Change of form of septate and gap junctions during development of the insect midgut

In insects, smooth septate junctions join cells derived from the embryonic midgut, and pleated septate junctions are found in all other tissues. Relatively little is known about either type of septate junction or the relationship between them, but they have been treated as two different junctions in the literature. The gap junctions which are associated with these septate junctions also differ. Crystalline gap junctions are found in the midgut, associated with smooth septate junctions, and irregular gap junctions are found in tissues where pleated septate junctions are located. We have examined the development of smooth septate junctions and crystalline gap junctions and the relationship between them, by studying the embryogenesis of the midgut in Manduca sexta (tobacco hornworm). At 56 hr of development (hatching is at 104 hr) pleated septate junctions and irregular gap junctions joined the midgut epithelial cells. At 65 hr, the septate junctions had disappeared, but gap junctions persisted. At 70 hr, smooth septate junctions had replaced the earlier pleated septate junctions and gap junctions associated with these smooth septate junctions were often of the crystalline form. In later embryos, the smooth septate junctions matured and enlarged, while all gap junctions became crystalline in form.     

肌细胞发育过程中间隙连接的变化

The appearance and changes of gap junctions during the development of striated muscle cells of Cynops orientalis have been studied by paraffin section, ultrathin-section and freeze-etching techniques. Gap junctions first appear between the somitic mesoderm cells in the late gastrula. A marked increase of the number of gap junctions occurs at the end of gastrulation. The number of gap junctions remains at a high level from neural plate stage up to nasal pit stage. After the stage of muscle contraction, the number of gap junctions decreases. Gap junctions do not disappear until muscle cells have attained their final differentiation and neuromuscular junctions have fully developed. The changes of size of gap junctions parallel with the changes in number. In addition, the number and size of gap junctions are both at the high level before cell fusion. Therefore, it is possible to conclude that cell communication is closely correlated with the development of striated muscle cells. The role of communications in cell determination and differentiation and in cell fusion of muscle cells are discussed.     

Evidence for high molecular weight proteins in arthropod gap and smooth septate junctions

The proteins that make up arthropod gap and septate junctions have not been identified with any certainty. Several candidate proteins for both types of junctions have been proposed in the literature, but there has been no agreement on any of these. Arthropod gap junctions do not label with antibodies to vertebrate gap junction connexins; it thus appears that unrelated proteins form these rather similar structures. Gap junctions inManduca sextamidgut epithelium are unusual since they function only during the molt and are non-functioning during the larval instars. We have developed a preparation from this tissue that is highly enriched in both gap and smooth septate junctions when examined by electron microscopy. SDS-PAGE gels of this preparation have two major protein bands, at 75 and 90 kDa. The presence of gap junctions correlates best with the 75 kDa protein and smooth septate junctions with the 90 kDa protein. Further, the 75 kDa band is stained by an antibody to a putative gap junction protein fromC. elegans. We propose that the 75 kDa protein is a major structural component of gap junctions inManduca sextamidgut epithelium and that the 90 kDa protein forms the smooth septate junctions.     

Increasing Gap Junctional Coupling: A Tool for Dissecting the Role of Gap Junctions

Much of our current knowledge about the physiological and pathophysiological role of gap junctions is based on experiments where coupling has been reduced by either chemical agents or genetic modification. This has brought evidence that gap junctions are important in many physiological processes. In a number of cases, gap junctions have been implicated in the initiation and progress of disease, and experimental uncoupling has been used to investigate the exact role of coupling. The inverse approach, i.e., to increase coupling, has become possible in recent years and represents a new way of testing the role of gap junctions. The aim of this review is to summarize the current knowledge obtained with agents that selectively increase gap junctional intercellular coupling. Two approaches will be reviewed: increasing coupling by the use of antiarrhythmic peptide and its synthetic analogs and by interfering with the gating of gap junctional channels.     

Innexins: members of an evolutionarily conserved family of gap-junction proteins

Gap junctions are clusters of intercellular channels that provide cells, in all metazoan organisms, with a means of communicating directly with their neighbours. Surprisingly, two gene families have evolved to fulfil this fundamental, and highly conserved, function. In vertebrates, gap junctions are assembled from a large family of connexin proteins. Innexins were originally characterized as the structural components of gap junctions in Drosophila, an arthropod, and the nematode Caenorhabditis elegans. Since then, innexin homologues have been identified in representatives of the other major invertebrate phyla and in insect-associated viruses. Intriguingly, functional innexin homologues have also been found in vertebrate genomes. These studies have informed our understanding of the molecular evolution of gap junctions and have greatly expanded the numbers of model systems available for functional studies. Genetic manipulation of innexin function in relatively simple cellular systems should speed progress not only in defining the importance of gap junctions in a variety of biological processes but also in elucidating the mechanisms by which they act.     

Innexins: members of an evolutionarily conserved family of gap-junction proteins

Gap junctions are clusters of intercellular channels that provide cells, in all metazoan organisms, with a means of communicating directly with their neighbours. Surprisingly, two gene families have evolved to fulfil this fundamental, and highly conserved, function. In vertebrates, gap junctions are assembled from a large family of connexin proteins. Innexins were originally characterized as the structural components of gap junctions in Drosophila, an arthropod, and the nematode Caenorhabditis elegans. Since then, innexin homologues have been identified in representatives of the other major invertebrate phyla and in insect-associated viruses. Intriguingly, functional innexin homologues have also been found in vertebrate genomes. These studies have informed our understanding of the molecular evolution of gap junctions and have greatly expanded the numbers of model systems available for functional studies. Genetic manipulation of innexin function in relatively simple cellular systems should speed progress not only in defining the importance of gap junctions in a variety of biological processes but also in elucidating the mechanisms by which they act.     

Nuclear roles in the postconjugant development of the ciliated protozoan Euplotes aediculatus. 1. Evidence for sequential roles of the differentiating macronucleus in exconjugant development

Thin-section and freeze-fracture data retrieved from developing fetal adrenals of mouse, rabbit, and rat reveal that gap junctions which appear as cortical and medullary cells morphogenetically assort themselves. In the presumptive zona fasciculata and reticularis communicating junctions develop between neighboring cells just prior to the onset of steroidogenesis. In the rodents, gap junctions assemble in large, particle-free formation plaques 1 day before the proliferation of smooth endoplasmic reticulum and the vesiculation of mitochondria (morphological features which are indicative of steroidogenesis). Similar observations have been obtained in fetal rabbits, but here gap junctions apparently develop in the absence of formation plaques. In each species, gap junctions apparently develop 1 or 2 days prior to a surge in corticosteroid production, suggesting that the formation of communicating junctions during the early phases of adrenal cortical differentiation may provide an avenue to coordinate steroidogenesis in the developing fetal cortex.     

Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures

The topological organization of the major rat liver gap junction protein has been examined in intact gap junctions and gap junction-derived single membrane structures. Two methods, low pH and urea at alkaline pH, were used to "transform" or "split" double membrane gap junctions into single membrane structures. Low pH treatment "transforms" rat liver gap junctions into small single membrane vesicles which have an altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after digestion with L-1-to-sylamido-2-phenylethylchloromethyl ketone-trypsin. Alkaline pH treatment in the presence of 8 M urea can split isolated rat liver gap junctions into single membrane sheets which have no detectable structural alteration or altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after proteolytic digestion, suggesting that these single membrane sheets may be useful for topological studies of the gap junction protein. Proteolytic digestion studies have been used to localize the carboxyl terminus of the molecule on the cytoplasmic surface of the intact gap junction. However, the amino terminus does not appear to be accessible to proteases or to interaction with an antibody that is specific for the amino-terminal region of the molecule in intact or split gap junctions. Binding of antibodies, that block junctional channel conductance, can be eliminated by proteolytic digestion of intact gap junctions, suggesting that all antigenic sites for these antibodies are located on the cytoplasmic surface of the intact gap junction. In addition, calmodulin gel overlays indicate that at least two calmodulin binding sites exist on the cytoplasmic surface of the junctional protein. The information generated from these studies has been used to develop a low resolution two-dimensional model for the organization of the major rat liver gap junctional protein in the junctional membrane.     

Junctional structures in digestive epithelia of a cephalopod

Intercellular junctions have been studied in the epithelia of digestive organs of Sepia officinalis (digestive gland, digestive duct appendages and caecum) by conventional staining, lanthanum tracer and freeze-fracturing techniques. In the three organs studied the same junctional complex occurs, consisting of a belt desmosome, a septate junction and gap junctions. The septate junction is of pleated-sheet type and the gap junction has its particles on the P face of the fracture. Circular structures have been found in the digestive gland septate junctions. Neither continuous nor tight junctions have been found. These results show that Cephalopods have junctional structures very close to those of other Molluscs and of Annelids. Some small differences between the septate junctions of the three organs could be related to their different physiology.     

Morphologic characteristics of intercellular junctions in early embryogenesis of mice

Dynamics concerning certain intercellular junctions have been followed during the preimplantation period of development in mouse embryos. The morphological analysis of the preimplantational embryos has demonstrated, that at the initial stages of cleavage (2-4 blastomeres) the cells make contacts by means of nonspecific junctions. Specialized intercellular junctions appear at the stage of 8 blastomeres and are presented as dotted tight and gap junctions. When the embryo is developing from the stage of 8 up to the stage of 16 blastomeres, certain connective complexes appear, consisting of dotted or cord-like tight and gap junctions. At the late morula stage, the external blastomeres in the apical part have contacts with each other by means of cingular tight junctions. In this place a connective complex might emerge; it is displayed as a tight junction and one or two gap junctions. At the blastocyst stage desmosomes and adhision zones appear. Between trophectodermal cells a connective complex arises; it is presented in the slice as a tight cingular junction, desmosomes (as a rule two) and an adhision zone. Between cells of the internal cellular mass the intercellular junctions are presented as dotted tight and gap junctions. Cells of the polar trophoectoderm and cells of the internal cellular mass could have contacts by means of gap and dotted tight junctions.     

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。     

神经组织缝隙连接

近年来神经组织中缝隙连接的分布和功能研究取得了一些显著进展。分子生物学方法的应用促进了ＧＪ结构,亚型及生物物理特性的揭示,染料偶联实验和Ｃａ＾２＋成像技术为ＧＪ的功能研究提供了直观有效的手段。ＧＪ的调控涉及ＧＪ的表达,导通性的改变等环节。ＧＪ胞间通讯的基本形式是交换第二信使和电偶联。     

Relationship of Cytoskeletal Filaments to Annular Gap Junction Expression in Human Adrenal Cortical Tumor Cells in Culture

In addition to the well-characterized surface gap junctions expressed at contact sites between cells, annular gap junction profiles have been localized within the cytoplasm of some cell populations. To study and characterize these annular profiles, gap junction protein type was demonstrated with Western blot and immunocytochemistry. The distribution of annular gap junctions and the relationships to cytoskeletal elements were demonstrated with immunocytochemical, transmission electron microscopic, or image analysis with confocal microscopy techniques. SW-13 adrenal cortical tumor cells expressed α₁gap junctions at areas of cell to cell contact. In addition, α₁gap junction annular profiles were seen within the cytoplasm. Actin and myosin II were found closely associated with these annular gap junctions, while no physical association between tubulin- or vimentin-containing fibers and gap junction protein could be established. Disruption of microfilaments with cytochalasin B treatment (10 μg/ml, 1 h) resulted in a decrease in the average number and an increase in the average size of annular gap junctions compared to control populations. The results are consistent with a role for cytoskeletal elements containing actin and myosin II in annular gap junction turnover.     

A structural characterization of gap junctions isolated from mouse liver

Mouse liver gap junctions have been isolated by using an anionic detergent, n-dodecanoyl sarcosine, in combination with non-ionic polyoxyethylene ethers (Brij 35 and Brij 58) and (W-1) detergents. Purified gap junctions are obtained in a sucrose step gradient containing 1-o-n-octyl-beta-D-glucopyranoside. This procedure is aimed at minimizing proteolysis. The junctions thus isolated have a hexagonal lattice of connexons with a lattice constant of 7.6-8.4 nm. Presence of a major Mr 26,000 gap junctional protein has been confirmed by SDS-PAGE.     

Genes, gene knockouts, and mutations in the analysis of gap junctions

Gop junctions are cell junctions found between most cells and tissues. They contain membrane channels that mediate the cell-to-cell diffusion of ions, metabolites, and small cell signaling molecules. Cell-cell communication mediated by gap junctions has been proposed to have a variety of functions, including roles in regulating events in development, cell differentiation, and cell growth and proliferation. The analysis of these possibilities has been confounded by the fact that there are over a dozen connexin genes encoding polypeptides that make up vertebrate gap junctions. This complexity, coupled with the fact that most cells express multiple connexin isotypes, likely explains why recent studies using reverse genetic and genetic approaches to disrupt connexin gene function have yielded only limited insights into the physiological roles of gap junctions. Nevertheless, studies in vivo and in vitro together have provided evidence for gap junctions being involved in the regulation of cell metabolism, growth, and differentiation in restricted cell and tissue types. Surprisingly, studies in invertebrates suggest that their gap junctions are encoded not by connexins, but by a family of proteins referred to as innexins. Analysis of various Drosophila and C. elegans mutants suggest that innexins may be functional homologs to the connexins. However, whether innexins are the elusive invertebrate gap junction proteins or, rather, accessory proteins that facilitate gap junction formation remains an open question. Given the rapid progress being made in the cloning and functional analysis of gap junctions in many diverse species, confusion and difficulties with nomenclature are coming to a head in this rapidly expanding field. It may be timely to form a Nomenclature Committee to establish a uniform classification scheme for naming gap junction proteins.     

Isolation and characterization of gap junctions from tissue culture cells.

The purification of membrane proteins in a form and amount suitable for structural or biochemical studies still remains a great challenge. Gap junctions have long been studied using electron microscopy and X-ray diffraction. However, only a limited number of proteins in the connexin family have been amenable to protein or membrane purification techniques. Molecular biology techniques for expressing large gap junctions in tissue culture cells combined with improvements in electron crystallography have shown great promise for determining the channel structure to better than 10 A resolution. Here, we have isolated two-dimensional (2D) gap junction crystals from HeLa Cx26 transfectants. This isoform has never been isolated in large fractions from tissues. We characterize these preparations by SDS-PAGE, Western blotting, negative stain electron microscopy and atomic force microscopy. In our preparations, the Cx26 is easily detected in the Western blots and we have increased expression levels so that connexin bands are visible on SDS-PAGE gels. Preliminary assessment of the samples by electron cryo-microscopy shows that these 2D crystals diffract to at least 22 A. Atomic force microscopy of these Cx26 gap junctions show exquisite surface modulation at the extracellular surface in force dissected gap junctions. We also applied our protocol to cell lines such as NRK cells that express endogenous Cx43 and NRK and HeLa cell lines transfected with exogenous connexins. While the gap junction membrane channels are recognizable in negatively stained electron micrographs, these lattices are disordered and the gap junction plaques are smaller. SDS-PAGE and Western blotting revealed expression of connexins, but at a lower level than with our HeLa Cx26 transfectants. Therefore, the purity and morphology of the gap junction plaques depends the size and abundance of the gap junctions in the cell line itself.     

Androgen-regulated formation and degradation of gap junctions in androgen-responsive human prostate cancer cells

The constituent proteins of gap junctions, called connexins (Cxs), have a short half-life. Despite this, the physiological stimuli that control the assembly of Cxs into gap junctions and their degradation have remained poorly understood. We show here that in androgen-responsive human prostate cancer cells, androgens control the expression level of Cx32-and hence the extent of gap junction formation-post-translationally. In the absence of androgens, a major fraction of Cx32 is degraded presumably by endoplasmic reticulum-associated degradation, whereas in their presence, this fraction is rescued from degradation. We also show that Cx32 and Cx43 degrade by a similar mechanism. Thus, androgens regulate the formation and degradation of gap junctions by rerouting the pool of Cxs, which normally would have been degraded from the early secretory compartment, to the cell surface, and enhancing assembly into gap junctions. Androgens had no significant effect on the formation and degradation of adherens and tight junction-associated proteins. The findings that in a cell culture model that mimics the progression of human prostate cancer, degradation of Cxs, as well as formation of gap junctions, are androgen-dependent strongly implicate an important role of junctional communication in the prostate morphogenesis and oncogenesis.     

Cell junctions in the early chick embryo--a freeze etch study

Cell junctions in the early chick embryo have been examined in freeze-etch specimen. Well developed zonulae occludentes are found in the epiblast as early as stage 4. Large gap junctions are also found in the epiblast at this stage. In those cells which have left the surface to form mesenchymal structures (Hensen's node, juxtanodal mesenchyme, primitive streak mesenchyme), one finds not only gap, but also tight, junctions. These junctions do not form continuous belts, but appear as fragments, often reduced to single strands, of typical tight junctions. They probably correspond to the focal tight junctions described earlier in sectioned material. The origin and possible significance of these contacts is discussed, and it is suggested that they represent remnants of junctions between neighboring cells in the epiblast. These junctional remnants slowly disappear by “dilution,” either through cell division and/or cell movement. The appearance of newly formed gap junctions is also described.     

Two different forms of gap junctions within the same organism, one with cytoskeletal attachments, in tunicates

The cells of the intestinal tract and the stigmatal cells of the branchial basket have been studied in a range of tunicates including phlebobranch, aplousobranch and stolidobranch ascidians, as well as the doliolid and pyrosomatid thaliaceans. The intercellular gap junctions between gut cells appear conventional in thin section as do those found in the lower part of adjacent stigmatal cells. However, save for the stolidobranchs, the stigmatal cells also have a second kind of gap junction which exhibit an unusual fibrous density in association with their junctional cytoplasmic surfaces; these are found in the apical region of the cells. The fibrous density is particularly well demonstrated in specimens treated with tannic acid during fixation, and subsequent en bloc uranyl acetate staining. In the branchial basket the position of these apical gap junctions is at regular intervals between adhaering junctions, which have a more substantial paramembranous fibrous mat; these two kinds of junctions alternate along deeply undulating membrane appositions. With freeze-fracture, after chemical or cryo-fixation, the gap junctions of the gut and those of the lower part of the stigmatal cells appear typical, with P-face connexons, while in the apical part of cells of the branchial basket the two faces of the gap junctions are very difficult to cleave apart. Frequently the P- and E-faces are found to adhere together in replicas, so that in these apical gap junctional regions, plaques of E-face with pits overlie the PF particles. In addition, regions of cytoplasm, into which the dense fibres project, often cleave over these adhaering E-faces of the apical gap junctions. The presence of these unusual gap junctional features in the apical region of the stigmata in the vicinity of cilia is discussed as regards their functional role.     

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.