describe here the subcellular distributions of three junctional proteins in different adherens-type contacts. The proteins examined include vinculin, talin, and a recently described 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 10:2249-2260). Immunofluorescent localization of the three proteins indicated that while vinculin was ubiquitously present in all adherens junctions, the other two showed selective and mutually exclusive association with either cell-substrate or cell-cell adhesions. Talin was abundant in focal contacts and in dense plaques of smooth muscle, but was essentially absent from intercellular junctions such as intercalated disks or adherens junctions of lens fibers. The 135-kD protein, on the other hand, was present in the latter two loci and was apparently absent from membrane-bound plaques of gizzard or from focal contacts. Radioimmunoassay of tissue extracts and immunolabeling of cultured chick lens cells indicated that the selective presence of talin and of the 135-kD protein in different cell contacts is spatially regulated within individual cells. On the basis of these findings it was concluded that adherens junctions are molecularly heterogeneous and consist of at least two major subgroups. Contacts with noncellular substrates contain talin and vinculin but not the 135-kD protein, whereas their intercellular counterparts contain the latter two proteins and are devoid of talin. The significance of these results and their possible relationships to contact-induced regulation of cell behavior are discussed.     

A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity

Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin-mediated epithelial cell-cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell-cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors) did not inhibit cell-cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell-cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell-cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell-cell contacts and immediately initiate cell surface polarity.     

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.     

Immunological identification of types I and III collagen in bovine lens epithelium and its anterior lens capsule

To define the molecular structure of bovine lens epithelium and its anterior lens capsule, we investigated the composition of lens capsule basement membrane proteins. Immunofluorescence and immunogold techniques were used to demonstrate the presence of type I and type III collagen in the lens capsule and in primary explant epithelial cultures grown on protein-binding membranes. Immunofluorescence staining with specific antibodies indicated that type I and type III collagen were constituents of lens basement membrane. We observed that deposition of type III collagen was more than type I collagen. The synthesis of fibrillar collagen by lens epithelium and its deposition in the lens capsule was established by localization of fibrillar collagen by transmission immunoelectron microscopy. These results demonstrate for the first time that normal lens epithelium synthesize fibrillar collagen which is an intrinsic component of the anterior lens capsule basement membrane.     

Direct perturbation of lens membrane structure may contribute to cataracts caused by U18666A, an oxidosqualene cyclase inhibitor

Induction of cataracts in experimental animals is a common toxic feature of oxidosqualene cyclase (OSC) inhibitors. U18666A has been shown to produce irreversible lens damage within a few weeks of treatment. Drug actions, besides reducing the availability of cholesterol, could contribute to cataract formation. Cholesterol added to cultures of lens epithelial cells could only partially overcome the growth-inhibiting effects of U18666A. In view of this finding and the fact that U18666A and other OSC inhibitors are highly lipophilic cationic tertiary amines, we tested the hypothesis that the cataractogenic effect of U18666A is related to direct perturbation of lens membrane structure and function. Based on changes in the anisotropy of fluorescent probes, U18666A incorporated into bovine lens lipid model membranes increased membrane structural order and, using small-angle x-ray diffraction, U18666A was shown to intercalate into the lens lipid model membranes and produce a broad condensing effect on membrane structure. Also, exposure of cultured lens epithelial cells and intact rat lenses to U18666A induced apoptosis. Induction of apoptosis may begin by intercalation of U18666A into cell membranes. By increasing membrane structural order, U18666A may also increase light scatter, thus directly contributing to lens opacification.