Ocular lens gap junctions: protein expression, assembly, and structure-function analysis.

Recent advances in understanding lens fiber gap junction formation are reviewed. These include studies of junctional protein expression in the embryonic lens, and of age related changes affecting gap junction structure and composition in the adult lens. An in vitro assembly system based on detergent solubilized pore complexes and endogenous lipids has been developed to provide information on the molecular interactions involved in gap junction formation and to provide material for structure analysis. Important information on the electrical properties of lens gap junction channels is obtained using electrophysiological techniques including planar lipid bilayer analysis and patch clamping.

Differential expression of two gap junction proteins in corneal epithelium.

Two distinct gap junction proteins (connexins) are expressed in rat corneal epithelium in a way which parallels cellular differentiation processes in this tissue. Connexin43 is restricted predominantly to the basal cells of the corneal epithelium and is present in significantly reduced amounts compared to the situation in the adjacent conjunctival epithelium. In contrast, a gap junction protein recognized by antibodies against MP70 which is the ovine homolog of mouse connexin50, is strongly expressed in the corneal epithelium and is present in the basal cells, wing cells and surface cells. While the functional significance of this differential expression of corneal epithelial connexins has yet to be established, the corneal epithelium is the third avascular tissue besides lens and heart valves which expresses a gap junction protein recognized by anti-MP70 antibodies.

Gap junction formation during development of the mouse lens.

We have identified a 60 kDa membrane protein (MP60) as a component of the mouse cortical lens fiber gap junction and a monoclonal antibody recognizing this protein has been used to establish the temporal and spatial patterns of gap junction formation during development of the mouse lens. The initial expression of MP60 during embryonic development of the mouse lens correlates with primary fiber elongation and is first seen on the luminal aspect of the extending cells. About 2 days after birth, the relatively large, antibody-positive macular structures characteristic of late embryonic fiber cells begin to disperse into progressively smaller structures within a centrally located region of the lens. This change in the staining pattern with antibody directed against MP60 is consistent with the dispersion of gap junction plaques as confirmed by freeze fracture analysis. Around 5 days after birth, the 60 kDa gap junctional protein in this central region of the lens undergoes a modification resulting in the alteration to the epitope for the monoclonal antibody and a consequent loss of immunorecognition. Our results suggest that gap junctions in the central region of the developing mouse lens undergo sequential changes in immunoreactivity which may reflect potentially distinct functional phases of intercellular communication.

An unusual symbiont from the gut of surgeonfishes may be the largest known prokaryote.

Symbionts first reported from the gut of a Red Sea surgeonfish, Acanthurus nigrofuscus (family Acanthuridae), were subsequently described as Epulopiscium fishelsoni. The taxonomic position of this very large (up to 576 microns in length) microorganism has previously been designated in the literature as either uncertain or eukaryotic. We suggest that similar symbionts from Great Barrier Reef surgeonfish may be prokaryotes, which together with E. fishelsoni from the Red Sea may represent the largest known forms of this cell type. Features identifying the symbionts as prokaryotes include the presence of bacterial-type flagella and a bacterial nucleoid and the absence of a nucleus or any other membrane-bound organelle.

Rotavirus spike structure and polypeptide composition.

Negatively stained preparations of rotavirus imaged with a low dose of electrons provide sufficient contrast to reveal surface projections or spikes. The number of spikes found projecting from different particles indicates that not all 60 peripentonal sites are occupied. Treatment at pH 11.2 with 250 mM ammonium hydroxide specifically removes the spikes, yielding smooth double-shelled particles of the same diameter as that of the native virus. Protein analysis confirms that the released spikes are composed of polypeptide VP4 (or its two cleavage products VP5* and VP8*) and that the smooth particle retains the other major outer shell protein VP7. Spikeless particles can be decorated by a monoclonal antibody specific for the major immunodominant neutralizing domain of VP7, implying that removal of the spikes does not denature the VP7 that is retained on the surface of the smooth particle.

Molecular portrait of lens gap junction protein MP70.

A 70-kDa membrane protein (MP70) is a component of the lens fiber gap junctions. Its membrane topology and its N-terminal sequence are similar to those of the connexin family of proteins. Some features of MP70 containing fiber gap junctions are, however, distinct from gap junctions in other mammalian tissues: (i) Lens connexons form crystalline arrays only after cleavage of junctional proteins in vitro. These hexagonal arrays have a periodicity of 13.6 nm which is significantly larger than the 8- 9-nm spacing of liver and heart gap junctions. (ii) Lens fiber gap junctions dissociate in low concentrations of nonionic detergent and this provides an avenue to purify MP70 directly from a membrane mixture. Isolated MP70 in the form of 17 S structures has an appearance consistent with connexon pairs. (iii) The C-terminal half of MP70 is cleaved in situ by a lens endogenous calcium-dependent protease. The processed from MP38 remains in the membrane and is abundant in the central region of the lens. A testable hypothesis for MP70 function is presented.

Structural and molecular biology of the eye lens membranes.

Lens transparency is associated with a unique design in tissue development and architecture. The fiber plasma membrane has domains which link with the cytoskeleton, thus maintaining cell shape. Other membrane regions form processes which interlock adjacent lens fibers, and intercellular junctions contain transmembrane pores which allow passage of metabolites between cells. Much interest has recently focused on the study of lens membrane structure and function, mainly because membrane dysfunction may be associated with cataract formation. This article reviews what is known about the structure of membrane domains, about the identification of domain-specific proteins, and describes current attempts to relate these results to function. Much of the presently available data is controversial, and an attempt will be made to reconcile them in revised models and testable hypotheses.

Dissociation of lens fibre gap junctions releases MP70.

MIP and MP70 are putative gap junction components in the plasma membranes of the mammalian lens fibre cells. We show now that MP70 can be solubilized separately from MIP in mild detergent solutions, and that this treatment results in the dissociation of the fibre gap junctions. Solubilized MP70 was isolated as 16.9 S particles by velocity gradient centrifugation and in the electron microscope had the appearance of short double-membrane structures consistent with connexon-pairs. These observations open a new experimental avenue in which to characterize separately the two putative lens gap junction proteins structurally and functionally.

The epithelial tight junction: structure, function and preliminary biochemical characterization.

The tight junction, or zonula occludens (ZO), forms a semi-permeable barrier in the paracellular pathway in most vertebrate epithelia. The ZO is the apical-most member of a series of intercellular junctions, collectively known as the junctional complex, found at the interface of the apical and lateral cell surface. This structure not only restricts movement of substances around the cells, but may also serve as a 'fence' acting to maintain the cell surface compositional polarity characteristic of epithelial cells. The morphology and physiology of the ZO have been well documented and are briefly reviewed here. The biochemistry of this important intercellular junction remains largely unknown, although a tight junction-specific polypeptide called 'ZO-1' has recently been identified. Preliminary observations regarding the role of this peripheral phosphoprotein in the biology of the ZO are presented.

Homologies between gap junction proteins in lens, heart and liver.

The cells in the mammalian lens are electrically and metabolically coupled with each other by a network of gap junctions. These are clusters of transmembrane channels by which the fibre cells situated deeper in the lens communicate through the epithelium with the aqueous humour, the source of nutrients for the lens. Hence gap junctions are important for lens transparency. The gap junction proteins in the mammalian lens have not yet been identified with certainty. A putative fibre gap junction protein of relative molecular mass 26,000 (26K) is not related to those from other tissues, such as the liver 28K junction component. Another lens membrane protein with Mr 70K (MP70) has also been localized in the lens fibre gap junctions. Here we demonstrate by amino-terminal sequence analysis that MP70 and its in vivo-processed form, MP38 (ref. 8), belong to a wider family of gap junction proteins. With this new data on the lens, homologies between gap junction proteins now extend to organs derived from all three embryonal layers, endoderm (liver), mesoderm (heart) and ectoderm (lens).

Protein processing in lens intercellular junctions: cleavage of MP70 to MP38.

Membrane protein MP70 is a component specifically of lens fiber 16-17 nm junctions. SDS-PAGE analysis of membrane preparations made separately from the sheep lens equatorial region (outer cortex), inner cortex and central region (nucleus) showed abundant MP70 in the young fibers in the outer cortex and drastically reduced levels of MP70 in the older fibers deeper in the lens. MP70 was cleaved to MP38 and thereby lost the epitope to monoclonal anti MP70 (6-4-B2-C6, Kistler et al J Cell Biol 101:28, 1985). This cleavage is fiber age-related and is effected by a calcium-dependent, lens-endogenous protease.

Formation, distribution and dissociation of intercellular junctions in the lens.

A 70,000 Mr membrane protein (MP70) has previously been identified as a specific component of lens intercellular junctions. In this paper we use anti-MP70 immunofluorescence microscopy of dissected fibre bundles to study the formation, distribution and dissociation of junctional plaques in the outer cortex region of the sheep lens. Abundant, small junctional plaques are assembled de novo in the broad sides of the elongating fibres near the equatorial lens periphery. In fully elongated, pole-to-pole fibres, junctional plaques are generally larger, and while dispersed on the broad sides of the fibres in the equatorial lens plane, these junctions line up in the middle of the broad and narrow sides of the fibres in the lens polar regions. This precisely defined positioning is independent of junction size and hence cannot solely be explained by the constraints of fibre width. Junctional plaques fragment to smaller sizes and MP70 is cleaved to MP38 in mature, enucleated fibres located in the deeper portions of the lens outer cortex. These results demonstrate a dynamic aspect of lens intercellular junctions and show that they are positioned in a precise fashion, possibly in association with other membrane or cytoskeletal components.

Frankia vesicles provide inducible and absolute oxygen protection for nitrogenase.

When Frankia HFPCcI3 was grown in culture at oxygen O(2) levels ranging from 2 to 70 kilopascals O(2), under nitrogen fixing conditions, nitrogenase activity adapted to ambient pO(2) and showed a marked optimum close to growth pO(2). Vesicles were thin walled at low pO(2) and very thick walled at high pO(2). Freeze fracture transmission electron microscopy confirmed that Frankia produces vesicles with outer walls thickened by multiple lipid-like monolayers, in proportion to ambient pO(2).

Immunolocalization of MP70 in lens fiber 16-17-nm intercellular junctions.

Thin section electron microscopy reveals two different types of membrane interactions between the fiber cells of bovine lens. Monoclonal antibodies against lens membrane protein MP70 (Kistler et al., 1985, J. Cell Biol., 101:28-35) bound exclusively to the 16-17-nm intercellular junctions. MP70 localization was most dramatic in the lens outer cortex and strongly reduced deeper in the lens. In contrast, the 12-nm double membrane structures and single membranes were consistently unlabeled. In freeze-fracture replicas with adherent cortical fiber membranes, MP70 was immunolocalized in the junctional plaques which closely resemble the gap junctions in other tissues. MP70 is thus likely to be associated with intercellular communication in the lens.

Membrane interlocking domains in the lens.

"Ball and socket"-like membrane processes interlock fiber cells in the sheep lens cortex, but appear reduced deeper in the lens. Wheat germ agglutinin (WGA) binds preferentially to these ball and socket structures, and more weakly to other membrane regions. On protein blots, 125I WGA binds to glycoproteins with 140,000 and 32,000 apparent molecular weight, the smaller protein also binding 125I fibronectin. In two animal cataract models, the intense WGA labeling of globular bodies replaces the spotty WGA staining pattern associated with the ball and sockets in the normal lens.

Aging of lens fibers. Mapping membrane proteins with monoclonal antibodies.

Lens fiber proteins with apparent molecular weights 57,000, 70,000, 82,000, and 100,000 were components in urea insoluble, membrane-rich fractions. Monoclonal antibodies against these proteins labeled membranes by immunofluorescence microscopy of sheep lens cryosections and are thus referred to as membrane proteins MP57, MP70, MP82, and MP100. MP70 has previously been localized in fiber junctional membranes (Kistler et al 1985, J Cell Biol 101:28-35). Using radioimmunoassays, the authors found a different membrane protein composition for the cortical and nuclear sheep lens regions. In addition, the membrane protein composition altered with the overall lens age. All the above membrane antigens were eventually cleaved by proteolysis in older fibers, and their degradation patterns could be grouped into distinct classes. The results are of basic importance for cataract research.

Identification of a 70,000-D protein in lens membrane junctional domains.

A 70,000-D membrane protein (MP70), which is restricted to the eye lens fibers and is present in immunologically homologous form in many vertebrate species, has been identified. By use of anti-MP70 monoclonal antibodies for immunofluorescence microscopy and electron microscopy, this polypeptide was localized in lens membrane junctional domains. Both immunofluorescence microscopy and SDS PAGE reveal an abundance of MP70 in the lens outer cortex that coincides with a high frequency of fiber gap junctions in the same region.