Passover eliminates gap junctional communication between neurons of the giant fiber system in Drosophila

The Passover-related gene family plays significant roles in cellular connectivity. Mutations in three family members from Drosophila and from Caenorhabditis elegans alter a few specific electrical synapses. The passage of cobalt between Drosophila neurons was used to assay the presence of gap junctional connections. The giant fiber in the wild type has specific gap junctional connections in the brain and in the thorax. In flies mutant for Passover, cobalt cannot pass into or out of the giant fiber in either the anterograde or the retrograde directions. A large number of other gap junctional connections remain unaffected. This demonstrates that the Passover gene is necessary for gap-junctional communication between the neurons of the Drosophila giant fiber system. © 1996 John Wiley & Sons, Inc.     

Supramolecular dynamics of gap junctions

During the life cycle of a membrane protein its molecular structure may change and for aggregated proteins this process may be observed on the supramolecular level. Here we demonstrate that this is the case for gap junction channels which maintain cell-cell communication. Freshly synthesized connexins are integrated as hexamers (connexons) into the plasma membrane where they form plaques after pairing with connexons of an attached cell. We inhibited protein trafficking by applying the fungal metabolite brefeldin A (BFA), quantified cell-cell coupling by calcein transfer and fluorescence-activated flow cytometry, and examined the degradation and formation of gap junction plaques by indirect immunofluorescence and immunogold labeling. Under control conditions 50% of the detected plaques were ubiquitylated and less than 10% showed a two-dimensional crystalline packing. One hour after BFA reversal about 60% of the plaques were crystalline and ubiquitylation dropped to 14%. Label for ubiquitin was predominantly found on non-crystalline plaques. We, therefore, conclude that newly formed gap junction plaques are of crystalline morphology which changes to a pleomorphic structure when individual channels are modified during their aging process. This dynamic in plaque morphology correlates with channel inactivation and plaque ubiquitylation.     

Identification and characterization of two novel splice forms of GRIP1 in the rat brain

We cloned two novel alternatively-spliced mRNA isoforms of glutamate receptor interacting protein 1 (GRIP1) which we named GRIP1d and GRIP1e 4-7. GRIP1d is a 135 kDa, 7-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 4-PDZ-domain GRIP1c 4-7. GRIP1e 4-7 is a 75 kDa 4-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 7-PDZ-domain GRIP1a/b. Northern blots indicated that GRIP1d mRNA is 5.1 kb long and abundant in brain. An antibody to the C-terminus of the 75 kDa GRIP1c 4-7 also recognized an abundant 135 kDa protein, consistent with the predicted size of GRIP1d. Similarly, an antibody to the C-terminus of the 135 kDa GRIP1a/b also recognized a low abundance 75 kDa protein, consistent with the predicted size of GRIP1e 4-7. Immunocytochemistry of hippocampal cultures and intact brain using these antibodies showed that (i) these isoforms are present in both GABAergic and glutamatergic synapses, and (ii) the isoforms co-localize in individual synapses. While GRIP1a/b isoforms are abundant in interneurons and highly concentrated in GABAergic presynaptic terminals, the isoforms recognized by the antibody to the C-terminus common to GRIP1c 4-7 and GRIP1d are much less abundant in interneurons and preferentially concentrate at the postsynaptic complex.     

Evolutionary origin of synapses and neurons – Bridging the gap

The evolutionary origin of synapses and neurons is an enigmatic subject that inspires much debate. Non‐bilaterian metazoans, both with and without neurons and their closest relatives already contain many components of the molecular toolkits for synapse functions. The origin of these components and their assembly into ancient synaptic signaling machineries are particularly important in light of recent findings on the phylogeny of non‐bilaterian metazoans. The evolution of synapses and neurons are often discussed only from a metazoan perspective leaving a considerable gap in our understanding. By taking an integrative approach we highlight the need to consider different, but extremely relevant phyla and to include the closest unicellular relatives of metazoans, the ichthyosporeans, filastereans and choanoflagellates, to fully understand the evolutionary origin of synapses and neurons. This approach allows for a detailed understanding of when and how the first pre‐ and postsynaptic signaling machineries evolved.     

Electron-microscopic studies on the presence of gap junctions between corneal fibroblasts in rabbits

Summary Corneal fibroblasts, major cellular components of the corneal stroma, are loosely arrayed between collagen lamellae. They play an important role in the metabolic and physiological homeostasis mechanisms by which the cornea is kept transparent. This paper deals with the demonstration of the gap junctions between the corneal fibroblasts of rabbits by transmission electron microscopy of thin sections and of freeze-fracture specimens. Under the transmission electron microscope, the corneal fibroblasts are seen between the lamellae of collagen fibers of the corneal stroma. Their long cytoplasmic processes are in contact with those of neighboring fibroblasts. Typical gap junctions are found between these cytoplasmic processes. In the freeze-fracture images, intramembrane particles with a diameter of 10.3 nm form polygonal aggregates on P faces. These findings suggest that corneal fibroblasts, coupled with each other, might function synchronously through gap junctions responsible for metabolic activities essential for the maintenance of corneal transparency.A part of this study was published in Kinki Daigaku Igaku Zasshi in Japanese as the thesis for Atsuko Ueda, M.D. This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan, from Osaka Eye Bank, Osaka, Japan, and from an intramural research fund of Kinki University     

Rare and spatially segregated release sites mediate a synaptic interaction between two identified network neurons

Laser‐scanning confocal microscopy (LSCM), electron microcopy (EM), and cellular electrophysiology were used in combination to study the structural basis of an inhibitory synapse between two identified neurons of the same network. To achieve this, we examined the chemical inhibitory synapse between identified neurons belonging to the lobster (Homarus gammarus) pyloric network: the pyloric dilator (PD) and the lateral pyloric (LP) neurons. In order to visualize simultaneously these two neurons, we used intrasomatic injection of Lucifer Yellow (LY) in one and rhodamine/horseradish peroxydase (HRP) in the other. Under LSCM, we found only two zones of close apposition in a restricted part of the neuritic tree of the two network neurons. Then, within these two zones, the synaptic release sites were searched using EM. To this end, photoconversion of LY with immunogold and development of HRP with DAB were performed on the previously observed preparations. Structural evidence was found for only one release site per zone. To confirm this result, and because the zones of contact were always segregated in a restricted part of the dendrites, we used laser photoablation to selectively delete, either pre‐ or postsynaptically, the branches on which the release sites were located. In both cases, such restrictive ablation completely abolished the functional interaction between these neurons. Our results therefore demonstrate that an inhibitory synapse that is essential for the operation of a neural network relies on only very few sites of contact localized in a highly restricted part of each neuron's dendritic arbor. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 150–163, 2002; DOI 10.1002/neu.10023     

Ultrastructural differentiation during embryonic Drosophila myogenesis in vitro

Summary Cultures of embryonicDrosophila melanogaster cells were examined by electron microscopy and events in myogenesis were recorded. Thick and thin myofilaments, T-tubules and sarcoplasmic reticulum all appeared at about the same time, 10.5 hr. This was about 5 hr after the final division of myoblasts and about the time that muscle cells were elongating, aligning and fusing. Sarcoplasm typical of insect muscle was detected by 18.5 hr, as were myotendonal and tendocuticular junctions. Two populations of myocytes were detected, the cytoplasm of one more electron-dense than the other. The only previous report of myofibrilogenesis in invertebrate embryos had described novel mechanisms. InDrosophila embryonic material, however, the sequence of myofibrilogenesis resembled that in post-embryonic insect or vertebrate material. Mrs. Pilar Toribio-Fiorio provided excellent technical assistance, and Patricia Minter, the secretarial expertise. This investigation was supported, in part, by NIH Grant NS9330 and the James Douglas Research Fund to Robert L. Seecof and NIH Grant No. 1 RO1 CA17223-01 to Raymond L. Teplitz.     

Heterotypic gap junctions between two neurons in the drosophila brain are critical for memory

Gap junctions play an important role in the regulation of neuronal metabolism and homeostasis by serving as connections that enable small molecules to pass between cells and synchronize activity between cells. Although recent studies have linked gap junctions to memory formation, it remains unclear how they contribute to this process. Gap junctions are hexameric hemichannels formed from the connexin and pannexin gene families in chordates and the innexin (inx) gene family in invertebrates. Here we show that two modulatory neurons, the anterior paired lateral (APL) neuron and the dorsal paired medial (DPM) neuron, form heterotypic gap junctions within the mushroom body (MB), a learning and memory center in the Drosophila brain. Using RNA interference-mediated knockdowns of inx7 and inx6 in the APL and DPM neurons, respectively, we found that flies showed normal olfactory associative learning and intact anesthesia-resistant memory (ARM) but failed to form anesthesia-sensitive memory (ASM). Our results reveal that the heterotypic gap junctions between the APL and DPM neurons are an essential part of the MB circuitry for memory formation, potentially constituting a recurrent neural network to stabilize ASM.     

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.     

On the structure of isolated junctions between communicating cells

Summary Gap junctions are specialized regions of contact between apposed plasma membranes of communicating cells. They are composed of hexagonally arranged units (connexons) embedded in plasma membranes and linked together in the extracellular space. The three-dimensional structure of the connexon, was obtained by Fourier analysis on specimens of isolated rat liver gap junctions. The connexon is an annular oligomer, composed of six subunits, that protrudes from both sides of the plasma membrane. The subunits are tangentially displaced about the connexon axis. A narrow channel is located along the connexon, axis spanning the thickness of the junction, but it is greatly reduced in the hydrophobic zones of the membranes. Two closely related forms of isolated gap junctions which have different connexon subunit structures but the same hexagonal lattice, were obtained. The transition between the two forms of communicating junctions seen in isolation is produced by radial inward motion of the connexon subunits near their cytoplasmic surfaces and a reduction of their inclination tangential to the 6-fold axis. Similar rearrangement of essentially rigid subunits embedded in the membrane could provide a mechanism for modulation of the junction permeability. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center. This work was supported by NH Grants 5P1GM23911-07 and 5T32-6M07403-04.     

Functional alterations in gut contractility after connexin36 ablation and evidence for gap junctions forming electrical synapses between nitrergic enteric neurons

Neurons in the enteric nervous system utilize numerous neurotransmitters to orchestrate rhythmic gut smooth muscle contractions. We examined whether electrical synapses formed by gap junctions containing connexin36 also contribute to communication between enteric neurons in mouse colon. Spontaneous contractility properties and responses to electrical field stimulation and cholinergic agonist were altered in gut from connexin36 knockout vs. wild-type mice. Immunofluorescence revealed punctate labelling of connexin36 that was localized at appositions between somata of enteric neurons immunopositive for the enzyme nitric oxide synthase. There is indication for a possible functional role of gap junctions between inhibitory nitrergic enteric neurons.     

Presynaptic dense bars at neuromuscular synapses of the lobster,Homarus americanus

Summary The threedimensional ultrastructure of presynaptic dense bars was examined by serial section electron microscopy in the excitatory neuromuscular synapses of the accessory flexor muscle in the limbs of larval, juvenile, and adult lobsters. The cross-sectional profile of the dense bar resembles an asymmetric hourglass, the part contacting the presynaptic membrane being larger than that projecting into the terminal. The bar has a height of 55–65 nm and varies in length from 75–600 nm. In its dimensions it resembles the dense projections in the synapses of the CNS of insects and vertebrates. The usual location of these dense bars is at well defined synapses, though a few are found at extrasynaptic sites either in the axon or terminal. In the latter case the bars are close to synapse-bearing regions, particularly in the larval terminals, suggesting that the extrasynaptic bars denote early events in synapse formation. In all cases the bars are intimately associated with electron lucent, synaptic vesicles located on either side, in the indentation of its hourglass-shaped cross sectional profile. The vesicles occur along the length of the bar and contact the presynaptic membrane. Consequently the dense bar may serve to align the vesicles at the presynaptic membrane prior to exocytosis. A similar role has been suggested for the presynaptic dense bodies at the neuromuscular junction of the frog, where synaptic vesicles form a row on either side of this structure.Supported by Muscular Dystrophy Association of Canada and NSERCC. Generous use of laboratory facilities at Woods Hole was provided by the late Fred Lang     

Biochemical and genetic investigations on gap junctions from mammalian cells

Gap junction protein (26K) in mouse or rat liver has been studied using a rabbit antiserum directed against the sodium dodecylsulfate denatured 26K protein from mouse liver. The liver 26K protein has been localized in gap junction plaques of hepatic plasma membranes by immuno electron microscopy. Affinity purified anti-26K antiserum showed weak cross reactivity with mouse or bovine lens gap junction protein (MIP26). This result suggests some structural homology between the different gap junction proteins in liver and lens. After partial hepatectomy of young rats the liver 26K protein appears to be degraded and later resynthesized. A variant of established Chinese hamster fibroblastoid cells has been isolated and shown to be defective in metabolic cooperation via gap junctions.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Ultrastructural changes of Drosophila larval and prepupal salivary glands cultured in vitro with ecdysone

Summary Alterations in the ultrastructure of in vitro cultured larval salivary glands of Drosophila melanogaster in response to the steroid hormone ecdysone were studied in relation to complex changes in puffing patterns. We found that the changes in the fine structure of cultured glands reflected progression of the puffing pattern, and they paralleled those seen in vivo. We observed that glue secretion by exocytosis, the main function of salivary glands, took place between puff stage 5 (PS5) and PS7. Glue could not be expectorated under culture conditions but was slowly released from the lumen through a duct into the medium. After the cultured glands reached PS13/PS14, further progress of puffing and fine structural alterations required that the ecdysteroid titer be transiently extremely low or absent. Under in vitro conditions we did not observe the putative new secretory program(s) described for glands in vivo after PS12. However, ultrastructural changes which unambiguously indicated that an autohistolytic process had begun in vitro started to appear after PS17. Many salivary gland cells developed numerous features of progressive self-degradation between PS18 and PS21. Actual degradation of salivary glands in vivo seemed to be rapid, but in vitro degradation was never completed, probably due to a lack of exogenous factors from the hemolymph. Manipulations of ecdysone titer in vitro in the culture medium, known during the larval puffing cycle to cause premature induction of developmentally specific puffing patterns, did not affect the normal development of ultrastructural features of the cytoplasm and nucleus.     

Electron microscope immunolocation of gap junctions in Drosophila

Gap junction immunolocation was carried out in thin sections of Lowicryl K4M-embedded Drosophila imaginal wing discs using an affinity-purified polycolonal anti-18 kD gap junction protein anitbody and a colloidal gold-conjugated secondary antibody. Colloidal gold labelling was predominantly associated with obliquely-sectioned gap junctions, the ends of junctional profiles and other regions in which the adjacent junctional membranes were separated or distorted. The pattern of staining suggests that the determinant recognized by the antibody is relatively inaccessible, probably with a topological location in the transmembrane or extracellular domain of the membrane-spanning connexin protein.     

Differential sensitivity of cholinergic and GABAergic neurons in chick embryos treated intracerebrally with ethanol at 8 days of embryonic age

We have shown that in embryos treated with ethanol in ovo during days 1–3, a critical period of neuroembryogenesis, cholinergic neuronal phenotypic expression is decreased whereas GABAergic and catecholaminergic neuronal populations are increased as assessed by neuronal markers choline acetyltransferse (ChAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) respectively. In this study, ethanol was administered intracerebrally to embryos at embryonic day 8, embryos were sacrificed at day 9 and ChAT and GAD activities assayed separately in cerebral hemispheres and remaining brain (diencephalon-midbrain and optic lobes). We found that ChAT activity was enhanced in the cerebral hemispheres only, whereas GAD activity was decreased in both cerebral hemispheres and remaining brain. We have concluded that the differential responses of neuronal phenotypes to ethanol may reflect compensatory mechanisms to ethanol insult. Moreover, these findings emphasize the vulnerability of the GABAergic neuronal phenotypes to ethanol neurotoxicity during early brain development in the chick.     

Reaching out: junctions between cardiac telocytes and cardiac stem cells in culture

Telocytes (TCs) were previously shown by our group to form a tandem with stem/progenitor cells in cardiac stem cell (CSC) niches, fulfilling various roles in cardiac renewal. Among these, the ability to ‘nurse’ CSCs in situ, both through direct physical contact (junctions) as well as at a distance, by paracrine signalling or through extracellular vesicles containing mRNA. We employed electron microscopy to identify junctions (such as gap or adherens junctions) in a co‐culture of cardiac TCs and CSCs. Gap junctions were observed between TCs, which formed networks, however, not between TCs and CSCs. Instead, we show that TCs and CSCs interact in culture forming heterocellular adherens junctions, as well as non‐classical junctions such as puncta adherentia and stromal synapses. The stromal synapse formed between TCs and CSCs (both stromal cells) was frequently associated with the presence of electron‐dense nanostructures (on average about 15 nm in length) connecting the two opposing membranes. The average width of the synaptic cleft was 30 nm, whereas the average length of the intercellular contact was 5 μm. Recent studies have shown that stem cells fail to adequately engraft and survive in the hostile environment of the injured myocardium, possibly as a result of the absence of the pro‐regenerative components of the secretome (paracrine factors) and/or of neighbouring support cells. Herein, we emphasize the similarities between the junctions described in co‐culture and the junctions identified between TCs and CSCs in situ. Reproducing a CSC niche in culture may represent a viable alternative to mono‐cellular therapies.