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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.

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Human amylin forms fibrillar amyloid between pancreatic islet cells in patients with non-insulin-dependent (type 2) diabetes mellitus. Fibrillar assemblies also form in vitro in aqueous solutions of synthetic human amylin. We now report on the structural polymorphism of these fibrils. The thinnest fibril, referred to as the protofibril, has an apparent width of 5 nm but is only rarely observed by itself. These protofibrils spontaneously assemble into higher order fibrillar structures with distinct morphologies. Prominent among these is an 8-nm fibril with a distinct 25-nm axial crossover repeat which is formed by left-handed coiling of two 5-nm protofibrils. Coiling of more than two 5-nm protofibrils results in cable-like structures of variable width depending on the number of protofibrils involved. Lateral (side-by-side) assembly of 5-nm protofibrils is also observed and produces ribbons which may contain two, three, four, or more protofibrils and occasionally large single-layered sheets. The mass-per-length (MPL) of the 5-nm protofibril is 10 kDa/nm. This has been established in two ways: first, the 8-nm fibril, which is formed by coiling two 5-nm protofibrils around each other, has an MPL of 20 kDa/nm. Second, higher order fibrils differ by increments of 10 kDa/nm. Hence, about 2.6 human amylin molecules (3904 Da) are packed in 1 nm of protofibril length. Similarities exist between amylin fibrils and those formed from other amyloid proteins, suggesting that the in vitro assembly of synthetic protein may serve as a useful model system in advancing our understanding of amyloid formation in disease.

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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.

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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.

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New immunolocalization data put the role of the lens MP26 (MIP) protein in a new perspective. During maturation of lens fibre cells, MIP is found to associate specifically with two structures, gap junctions and cell interlocking processes (known as ball and socket domains). It is significant that the zone in which these associations are most striking is discrete, coinciding with the zone of rapidly enlarging junctional plaques and newly forming ball and socket domains. Observation of domain-specific interactions of MIP with forming gap junctions and ball and socket domains suggests that MIP may be involved in the formation of close membrane appositions. Furthermore, previous ambiguities in the literature over the presence of MIP in gap junctions are clarified by the knowledge that, in situ, MIP associates strongly with gap junctions for only a brief period (with less than about 5% of all lens gap junctions at any one time) during the assembly of junctional plaques.

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MP17 is the second most abundant integral membrane protein in the mammalian lens. It has some common features with the major intrinsic polypeptide MIP26, but amino terminal sequencing shows that MP17 is a separate gene product. Both MP17 and MIP26 are abundant in isolated lens fibre membrane vesicles and are not detectable in the fibre gap junctions.

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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.

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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).

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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.

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