Freeze-fracture analysis of gap and septate junctions in embryos of a moth

Gap and septate junctions were examined in embryos of Manduca sexta (tobacco hornworm). The junctions observed were similar in structure to those reported for adult insect tissues. In the epidermis typical pleated septate junctions were found. Associated with the pleated septate junctions were inverted gap junctions which had irregularly arranged particles and pits. In the midgut typical smooth septate junctions were found. Associated with these septate junctions were gap junctions which had a regular hexagonal packing pattern. This codistribution of gap and septate junction types is discussed in light of current theories that the gap junction types are alternative forms of the same structure in different metabolic environments. In addition to these gap and septate junctions a new junction, perhaps a modified pleated septate junction, is described.     

Evidence mounts for the role of gap junctions during development

While evidence for the role of gap functions, particularly during development, has been mounting, it has remained largely correlative, linking structure with presumed functions. With the recent advent of functional antibodies raised to the junctional protein, however, it has become possible to study the role of gap junctions more directly. There is now considerable evidence indicating that they play a vital role in tissue pattern formation and differentiation by allowing direct cell-to-cell transfer of developmental signals or morphogens.     

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.     

Organization and function of septate junctions

In most cell types, distinct forms of intercellular junctions have been visualized at the ultrastructural level. Among these, the septate junctions are thought to seal the neighboring cells and thus to function as the paracellular barriers. The most extensively studied form of septate junctions, referred to as the pleated septate junctions, is ultrastructurally distinct with an electron-dense ladder-like arrangement of transverse septa present in invertebrates as well as vertebrates. In invertebrates, such as the fruit fly Drosophila melanogaster, septate junctions are present in all ectodermally derived epithelia, imaginal discs, and the nervous system. In vertebrates, septate junctions are present in the myelinated nerves at the paranodal interface between the myelin loops and the axonal membrane. In this review, we present an evolutionary perspective of septate junctions, especially their initial identification across phyla, and discuss many common features of their morphology, molecular organization, and functional similarities in invertebrates and vertebrates.     

Permeability of gap junctions at the segmental border in insect epidermis

We have examined cell-cell communication between epidermal cells of fifth-instar larvae of the milkweed bug Oncopeltus fasciatus and those of maggots of the blowfly Calliphora erythrocephala. Ionic coupling and the transfer of injected Lucifer Yellow (molecular weight 450) and lead-EDTA (molecular weight 374) were used to map the pattern of communication. All epidermal cells, regardless of their position with respect to the segmental border, were ionically coupled. In both species Lucifer Yellow was transferred freely between cells lying in the same segment—that is, in the same developmental compartment as defined by cell lineage. Dye injections close to the segmental border showed that Lucifer Yellow was not transferred between cells in adjacent segments—that is, across the compartmental border. In Calliphora failure of Lucifer Yellow transfer at the segmental border was always observed; in Oncopeltus Lucifer Yellow was not transferred in 90% of preparations examined. Injections of PbEDTA^2? in Calliphora showed that this anion was transferred freely from cell to cell and did not respect the segmental boundary. Previous studies of the distribution of gap junctions at and away from the segmental border make it unlikely that the failure of Lucifer Yellow to cross from segment to segment is due to a reduced number of gap-junctional channels at the border. We conclude that gap junctions at the segmental borders may have different permeability properties from those between cells in the same segment.     

Neuritic growth cone and ependymal gap junctions in the feline subfornical organ during early development

Summary Intercellular contacts in the subfornical organ (SFO) of kittens 3, 16, and 29 days old were studied in thin sections and by the freeze-etch method. Gap junctions appeared between growing nerve processes and target cells. The junctions were interspersed between immature synapses lacking mitochondria as well as full preand postsynaptic membrane specializations. Gap junctions were seen on filopodia as well as on more mature processes. The morphology of these junctions was typical of those described earlier but they were of small size (0.2–0.3 m).Gap junctions of peculiar form were also seen between ependymal elements in the SFO at 16 days. These were of large size (0.5–0.8 m) and were often of segmented character. This segmentation consisted of bands 3–4 particles in width with a center-to-center spacing of 90 nm with particle free corridors between corresponding to the width of about two rows of particles. The margin of the group might be circumscribed by a row of particles. Although gap junctions of large size were seen between ependymal cells in thin section, features corresponding to the particle free corridors have not been observed to date.On leave of absence from the National Institute of Neurological and Communicative Disorders and Stroke, Section of Functional Neurosurgery, Branch of Clinical Neuroscience, Bethesda, Maryland 20014, USAThis work was supported by grants from the Swiss National Foundation for Scientific Research Nos. 3.636.76 and 3.611.0.75, the EMDO Stiftung and the Dr. Eric Slack-Gyr Stiftung     

Involvement of gap junctions in the development of the neocortex

Gap junctions play an important role during the development of the mammalian brain. In the neocortex, gap junctions are already expressed at very early stages of development and they seem to be involved in many processes like neurogenesis, migration and synapse formation. Gap junctions are found in all cell types including progenitor cells, glial cells and neurons. These direct cell-to-cell connections form clusters consisting of a distinct number of cells of a certain type. These clusters can be considered as communication compartments in which the information transfer is mediated electrically by ionic currents and/or chemically by, e.g., small second messenger molecules. Within the neocortex, four such communication compartments can be identified: (1) gap junction-coupled neuroblasts of the ventricular zone and gap junctions in migrating cells and radial glia, (2) gap junction-coupled glial cells (astrocytes and oligodendrocytes), (3) gap junction-coupled pyramidal cells (only during the first two postnatal weeks) and (4) gap junction-coupled inhibitory interneurons. These compartments can consist of sub-compartments and they may overlap to some degree. The compartments 1 and 3 disappear with ongoing develop, whereas compartments 2 and 4 persist in the mature neocortex. Gap junction-mediated coupling of glial cells seems to be important for stabilization of the extracellular ion homeostasis, uptake of neurotransmitters, migration of neurons and myelination of axons. Electrical synapses between inhibitory interneurons facilitate the synchronization of pyramidal cells. In this way, they contribute to the generation of oscillatory network activity correlated with higher cortical functions. The role of gap junctions present in neuroblasts of the ventricular zone as well as the role of gap junctions found in pyramidal cells during the early postnatal stages is less clear. It is assumed that they might help to form precursors of the functional columns observed in the mature neocortex. Although recent developments of new techniques led to the solution of many problems concerning gap junction-coupling between neurons and glial cells in the neocortex, there are many open questions which need to be answered before we can achieve a comprehensive understanding of the role of gap junctions in the development of the neocortex.     

Involvement of gap junctions in placental functions and development

Connexin (Cx) expression and gap junctional intercellular communication (GJIC) are involved in development and differentiation processes. Mediating exchanges between mother and fetus, the placenta is formed when fetal membranes are apposed or even fusing or destroying the uterine mucosa. Therefore, an extraordinary variability of placental structures is observed throughout the mammalian species. This variability affect mainly, the maternofetal blood flow interrelationships, the kind and number of tissue layers separating maternal and fetal bloods, the trophoblast invasiveness and the formation of a syncytium (syncytiotrophoblast). Here, the expression, the localisation and the possible role of Cx and GJIC in placental functions and development are discussed. In rodents, gene knock out in mice have vastly improved our understanding of the role of Cx genes in mouse placental development: Cx26 in transplacental uptake of glucose, Cx31 in the proliferative process of trophoblastic cells and Cx45 in placental vascularisation. In human, it appears that Cx43 allows a GJIC required for the fusion process of cytotrophoblastic cells leading to the formation of the syncytiotrophoblast, the site of the numerous placental functions. On other hands, Cx40 plays a critical role in the switch from a proliferative to an invasive phenotype of the trophoblastic cells invading the endometrium. Owing to the striking diversity of Cx expression in placental structures, we must be careful when extrapolating findings from one species to another.     

The fine structure of polychaete septate junctions

Summary Epidermal septate junctions of Nereis sp. and Cirriformia sp. fixed with OsO₄ or glutaraldehyde/OsO₄ display variable structure in electron micrographs. In transverse section the septa are often indistinct and obscured by opaque material that fills the junctional cleft. Septa (spaced at 180–280 Å) are more clearly defined in slightly oblique transverse section; they exhibit an electron lucent center and appear to be linked by arms. En face views of the junction show a honeycomb pattern. Cytoplasmic faces of junctional membranes are backed with plaques opposite the septa. Lanthanum used as a tracer delineates junctional structure in negative contrast. In transverse section a chain-like lattice is present in the junctional cleft. En face views show parallel rows of pleated elements often linked by arms into honeycomb arrays. Oblique sections demonstrate that these pleated elements are continuous with the chain-like lattice seen in transverse sections. Lanthanum does not pass entirely through the junction. Lanthanum reveals that the septa have a very intricate substructure, but it is difficult to visualize the architecture that could generate the various images presented by these junctions when seen in different orientations. However, it is clear that these junctions possess some features that are diagnostic of several supposedly different types of septate junctions in invertebrates.Supported by USPHS grants NIH 5 P01 NS-07512, NIH 2701 GM-00102, and NB-00840, and by a grant from the Pomona College Research CommitteeI thank Sarah Wurzelmann, Stanley Brown, Nancy Kelly, and Gerhard Ott for excellent technical assistance. Portions of this study were carried out while I was a Postdoctoral Fellow in the Department of Anatomy, Albert Einstein College of Medicine. I dedicate this article to Berta Scharrer as a token of appreciation and affection for her guidance, encouragement, inspiration, and example of excellence     

The stage-specific function of gap junctions during tumourigenesis

Tumour development is a process resulting from the disturbance of various cellular functions including cell proliferation, adhesion and motility. While the role of these cell parameters in tumour promotion and progression has been widely recognized, the mechanisms that influence gap junctional coupling during tumorigenesis remain elusive. Neoplastic cells usually display decreased levels of connexin expression and/or gap junctional coupling. Thus, impaired intercellular communication via gap junctions may facilitate the release of a potentially neoplastic cell from the controlling regime of the surrounding tissue, leading to tumour promotion. However, recent data indicates that metastatic tumour cell lines are often characterized by relatively high levels of connexin expression and gap junctional coupling. This review outlines current knowledge on the role of connexins in tumorigenesis and the possible mechanisms of the interference of gap junctional coupling with the processes of tumour invasion and metastasis. Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7–11, 2007, “The Cell and Its Environment”. Publication costs were covered by the organisers of this meeting.     

Improved enrichment of insect gap junctions by filtration and sonication of NaOH-extracted subcellular fractions

The purification of gap junctions from insects has been hampered by low yields when starting with dissected tissues or by contamination with non-junctional structures when starting with intact insects. A method is described here involving filtration and sonication of NaOH-extracted crude membrane fractions from larvae of the lepidopteran Heliothis virescens which yields fractions containing approximately 50% gap junctions and approximately 7 microg total protein per g of larvae and represents an estimated 10-100 fold increase in gap junction enrichment compared with a previously described procedure (Cell Tissue Res. 274: 393-403, 1993). The remaining structures in the fractions consist of non-junctional membrane, septate junctions, lamellate bodies and amorphous material.     

Diverse gap junctions modulate distinct mechanisms for fiber cell formation during lens development and cataractogenesis

Different mutations of alpha3 connexin (Cx46 or Gja8) and alpha8 connexin (Cx50 or Gja8), subunits of lens gap junction channels, cause a variety of cataracts via unknown mechanisms. We identified a dominant cataractous mouse line (L1), caused by a missense alpha8 connexin mutation that resulted in the expression of alpha8-S50P mutant proteins. Histology studies showed that primary lens fiber cells failed to fully elongate in heterozygous alpha8(S50P/+) embryonic lenses, but not in homozygous alpha8(S50P/S50P), alpha8-/- and alpha3-/- alpha8-/- mutant embryonic lenses. We hypothesized that alpha8-S50P mutant subunits interacted with wild-type alpha3 or alpha8, or with both subunits to affect fiber cell formation. We found that the combination of mutant alpha8-S50P and wild-type alpha8 subunits specifically inhibited the elongation of primary fiber cells, while the combination of alpha8-S50P and wild-type alpha3 subunits disrupted the formation of secondary fiber cells. Thus, this work provides the first in vivo evidence that distinct mechanisms, modulated by diverse gap junctions, control the formation of primary and secondary fiber cells during lens development. This explains why and how different connexin mutations lead to a variety of cataracts. The principle of this explanation can also be applied to mutations of other connexin isoforms that cause different diseases in other organs.     

Formaldehyde-induced appearance of septate junctions between digestive vacuoles

Tissue biopsies from (1) some chronic inflammatory diseases, (2) a necrotic tumoral process, (3) normal human lymphatic ganglia, and (4) two congenital diseases of the adrenal cortex were selected for study. A block from each biopsy was fixed in glutaraldehyde-paraformaldehyde; a second block was fixed in 10% formaldehyde. In all cases septate junctions between digestive vacuoles did occur in phagocytic cells and some adrenal cortex cells fixed in formaldehyde. These junctions were similar to those reported recently for malakoplakia phagocytes. Consistently, they were not found to attach organelles other than lysosomes derivatives. Both phagocytes and adrenal cortex cells in the material fixed in glutaraldehyde-paraformaldehyde did not display adhesive specializations between digestive vacuoles. This suggests that the septate junctions described herein are artifactuous structures induced by formaldehyde. There is, however, a certain degree of specificity of cells having the capability of developing these septate junctions. It is assumed that the coating material of digestive organelles in phogocytes and some other cells would be responsible for both cell specificity and organelle specificity of the formaldehyde-induced septate junctions.     

Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice

The importance of connexins (Cxs) in the cochlear functions has been indicated by the finding that mutations in connexin genes cause a large proportion of sensorineural deafness cases. However, functional roles of connexins in the cochlea are still unclear. In this study, we compared the relative expression levels of 16 different subtypes of mouse connexins in the cochlea. cDNA macroarray hybridizations identified four most prominently expressed connexins (listed in descending order): Cxs 26, 29, 30, and 43. Two of these connexins (Cx26 and Cx30), both belonging to the beta-group, were investigated for their molecular assemblies in the cochlea. Co-immunostaining showed expressions of Cxs 26 and 30 in the same gap junction plaques and their co-assembly was confirmed by co-immunoprecipitation of proteins extracted from the cochlear tissues. The heterologous molecular assembly of connexins is expected to produce gap junctions with biophysical characteristics appropriate for maintaining ionic homeostasis in the cochlea.     

Actin filaments are associated with the septate junctions of invertebrates

Septate junctions are almost ubiquitous in the tissues of invertebrates but are never found in those of vertebrates. In spite of their widespread occurrence and hence obvious importance to the invertebrates, their precise function has remained elusive although they have been variously considered to be regions of cell-cell coupling, permeability barriers or adhesion sites. This report demonstrates that elements of the cytoskeletal system insert into the cytoplasmic face of septate junctions. Actin filaments, identified by virtue of their capacity to bind the S1 subfragment of rabbit myosin, are associated with the membranes of septate junctions. Cytochalasin D, an actin depolymerizer, leads to disorganization of the intramembrane components of these junctions. These data suggest that a primary role of septate junctions could be to maintain intercellular cohesion and hence tissue integrity. The assembly and localization of these junctions may be mediated, directly or indirectly, by the cytoplasmic actin filaments associated with their lateral membranes.