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How a gap junction maintains its structure
Abstract
In gap junctions, identical membrane proteins are linked up in pairs (dyads) that bridge the extracellular space between two apposed cell membranes. Typically, several thousand of these dyads are aggregated in the plane of the membranes and form a junctional plaque with a distinct boundary. The question thus arises as to what maintains the dyads in an aggregated state. From a statistical mechanical analysis of the positions of dyads in a freeze-fracture electron micrograph, we report here that the aggregates are not maintained by an attractive force between pairs of dyads, but probably by the minimization of the repulsive force between apposed membranes. On the basis of this analysis we present a model for the structure of mature gap junctions as well as certain aspects of the formation and disassembly of gap junctions.
... Au même moment, paraissait une étude s'intéressant à l'agrégation des protéines d'adhésion dans les jonctions communicantes (Braun et al., 1984;Abney et al., 1987). Dans ces jonctions, une interaction homophilique s'établit entre protéines d'adhésion des membranes en vis-à-vis et résulte en la formation de paires de protéines interagissant de façon "trans" et s'agrégeant pour former les jonctions. ...
... L'éloignement d'un couple de protéines adhésives de l'agrégat (a → b) réduit la distance entre des membranes qui ont naturellement tendance à se repousser. b est donc énergétiquement moins favorable que a. Source : Braun et al. (1984). inclus dans une membrane qui se séparent en deux morceaux sous l'eet des forces qu'ils créent en déformant la membrane (Elliot et al., 1983). ...
Composition du jury : Pierre Bongrand (Rapporteur) Bertrand Fourcade (Examinateur) François Gallet (Examinateur) Daniel Riveline (Directeur de thèse) Olivier Thoumine (Rapporteur) Marcel Vallade (Directeur de thèse)
... Communication through the gap junction or hemichannel activity in olfactory receptor neuron critical role in maintaining olfactory sensitivity and order perception (Zhang 2010). Gap junctions, consisting of intercellular channels, facilitate the direct diffusion of ions and small molecules between neighboring cells [1][2][3][4][5] . Connexins (Cx), which are tetraspan integral membrane proteins, assemble into hexameric structures (connexons) that dock head-tohead, forming intercellular channels. ...
In insects, the antennal lobe (AL) serves as the primary processing center for olfactory signals, housing a complex network of neurons that analyze and integrate olfactory information. Gap junctions, an integral part of the AL neural network, have been shown to play a significant role in processing and transmitting olfactory information. However, their specific function within the AL system remains not fully understood. To bridge this knowledge gap, this study aims to investigate the role of gap junctions in the AL circuitry using experimental and computational methods. The focus is on developing a computational model of the gap junctions between local neurons and projection neurons within the AL. By employing a biologically accurate model to mimic the behavior of the AL neural network, the researchers aim to explore the impact of gap junctions on the processing and transmission of olfactory information. The study's findings highlight the critical role of gap junctions in synchronizing the activity of neurons within the AL circuitry. The computational model serves as a valuable framework for understanding the function of these junctions in the AL network and could potentially be applied to evaluate their impact on olfactory processing in other insects.
... IGJCs may be homotypic (if connexons are identical) or heterotypic (if the two connexons are different) [13]. IGJCs are thought to cluster into plaques via long-range protein aggregation driven from interparticle interactions as well as lateral pressures between the junction and the surrounding glycocalyx [14,15]. ...
Background
Connexin hemichannels have been implicated in pathology-promoting conditions, including inflammation, numerous widespread human diseases, including cancer and diabetes, and several rare diseases linked to pathological point mutations.
Methods
We analysed the literature focusing on antibodies capable of modulating hemichannel function, highlighting generation methods, applications to basic biomedical research and translational potential.
Results
Anti-hemichannel antibodies generated over the past 3 decades targeted mostly connexin 43, with a focus on cancer treatment. A slow transition from relatively unselective polyclonal antibodies to more selective monoclonal antibodies resulted in few products with interesting characteristics that are under evaluation for clinical trials. Selection of antibodies from combinatorial phage-display libraries, has permitted to engineer a monoclonal antibody that binds to and blocks pathological hemichannels formed by connexin 26, 30 and 32.
Conclusions
All known antibodies that modulate connexin hemichannels target the two small extracellular loops of the connexin proteins. The extracellular region of different connexins is highly conserved, and few residues of each connexins are exposed. The search for new antibodies may develop an unprecedented potential for therapeutic applications, as it may benefit tremendously from novel whole-cell screening platforms that permit in situ selection of antibodies against membrane proteins in native state. The demonstrated efficacy of mAbs in reaching and modulating hemichannels in vivo, together with their relative specificity for connexins overlapping epitopes, should hopefully stimulate an interest for widening the scope of anti-hemichannel antibodies. There is no shortage of currently incurable diseases for which therapeutic intervention may benefit from anti-hemichannel antibodies capable of modulating hemichannel function selectively and specifically.
... Indeed, these theoretical analyses predict that, in cases in which a negative (antiadhesive) process would not be present to limit the size of stochastically appearing clusters, these clusters would grow, coalesce or fuse indefinitely until available receptors are depleted. This negative potential could be because of membrane fluctuation or of the presence of molecules in the glycocalyx the size of which is at least one order of magnitude longer than the cadherin ectodomain, as proposed recently for the actomyosin-independent formation of integrin clusters at focal adhesions or much earlier for the formation of gap junctions (Braun et al. 1984;Paszek et al. 2014). ...
The cadherin-catenin adhesion complex is the key component of the intercellular adherens junction (AJ) that contributes both to tissue stability and dynamic cell movements in epithelial and nonepithelial tissues. The cadherin adhesion complex bridges neighboring cells and the actin-myosin cytoskeleton, and thereby contributes to mechanical coupling between cells which drives many morphogenetic events and tissue repair. Mechanotransduction at cadherin adhesions enables cells to sense, signal, and respond to physical changes in their environment. Central to this process is the dynamic link of the complex to actin filaments (F-actin), themselves structurally dynamic and subject to tension generated by myosin II motors. We discuss in this review recent breakthroughs in understanding molecular and cellular aspects of the organization of the core cadherin-catenin complex in adherens junctions, its association to F-actin, its mechanosensitive regulation, and dynamics.
... Une explication possible de l'agrégation observée des protéines d'adhésion serait l'existence d'interactions cis indirectes entre molécules d'adhésion appartenant à la même cellule. Les propriétés physiques de la membrane peuvent alors expliquer ce phénomène (Braun et al., 1984). Deux hypothèses sont confrontées dans le modèle de Braun. ...
We studied the growth of asymmetric cellular contacts : adherens junctions and focal contacts. We showed, by imaging, micromanipulation and cellular assembly modification experiments, that adherens junctions between two apparently identical cells exhibit a symmetry breaking due to different roles of actin cytoskeleton for both cells in contact : the “donor” cell polymerizes actin which leads to cell membranes contact; as a response, the “receiving” cell assembles acto-myosin bundles which promotes local force application on the junctions and a consequent increase of their length. Growth curves of adherens junctions when cell contractility is increased using controlled nocodazole concentrations have been measured. Fits of experimental growth curves allowed a determination of the contractile forces applied by the “receiving” cell. We were thus able to obtain for the first time the force-extension diagram for adherens junctions by a non-invasive method. The biological approaches used to name the key cytoskeletal players and the physical approaches used to fit growth laws have both shown that intercellular contacts exhibits local reinforcement in a manner similar to focal contact reinforcement. We have finally observed by TIRFM internal dynamics of growing focal contacts using different labelled proteins (fibronectin, integrin, vinculin, actin) in order to propose a mechanosensing mechanism.
... 2) The clustering of connexons can be initiated by an extrinsic signal (77). The signal that triggers connexon clustering is unknown, but statistical analysis of freeze-fracture electron microscopy images indicates that minimization of distance between apposing membranes (30 m) holds connexon aggregates together and traps them in a specific area on the cell membrane (9). ...
The eye lens is comprised of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin1 (Tmod1), an actin pointed-end capping protein, CP49, a lens-specific intermediate filament protein, or lacking both Tmod1 and CP49. Surprisingly, we found that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure and sodium concentration. There are no changes in the protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double knockout (DKO) lenses, and normal gap junctions are observed by electron microscopy. However, immunostaining and quantitative analysis of 3D confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers are unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micron-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance.
Copyright © 2015, American Journal of Physiology - Cell Physiology.
The gap junction is the plasma membrane specialisation responsible for electrical and chemical coupling of cardiac myocytes. Despite increasingly recognised significance, there is a paucity of data on gap junctions in the human heart. This thesis reports studies in which confocal microscopic immunohistochemistry of the principal gap-junctional protein, connexin43, and electron microscopy, were used to characterise gap-junctional organisation in developing and adult human ventricular myocardium, and to investigate altered gap-junctional organisation as a possible contributing factor to electromechanical dysfunction in congenital, ischaemic and hypertrophic myocardial diseases. In the adult left ventricle, connexin43-gap junctions occupy a surface area of 0.0051 μm2/μm3 myocardial volume, and are confined to the intercalated disks. In the neonate, connexin43-gap junctions are distributed over the entire surface of ventricular myocytes, with a progressive (linear) change to the adult pattern by six years of age. Recurrently ischaemic adult ventricular myocardium has gap junctions confined to intercalated disks of normal number and appearance, but with a reduced gap-junctional surface area (0.0027 μm2/μm3), as also found in chronically hypertrophied ventricular myocardium (0.0031 μm2/μm3). When expressed per myocyte, there is a 30% reduction in gap-junctional content in ischaemic ventricle compared with normal. The pattern of junction distribution is grossly disturbed at the surviving borders of myocardial infarcts, both early and after healing. In the early hypertrophic response to acute renovascular hypertension in an animal model, left ventricular myocytes show a 35% increase in gap- junctional surface area per unit myocyte volume. Hearts with congenitally anomalous morphology are not associated with abnormal gap junction distribution. The cells comprising accessory atrioventricular pathways have a junction distribution suggestive of ventricular myocytes, and of efficient coupling, despite vestigial contractile apparatus. Although the pattern of myocardial gap junction distribution is severely disrupted at infarct borders, the site of origin of many clinical arrhythmias, there are no widespread disturbances of gap-junctional distribution patterns, in the diseases investigated. Quantitative alterations of gap-junctional expression, however, may be a hitherto unrecognised determinant of mechanical and arrhythmic dysfunction in diseased hearts.
Subsequent to the first morphologic description of the gap junction by Revel and Karnovsky in 1967 (113), a plethora of reports has appeared describing the structure, function and pathologic changes of this plasma membrane structure. The gap junction appears to serve as a conduit for the cell-to-cell exchange of low molecular weight ions and molecules between adjacent cells (intercellular communication). A number of functions have been attributed to intercellular communication including the maintenance of normal cellular homeostasis. Another function that is clearly related to the neoplastic process is the control of cellular growth (81). Yotti, Chang, and Trosko (178) and Murray and Fitzgerald (99), working independently, reported in 1979 that tumor promoting phorbol ester compounds were capable of inhibiting gap junction-mediated intercellular communication between cells in culture. These findings resulted in the hypothesis that tumor promoters stimulate cell proliferation of initiated cells by inhibiting gap junctional intercellular communication in the initiated cells (146).
Six years ago, in 1989, the topic addressed in this series of NATO Advanced Study Institutes was “States and Phase Transitions in Soft Condensed Matter” [1]. At that time, the ASI confronted a broad spectrum of contemporary issues in complex fluid science including polymers, colloids, ferrofluids, membranes, microemulsions, and other macromolecular systems. This year the topic is “Physics of Biomaterials: Fluctuations, Self-Assembly and Evoluton”, which is one of the principal directions in which soft condensed matter has evolved. Once again, I’ve been asked to set the stage for a marvelous series of lecturers who will present material at the cutting edge of the field. The topics include protein folding, statistical and evolutionary aspects of DNA coding, biomolecular motor dynamics, neural nets, and coupled membrane-protein systems. Indeed I’m delighted to be here because there will be an enormous amount for me to learn. I, by no means, feel competant to provide any reasonable introduction to this broad array of subjects; neither do I possess the background in modern molecular and cellular biology that is necessary to place these topics in their proper context. Therefore these lectures will be restricted to a review of an area in which we in Santa Barbara have some experience, namely, lipid self-assembly to form membranes and their resulting interactions with various types of inclusions. This written extended abstract will discuss our rationale for treating this class of problems.
Adhesion of colloidal particles (synthetic or biological) to one another or to other surfaces, typically immersed in a fluid medium, is frequently a mesoscale phenomenon, intermediate between microscopic and macroscopic scales. Similar to other mesoscale phenomena, gaining a useful understanding based on ideas from either continuum or atomistic concepts alone is limited. Combination of experimental techniques like optical trapping and scanning probe techniques with simultaneous imaging provides direct insight into the space- and time-dependent dynamics of the adhesion process in the relevant scales. The strength of adhesion typically evolves with time; the details of the processes depend on the overall strength of attractive interaction among the adhering objects as well as its spatial fluctuations governed by the randomness of the surfaces involved and their own time-dependent reconstruction due to varying local stresses. This review focuses on situations where the adhesive inter-object interactions are “weak” compared to the cohesive intra-object interactions whereby all processes occur, to a good approximation, in the interfacial region. In these situations, the dynamics of adhesion is affected by both the “quenched” spatial disorder of the surfaces as well as the thermal fluctuations. A local microrheological characterization of the stress-coupling in the interfacial region provides a heuristic description of the adhesion process. The discrete, thermally assisted “punctuated descent” of the effective adhering system down a rough energy landscape through a hierarchy of finite metastable minima reflects the mesoscopic nature of adhesion. We review these experimental observations of particle adhesion in the weak limit and explore possible common mechanisms underlying apparently disparate phenomena and processes.
A model for protein-lipid interactions in bilayer membranes where the proteins are very dilute is extended to higher protein concentration, where appreciable lipid-mediated protein-protein interactions occur. It is found that proteins may change the lipid phase transition temperature and that they weaken the phase transition. There exists a critical protein concentration above which the sharp lipid phase transition is abolished. The model also qualitatively reproduces several experimental observations on the physical behavior of bilayers formed from mixtures of cholesterol and phosphatidylcholines.
We report here rapid assembly of gap junctions in prostate epithelial cells in vitro. Assembly of gap junctions can be induced by incubation at 0 degrees C followed by incubation at 37 degrees C. Colchicine (10(-5) M, 10(-3) M) and cytochalasin B (25 micrograms/ml), 100 micrograms/ml) at room temperature or at 37 degrees C also induce assembly of gap junctions. Assembly of the junctions proceeds even in the presence of a metabolic inhibitor (dinitrophenol) or of an inhibitor of protein synthesis (cycloheximide). We conclude that assembly of gap junctions can proceed from a pool of pre-existing precursors. The experimental conditions that result in gap-junction assembly involve perturbation of the cytoskeleton. Therefore, we propose that the assembly of gap junctions requires convergent migration of precursor molecules whose positional control in the membrane is released by perturbation of the cytoskeleton. Aggregates of particles and rugosities, whose distribution size and shape is similar to that of gap junctions, may represent intermediate assembly stages. This would indicate that the final stages in the assembly take place only after convergence of the precursor molecules to the junctional site and involve profound conformational changes required for establishment of fully assembled connexons.
The structure of gap junctions in the rabbit ciliary epithelium, corneal endothelium, and mouse stomach and liver was studied with the freeze-fracturing technique after rapid freezing to near 4 degrees K from the living state. In the ciliary epithelium, the connexons were randomly distributed, separated by smooth membrane matrix. In the corneal endothelium, both random and crystalline arrangements of the connexons were observed. In the stomach and liver, the connexons were packed but not crystalline. Experimental anoxia or lowered pH caused crystallization of the connexons within 20-30 min. In the ciliary epithelium, the effects of prolonged anoxia or low pH could not be reversed . In addition, invaginated or annular gap junctions increased in number, but their connexons were usually distributed at random. Rapid freezing thus demonstrates that gap junctions of different tissues are highly pleiomorphic in the living state, and this may explain their variations in structure after chemical fixation. The slow time-course and irreversibility of the morphological changes induced by prolonged anoxia or low pH suggest that connexon crystallization may be a long-term consequence rather than the morphological correlate of the switch to high resistance.
The ultrastructure of the gap junction may be visualized in both in situ and isolated preparations by using a variety of electron microscope techniques. The junction is composed of a lattice of subunits, called connexons, which show variable degrees of packing into a hexagonal lattice depending on a variety of poorly understood conditions. In general, it appears that more uncoupled and "dead" the junction, the more regular and condensed the hexagonal lattice becomes. It is not yet known whether these are "postmortem" changes or physiologically active and reversible changes involved in regulation of junctional permeability. Using a variety of techniques, it can be seen that the connexon extends completely across the junctional membranes, from the cytoplasmic surface of one cell to the cytoplasmic surface of the other, spanning the 2-nm "gap" between the apposed junctional membranes. Thus it is possible to implicate the connexon as a permeability channel from cytoplasm to cytoplasm, although the hydrophilic pore through the center of the connexon has not yet been demonstrated to span the full junction thickness. X-ray diffraction experiments support these conclusions, and the excellent correlation between the electron microscope and X-ray diffraction data lends great confidence to the interpretations of gap junction structure presented thus far. These data are summarized in the drawing in Figure 17. This is a scale drawing of two connexons, each of which is imagined to be composed of a dimer of hexamers. It must be emphasized that as yet there is no direct evidence for a sixfold symmetry within the connexon. Of special interest now are the types of protein-protein interactions that hold the two halves of the connexon together across the 2-nm gap and the lateral interactions between the connexons at the level of the lipid bilayers.
Maps of the spatial distribution of surface immunoglobulin on B lymphocytes obtained by freezeetch electron microscopy were analysed mathematically. The pictures were automatically scanned by a digital microdensitometer and the number and coordinates of the immunoglobulin molecules determined. A statistical measure, the radial distribution function, g(r), commonly used to study the structure of liquids, was computed for each map. The radial distribution function of surface immunoglobulin was found to closely resemble g(r) for fluids, indicating the presence of shortrange (but not long-range) order. It was determined that a given surface immunoglobulin molecule has a high probability of being surrounded by other immunoglobulins at distances approximately one-half the mean interparticle spacing. From this one can conclude that the distribution of surface immunoglobulin is non-random and is characterized by local clustering. The mean number of first nearest neighbors surrounding each surface immunoglobulin, computed from g(r), was found to be near two. This value is consistent with a degree of linear order in the topographic distribution of immunoglobulin. The radial distribution function provides a method of quantitating the amount of clustering in a spatial distribution. This function may prove useful in accessing the amount of receptor cross-linking necessary to trigger cellular responses, and in elucidating mechanisms for aggregation and movement of membrane macromolecules.
This study describes the effects ensuing from a non-specific interaction between membrane integral proteins and the surrounding lipids. The results are obtained using an appropriate molecular field theory to describe the ordering of membrane lipids. The modification of the lipid structure near a protein molecule, while most pronounced within the annulus of the first neighbour molecules, extends two or three layers beyond the annulus. The ordering of lipids within the annulus has a modified temperature dependence, and becomes a continuous function of temperature for low lipid/protein ratios. The change in order of lipid molecules surrounding a protein leads to an indirect, lipid-mediated interaction between membrane integral proteins. This interaction depends sensitively on the bulk lipid order. Under favourable circumstances, it gives rise to protein aggregation.
We have combined freeze-fracture and electrophysiological methods in a study of gap junction formation between reaggregated Novikoff hepatoma cells. Cell clumps are dissociated with EDTA, and the resulting single cells are allowed to reaggregate (5-180 min) in loose pellets in the presence of calcium at 37 degrees . The earliest electron microscopic evidence for the genesis of new junctions is the appearance of flattened regions of the plasma membrane with a relative paucity of small intramembranous particles. These regions contain instead loosely organized groupings of 9- to 11-nm intramembranous particles, which are seen on the A face of the fractured plasma membrane, while corresponding pits occur on the membrane B face. We have termed the specialized membrane regions "formation plaques." They are seen as early as 5 min after reaggregation and are quite numerous by 30 min. Larger plaques are observed at later times. Plaques seen at 30 min are consistently matched with other plaques on apposed cells, although the extracellular space separating these structures still exceeds 10 nm. By 60 min, some matched plaques display a reduced extracellular space, resembling that of normal gap junctions. Between 30 and 60 min, aggregates of closely packed particles on A faces and hexagonally arranged pits on B faces frequently appear in the formation plaques. The aggregates, which are indistinguishable from small gap junctions, appear to enlarge over the subsequent 2-hr period as the number of unaggregated 9- to 11-nm particles declines. Microelectrode studies demonstrate progressive increases in the percent of interfaces containing lowresistance junctions and in the degree of elctrical coupling in preparations incubated up to 2 hr. Coupling is first detected at about the same time as particle aggregates (or formation plaques with reduced extracellular spaces), and increases as aggregate sizes increase.
The pore patterns on a large number of nuclear and one non-nuclear membrane have been examined. Important qualitative features of the arrangement of pores can be described by computing a statistical measure, the radial distribution function, of the pore patterns. This measure distinguishes three different pore patterns: 1.1. Clumped patterns in which pores are found in clusters which are widely separated from one another.2.2. Patterns in which there is only a short range inhibitory region surrounding each pore with no evidence of a long range ordering.3.3. Patterns in which there is a local ordering in the region surrounding each pore, extending to several times the mean interpore spacing.Physical mechanisms which can lead to each of the observed patterns are discussed.
The pair distribution functions have been measured from freeze-fracture pictures of bacteriorhodopsin and rhodopsin recombinants with diacyl phosphatidylcholines (PC) of various hydrocarbon chain lengths. Pictures were used of samples that had been frozen from above the phase transition temperature of the lipid. Measured functions were compared with those calculated from two model interparticle potential energy functions, (a) a hard-disk repulsion only, and (b) a hard-disk repulsion plus electrostatic repulsion for a point charge buried in the membrane. The measured functions for bacteriorhodopsin di 12:0 PC, di 14:0 PC, and di 16:0 PC recombinants can be simulated using an interparticle hard-disk repulsion only. Bleached rhodopsin di 12:0 PC and di 18:1 trans-PC recombinants, and dark-adapted rhodopsin di 10:0 PC recombinants yield functions that are better simulated by assuming an additional repulsive interaction. The measured functions resemble those calculated using the hard-disk plus electrostatic repulsion model. The picture of dark-adapted rhodopsin in di 18:1 trans-PC frozen from 20 degrees C shows partial aggregation that is apparent in the measured pair distribution function. This attractive interaction persists even at 37 degrees C, where the measured function shows deviations from the hard-disk repulsive model, indicative of an attractive interparticle interaction. Implications of these results are discussed in terms of protein-lipid interactions.