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Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions
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Gap junctions allow direct communication between cells without recourse to the extracellular space and have been widely implicated as important mediators of cell-cell signalling. They are constructed from the connexin proteins, which form a large family, and individual connexins show complex spatial and temporal variations in their expression patterns. Understanding how this variation contributes to the control of intercellular signalling, both in the adult and during embryonic development, is an important problem that would be aided by reagents that interfere with gap junctional communication through specific connexins. We have begun to address this issue by raising antibodies to peptides derived from connexin43 and connexin32. Connexin43 peptides were located in the amino terminus, cytoplasmic loop and carboxytail. Connexin32 peptides came from the cytoplasmic loop and the first extracellular loop. Immunoblotting and immunostaining properties of purified IgGs were characterized on mouse heart, liver and the 8- to 16-cell mouse embryo. Effects on transfer through gap junctions were assessed in the fully compacted 8-cell mouse embryo by co-injection with Lucifer Yellow or Cascade Blue. Embryos were maintained in culture to assess the developmental consequences of injection. Peptide competition was used to confirm the specificity of immunostaining and inhibition of dye transfer. All connexin specific antibodies recognized their parent connexin on immunoblots and showed no 43/32 cross-reactivity. The connexin32 extracellular loop antibody recognized both connexin 32 and 43 on immunoblots, as predicted by the amino acid sequence homology in this region, but did not immunostain intact gap junctions. Connexin specific antibodies that immuno-stained showed the predicted connexin specificity. Antibodies to either connexin43 amino acids (AA) 1-16 (amino terminus) or AA 101-112 (cytoplasmic loop) neither immunostained nor prevented functional communication through 8-cell embryo gap junctions. Antibodies to AA 123-136 and AA 131-142 in the cytoplasmic loop immunostained heart and 8-cell embryo gap junctions and blocked transfer through them with high efficiency. Fab' fragments were equally effective. Peptide competition showed that both antibodies contained epitopes within AA 131-136 of connexin43. Antibodies against AA 313-324 in the carboxytail immunostained heart and the 8-cell embryo and, as IgGs, prevented dye transfer. Fab' fragments were ineffective. All connexin43 antibodies that blocked gap junctional communication between cells of the 8-cell mouse embryo induced non-communicating cells subsequently to withdraw from compaction.(ABSTRACT TRUNCATED AT 400 WORDS)
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... Connexin-mimetics initially were synthesized for use as epitopes for connexin antibody production [130,131]. The blocking antibodies raised using these peptides against the EL and IL domains of several connexins were tested as GJ blockers and demonstrated some effect in reducing dye transfer in chick embryos [116]. However, EL loop antibodies Gap7M (EL1/2), Gap11 (EL1/2 Cx32), Gap15 (IL of Cx43), and Gap17 (CT Cx40) were found to be ineffective in functional testing and were not able to reduce connexin-associated contractile responses in rabbit arterial tone [92]. ...
... Despite this, Dahl et al. described that peptides, used to generate antibodies, were effective in reducing Cx32 GJ formation and signaling in oocytes [132]. In 1995, Becker et al. found that connexin-mimetic peptides, mimicking the sequences of the IL domain, could effectively block GJ channels when directly injected into embryo cells [116]. In the subsequent decades, a number of connexin-mimetic peptides have been generated and tested with a multitude of proposed functions including blockade of hemichannel and GJ signaling, alterations in GJ formation, and disruption of protein localization and protein-protein interactions (Table 1). ...
... There is very little sequence homology in the connexin-IL, unlike the -EL regions, therefore peptides are presumably isoform-specific. Early peptide studies targeting the Cx43-IL, using peptides within the L2 region, GAP 13 and GAP 15 peptides, required direct injection of the peptides into cells to produce a functional block, due to the inability of the peptide to cross the plasma membrane [116]. Since then, many studies have linked cell-penetrating moieties that permit the membrane translocation of peptides. ...
Gap junctions (GJ) and connexins play integral roles in cellular physiology and have been found to be involved in multiple pathophysiological states from cancer to cardiovascular disease. Studies over the last 60 years have demonstrated the utility of altering GJ signaling pathways in experimental models, which has led to them being attractive targets for therapeutic intervention. A number of different mechanisms have been proposed to regulate GJ signaling, including channel blocking, enhancing channel open state, and disrupting protein-protein interactions. The primary mechanism for this has been through the design of numerous peptides as therapeutics, that are either currently in early development or are in various stages of clinical trials. Despite over 25 years of research into connexin targeting peptides, the overall mechanisms of action are still poorly understood. In this overview, we discuss published connexin targeting peptides, their reported mechanisms of action, and the potential for these molecules in the treatment of disease.
... Connexin-mimetics initially were synthesized for use as epitopes for connexin antibody production [127,128]. The blocking antibodies raised using these peptides against the EL and IL domains of several connexins were tested as GJ blockers and demonstrated some effect in reducing dye transfer in chick embryos [113]. However, EL loop antibodies Gap7M (EL1/2), Gap11 (EL1/2 Cx32), Gap15 (IL of Cx43), and Gap17 (CT Cx40) were found to be ineffective in functional testing and were not able to reduce connexin-associated contractile responses in rabbit arterial tone [89]. ...
... Despite this, Dahl et al. described that peptides, used to generate antibodies, were effective in reducing Cx32 GJ formation and signaling in oocytes [129]. In 1995, Becker et al. found that connexin-mimetic peptides, mimicking the sequences of the IL domain, could effectively block GJ channels when directly injected into embryo cells [113]. In the subsequent decades, a number of connexin-mimetic peptides have been generated and tested with a multitude of proposed functions including blockade of hemichannel and GJ signaling, alterations in GJ formation, and disruption of protein localization and protein-protein interactions ( Table 1). ...
... There is very little sequence homology in the connexin-IL, unlike the -EL regions, therefore peptides are presumably isoform specific. Early peptide studies targeting the Cx43-IL, using peptides within the L2 region, GAP 13 and GAP 15 peptides, required direct injection of the peptides into cells to produce a functional block, due to the inability of the peptide to cross the plasma membrane [113]. Since then, many studies have linked cell penetrating moieties that permit membrane translocation of peptides. ...
Gap junctions (GJ) and connexins play integral roles in cellular physiology and have been found to be involved in multiple pathophysiological states from cancer to cardiovascular disease. Studies over the last 60 years have demonstrated the utility of altering GJ signaling pathways in experimental models, which has led to them being attractive targets for therapeutic intervention. A number of different mechanisms have been proposed to regulate GJ signaling, including channel blocking, enhancing channel open state, and disrupting protein-protein interactions. The primary mechanism for this has been through the design of numerous peptides as therapeutics, that are either currently in early development or are in various stages of clinical trials. Despite over 25 years of research into connexin targeting peptides, the overall mechanisms of action are still poorly understood. In this overview, we discuss published connexin targeting peptides, their reported mechanisms of action and the potential for these molecules in the treatment of disease.
... The amino acid sequences of Cx43 ELs have been frequently used as templates for the design of mimetic peptide blockers. Significantly, 43Gap26 (VCYDKSFPISHVR) and 43Gap27 (SRPTEKTIFII) are among the first peptide mimetics of Cx43 ELs [32,33]. These peptides were initially shown to inhibit gap junctional intercellular communication (GJIC) by reducing gap junction formation presumptively through their complementary interaction with the ELs, thereby preventing the docking of oppos- ing Cx43HC [4,12,16,32,33]. ...
... Significantly, 43Gap26 (VCYDKSFPISHVR) and 43Gap27 (SRPTEKTIFII) are among the first peptide mimetics of Cx43 ELs [32,33]. These peptides were initially shown to inhibit gap junctional intercellular communication (GJIC) by reducing gap junction formation presumptively through their complementary interaction with the ELs, thereby preventing the docking of oppos- ing Cx43HC [4,12,16,32,33]. The blocking effects of 43Gap26 and 43Gap27 on Cx43HC have been tested in a broad variety of cells and were shown to occur within minutes after administration, suggesting that existing CxHC are indeed targeted by these peptide blockers [34,35]. ...
The connexin protein family consists of approximately 20 members, and is well recognized as the structural unit of the gap junction channels that perforate the plasma membranes of coupled cells and, thereby, mediate intercellular communication. Gap junctions are assembled by two preexisting hemichannels on the membranes of apposing cells. Non-junctional connexin hemichannels (CxHC) provide a conduit between the cell interior and the extracellular milieu, and are believed to be in a protectively closed state under physiological conditions. The development and characterization of the peptide mimetics of the amino acid sequences of connexins have resulted in the development of a panel of blockers with a higher selectivity for CxHC, which have become important tools for defining the role of CxHC in various biological processes. It is increasingly clear that CxHC can be induced to open by pathogen-associated molecular patterns. The opening of CxHC facilitates the release of damage-associated molecular patterns, a class of endogenous molecules that are critical for the pathogenesis of inflammatory diseases. The blockade of CxHC leads to attenuated inflammation, reduced tissue injury and improved organ function in human and animal models of about thirty inflammatory diseases and disorders. These findings demonstrate that CxHC may contribute to the intensification of inflammation, and serve as a common target in the treatments of various inflammatory diseases. In this review, we provide an update on the progress in the understanding of CxHC, with a focus on the role of these channels in inflammatory diseases.
... Another strategy to specifically inhibit Gj involves the development of connexin antibodies. Using fragments of antibodies such as Gap13 or Gap15 to target the C-terminus regions of connexin43 has demonstrated their efficiency at blocking Gj [201]. Interestingly, considering that connexin43 has also been reported to promote the bystander effect of chemotherapeutic drugs, targeting connexin43 could combined with treatment for chemotherapy. ...
Despite research and clinical advances during recent decades, bone cancers remain a leading cause of death worldwide. There is a low survival rate for patients with primary bone tumors such as osteosarcoma and Ewing’s sarcoma or secondary bone tumors such as bone metastases from prostate carcinoma. Gap junctions are specialized plasma membrane structures consisting of transmembrane channels that directly link the cytoplasm of adjacent cells, thereby enabling the direct exchange of small signaling molecules between cells. Discoveries of human genetic disorders due to genetic mutations in gap junction proteins (connexins) and experimental data using connexin knockout mice have provided significant evidence that gap-junctional intercellular communication (Gj) is crucial for tissue function. Thus, the dysfunction of Gj may be responsible for the development of some diseases. Gj is thus a main mechanism for tumor cells to communicate with other tumor cells and their surrounding microenvironment to survive and proliferate. If it is well accepted that a low level of connexin expression favors cancer cell proliferation and therefore primary tumor development, more evidence is suggesting that a high level of connexin expression stimulates various cellular process such as intravasation, extravasation, or migration of metastatic cells. If so, connexin expression would facilitate secondary tumor dissemination. This paper discusses evidence that suggests that connexin 43 plays an antagonistic role in the development of primary bone tumors as a tumor suppressor and secondary bone tumors as a tumor promoter.
... To facilitate direct quantitative comparison, all tissue generated from a single experimental run were stained in parallel on the same day. The protocol used was adapted from that described by Becker et al. (1995). Slides were defrosted at room temperature, a wax ring was drawn around the sections using a PAP pen and a drop of PBS placed on the sections to rehydrate them. ...
The albino visual system is characterised by a range of abnormalities including a severe rod deficit, an underdevelopment of the central retina and a misprojection of a portion of the temporal ganglion cells. During development of the retina levels of proliferation and apoptosis are elevated, and the maturational gradient is delayed. There is evidence from in vitro studies to suggest that L-DOPA, a precursor in both the melanin and catecholamine synthesis pathways, is able to regulate these abnormal patterns of cell proliferation in a manner which is consistent with its role in the regulation of retinal development. The aims of this thesis were; (1) to further characterise the developmental abnormalities associated with albinism, (2) to examine the effects of L-DOPA on patterns of cell division and neurogenesis within an in vivo context, and (3) to determine the mechanisms by which L-DOPA mediates its effects. Using both in vivo and in vitro methods, patterns of cell division and retinal differentiation were examined in pigmented and albino rat tissue. Results demonstrate that coincidental with abnormal levels of proliferation, the early postnatal albino retina is characterised by; a thickening of the neuroblastic layer, an excess of cells in the cell cycle, defects in the distribution of cleavage orientations within the apical-basal plane and elevations in gap junction expression. Furthermore, the vast majority of these parameters could be regulated by the introduction of L-DOPA to the retina and -preliminary experiments would suggest- through the addition of dopamine. These findings are consistent with a model in which reduced levels of L-DOPA in the albino retina lead to spatiotemporal defects in patterns of cell division, potentially as a result of secondary consequences on concentrations of retinal dopamine.
Die Rolle von Connexinen und Gap Junction-vermittelter Kommunikation in pluripotenten Stammzellen sowie der frühen Embryonalentwicklung sind bis heute nicht vollständig aufgeklärt. Mutationen in humanen Connexinen verursachen eine Vielzahl von Krankheiten. Connexin-defiziente iPS Zellen stellen eine gute Basis für die Erforschung der Rolle von Connexinen während der Embryonalentwicklung und bei der Krankheitsentstehung dar. Das Ziel der vorliegenden Arbeit war es, das CRISPR/Cas9-System in pluripotenten Stammzellen erfolgreich anzuwenden und ein Protokoll zur Erstellung verschiedener Cx43-Defektmutanten zu entwerfen. Nach der Etablierung der CRSIPR/Cas9-Methode in HEK293T-Zellen konnte in der vorliegenden Arbeit darüber hinaus erfolgreich eine Cx43-Defizienz in FSiPS-Zellen erzeugt werden. Weiterhin wurden mehrere Cx43-Mutanten geschaffen und initial auf Pluripotenzmarker und ihr Differenzierungspotential untersucht. Diese Arbeit bildet die Basis für weitere Untersuchungen des Cx43 in iPS-Zellklonen und davon abgeleiteten Zelltypen sowie artifiziellen 3D-Gewebekulturen. Darüber hinaus bildet sie die Grundlage für die Bildung weiterer Connexin-Defektmutanten sowie von iPS-Zellen mit krankheitsrelevanten Mutationen.
Connexin (Cx43)-formed channels have been linked to cardiac arrhythmias and diseases of the heart associated with myocardial tissue loss and fibrosis. These pathologies include ischemic heart disease, ischemia-reperfusion injury, heart failure, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and Duchenne muscular dystrophy. A number of Cx43 mimetic peptides have been reported as therapeutic candidates for targeting disease processes linked to Cx43, including some that have advanced to clinical testing in humans. These peptides include Cx43 sequences based on the extracellular loop domains (e.g., Gap26, Gap 27, and Peptide5), cytoplasmic-loop domain (Gap19 and L2), and cytoplasmic carboxyl-terminal domain (e.g., JM2, Cx43tat, CycliCX, and the alphaCT family of peptides) of this transmembrane protein. Additionally, RYYN peptides binding to the Cx43 carboxyl-terminus have been described. In this review, we survey preclinical and clinical data available on short mimetic peptides based on, or directly targeting, Cx43, with focus on their potential for treating heart disease. We also discuss problems that have caused reluctance within the pharmaceutical industry to translate peptidic therapeutics to the clinic, even when supporting preclinical data is strong. These issues include those associated with the administration, stability in vivo, and tissue penetration of peptide-based therapeutics. Finally, we discuss novel drug delivery technologies including nanoparticles, exosomes, and other nanovesicular carriers that could transform the clinical and commercial viability of Cx43-targeting peptides in treatment of heart disease, stroke, cancer, and other indications requiring oral or parenteral administration. Some of these newly emerging approaches to drug delivery may provide a path to overcoming pitfalls associated with the drugging of peptide therapeutics.
Anne Warner applied physiological techniques to developmental biology, elucidating the mechanisms of cell interaction and communication that pattern the early embryo. Through her determination and passion for science, she contributed crucial discoveries in the fields of muscle physiology, cellular differentiation and gap junction communication. She spent the majority of her career at University College London, which became her intellectual home and where she acquired a Royal Society Foulerton Research Professorship, becoming a highly respected and influential figure. In her work on gap junctions, Anne was the first to show that embryonic development and patterning required gap junctions, and that the restriction of junctional communication between cells played a key role in tissue differentiation. Anne excelled in her breadth of vision across research and its interdisciplinary possibilities. In 1998 she established the CoMPLEX Centre for systems biology at UCL, bringing her own group together with scientists from across the STEM subjects to build testable mathematical models of biological systems across multiple scales. Indefatigable in her capacity for leadership and committee work, she assumed an eclectic set of roles across a large span of research organizations and professional societies, and had a lifelong association with the Physiological Society. In 1984 she founded the Microelectrodes course at the Plymouth Marine Biology Laboratory, which has trained generations in the art of electrophysiology and still continues today. With uncompromisingly high standards, she inspired her mentees to be ambitious and fearless, and established postdoctoral fellowships to help the young scientists who followed after her.
Neocaridina davidi is a freshwater shrimp that originates from Taiwan and is commonly bred all over the word. Like all decapods, which develop indirectly, this species has pelagic larvae that may differ entirely in their morphology and habits from adult specimens. To fill a gap of knowledge about the developmental biology of freshwater shrimps we decided to document the 3D‐localization of the midgut inside the body cavity of larval stages of N. davidi using X‐ray microtomography, and to describe all structural and ultrastructural changes of the midgut epithelium (intestine and hepatopancreas) which occur during postembryonic development of N. davidi using light and transmission electron microscopy. We laid emphasis on stem cell functioning and cell death processes connected with differentiation. Our study revealed that while the intestine in both larval stages of N. davidi has the form of a fully developed organ, which resembles that of adult specimens, the hepatopancreas undergoes elongation and differentiation. E‐cells, which are midgut stem cells, due to their proliferation and differentiation are responsible for the above‐mentioned processes. Our study revealed that apoptosis is a common process in both larval stages of N. davidi in the intestine and proximal region of the hepatopancreas. In zoea III, autophagy as a survival factor is activated in order to protect cells against their death. However, when there are too many autophagic structures in epithelial cells, necrosis as passive cell death is activated. The presence of all types of cell death in the midgut in the zoea III stage confirms that this part of the digestive tract is fully developed and functional. Here, we present the first description of apoptosis, autophagy and necrosis in the digestive system of larval stages of Malacostraca and present the first description of their hepatopancreas elongation and differentiation due to midgut stem cell functioning.
This thesis investigates the roles of neurotransmitters and their receptors, changes in intracellular calcium ([Ca2+]i), and the retinal pigment epithelium (RPE) in the regulation of cell proliferation and migration in the developing chick retina. Confocal imaging of retinae is used to show that before synapses are formed, cells in the ventricular zone (VZ) display intermittent spontaneous [Ca2+]i transients that depend upon the endogenous release of neurotransmitters. Purinergic and muscarinic receptor- evoked transients occur in a mixed population of interphase and mitotic cells. Those produced by GABAergic and glutamatergic receptors are mostly restricted to the interphase population. Muscarinic receptor activation is shown to slow down, and purinergic activation to speed up, mitosis. These actions may result from the [Ca2+]i transients these agonists evoke. GABA and glutamate receptor activation are without effect on mitosis. The nuclei of retinal progenitor cells (PCs) migrate back-and-forth across the retina in a process called interkinetic nuclear migration (INM). To study the possible influence of neurotransmitter receptor activation on INM, a 'gene gun' technique was used to label cells in the VZ; the speed of movement of some cells is influenced by neurotransmitters. [Ca2+]i transients occur in cells during INM, which may be important in its regulation. Gap junctional communication between the RPE and the neural retina was investigated. Ca2+ -imaging experiments show that gap junctions support the spread of spontaneous Ca2+ signals between neighbouring cells. Whole-cell patch clamp recording was used to fill VZ cells with a combination of gap junction-permeable and impermeable dyes. These injections show that gap junctions couple PCs into clusters that largely exclude differentiated neurons. Coupling was also observed between cells in the RPE and the neural retina. These pathways may be important in the regulation of proliferation. The RPE is shown to express both purinergic and muscarinic receptors and to have a profound influence on the rate of cell proliferation in the neural retina. The RPE may speed mitosis in the retina through the release of ATP and other factors.
Analysis by SDS-PAGE of gap junction fractions isolated from heart suggests that the junctions are comprised of a protein with an Mr 43,000. Antibodies against the electroeluted protein and a peptide representing the 20 amino terminal residues bind specifically on immunoblots to the 43-kD protein and to the major products arising from proteolysis during isolation. By immunocytochemistry, the protein is found in ventricle and atrium in patterns consistent with the known distribution of gap junctions. Both antibodies bind exclusively to gap junctions in fractions from heart examined by EM after gold labeling. Since only domains of the protein exposed at the cytoplasmic surface should be accessible to antibody, we conclude that the 43-kD protein is assembled in gap junctions with the amino terminus of the molecule exposed on the cytoplasmic side of the bilayer, that is, on the same side as the carboxy terminus as determined previously. By combining proteolysis experiments with data from immunoblotting, we can identify a third cytoplasmic region, a loop of some 4 kD between membrane protected domains. This loop carries an antibody binding site. The protein, if transmembrane, is therefore likely to cross the membrane four times. We have used the same antisera to ascertain if the 43-kD protein is involved in cell-cell communication. The antiserum against the amino terminus blocked dye coupling in 90% of cell pairs tested; the antiserum recognizing epitopes in the cytoplasmic loop and cytoplasmic tail blocked coupling in 75% of cell pairs tested. Preimmune serum and control antibodies (one against MIP and another binding to a cardiac G protein) had no or little effect on dye transfer. Our experimental evidence thus indicates that, in spite of the differences in amino acid sequence, the gap junction proteins in heart and liver share a general organizational plan and that there may be several domains (including the amino terminus) of the molecule that are involved in the control of junctional permeability.
Analysis by SDS-PAGE of gap junction fractions isolated from heart suggests that the junctions are comprised of a protein with an Mr 43,000. Antibodies against the electroeluted protein and a peptide representing the 20 amino terminal residues bind specifically on immunoblots to the 43-kD protein and to the major products arising from proteolysis during isolation. By immunocytochemistry, the protein is found in ventricle and atrium in patterns consistent with the known distribution of gap junctions. Both antibodies bind exclusively to gap junctions in fractions from heart examined by EM after gold labeling. Since only domains of the protein exposed at the cytoplasmic surface should be accessible to antibody, we conclude that the 43-kD protein is assembled in gap junctions with the amino terminus of the molecule exposed on the cytoplasmic side of the bilayer, that is, on the same side as the carboxy terminus as determined previously. By combining proteolysis experiments with data from immunoblotting, we can identify a third cytoplasmic region, a loop of some 4 kD between membrane protected domains. This loop carries an antibody binding site. The protein, if transmembrane, is therefore likely to cross the membrane four times. We have used the same antisera to ascertain if the 43-kD protein is involved in cell-cell communication. The antiserum against the amino terminus blocked dye coupling in 90% of cell pairs tested; the antiserum recognizing epitopes in the cytoplasmic loop and cytoplasmic tail blocked coupling in 75% of cell pairs tested. Preimmune serum and control antibodies (one against MIP and another binding to a cardiac G protein) had no or little effect on dye transfer. Our experimental evidence thus indicates that, in spite of the differences in amino acid sequence, the gap junction proteins in heart and liver share a general organizational plan and that there may be several domains (including the amino terminus) of the molecule that are involved in the control of junctional permeability.
The membrane topology of connexin32, a principal polypeptide of gap junctions in diverse cell types, has been studied in rat and mouse hepatocyte gap junctions using site-specific antisera raised against synthetic oligopeptides corresponding to amino acid sequences deduced from cDNA clones. Based on published hydropathicity maps and identified protease-sensitive cleavage sites, oligopeptides were synthesized corresponding to two hydrophilic domains of connexin32, one predicted to face the cytoplasm, the other predicted to be directed extracellularly. Antisera were raised to keyhole limpet hemocyanin conjugates of the oligopeptides and used to map the distribution of their antigens using indirect immunocytochemistry on isolated gap junctions. The results directly demonstrated the cytoplasmic orientation of an antigen contained within amino acids 98-124 of the connexin32 sequence. The extracellular space in intact, isolated gap junctions is too small to permit binding of antibody molecules, necessitating the experimental separation of the junctional membranes to expose their extracellular surfaces using a urea/alkali procedure. While an antigen contained within amino acids 164-189 was visualized on the extracellular surfaces of some of the separated junctional membranes, variability in the observations and in the splitting procedure left ambiguities concerning the biological relevance of the observations after the denaturing conditions necessary to separate the junctional membranes. Using a different approach, however, the antigen could be exposed in intact liver using a hypertonic disaccharide junction-splitting procedure. The period of time of antigen exposure at the cell surface appears to peak at 30 s and disappear by 2-4 min. Taken together, these data demonstrate the extracellular orientation of an antigen contained within amino acids 164-189, which may be involved in cell-cell interaction within the gap junction.
Rat heart and other organs contain mRNA coding for connexin43, a polypeptide homologous to a gap junction protein from liver (connexin32). To provide direct evidence that connexin43 is a cardiac gap junction protein, we raised rabbit antisera directed against synthetic oligopeptides corresponding to two unique regions of its sequence, amino acids 119-142 and 252-271. Both antisera stained the intercalated disc in myocardium by immunofluorescence but did not react with frozen sections of liver. Immunocytochemistry showed anti-connexin43 staining of the cytoplasmic surface of gap junctions in isolated rat heart membranes but no reactivity with isolated liver gap junctions. Both antisera reacted with a 43-kD polypeptide in isolated rat heart membranes but did not react with rat liver gap junctions by Western blot analysis. In contrast, an antiserum to the conserved, possibly extracellular, sequence of amino acids 164-189 in connexin32 reacted with both liver and heart gap junction proteins on Western blots. These findings support a topological model of connexins with unique cytoplasmic domains but conserved transmembrane and extracellular regions. The connexin43-specific antisera were used by Western blots and immunofluorescence to examine the distribution of connexin43. They demonstrated reactivity consistent with gap junctions between ovarian granulosa cells, smooth muscle cells in uterus and other tissues, fibroblasts in cornea and other tissues, lens and corneal epithelial cells, and renal tubular epithelial cells. Staining with the anti-connexin43 antisera was never observed to colocalize with antibodies to other gap junctional proteins (connexin32 or MP70) in the same junctional plaques. Because of limitations in the resolution of the immunofluorescence, however, we were not able to determine whether individual cells ever simultaneously express more than one connexin type.
We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.
Fundamental to the understanding of mouse limb morphogenesis and pattern formation is the need to elucidate the spatial and temporal distribution of gap junction proteins (connexins, Cx) and cell-cell communication compartments. To this end, we used immunofluorescence and confocal microscopy together with 3-dimensional reconstruction software to map the distribution of Cx43 and Cx32 in 11–14.5 days postcoitum (dpc) mouse limbs. Cx43 was strictly localized to the apical ectodermal ridge (AER) and non-ridge ectoderm throughout all stages of mouse limb development studied. Cx32, on the other hand, was abundant in the mesenchyme with only low levels of expression in the 11–13.5 dpc ectoderm. However, at 14–14.5 dpc there was a clear increase in Cx32 expression in the ectoderm. Double labeling for connexins and confocal microscopy revealed Cx43 and Cx32 in the same optical section of the basal cells of the ectoderm but in separate plaques. Lucifer yellow dye injections showed that the cells of the AER were in direct communication with the nonridge ectoderm but dye was never observed to spread to the mesenchyme. Cells of the mesenchyme were coupled to each other but to a much lesser extent than cells of the ectoderm. Finally, although there was an increase in Cx32 expression in the ectoderm at 14–14.5 dpc, this was not correlated with any detectable change in communication compartments. Thus, the lack of dye transfer between the ectoderm and underlying mesenchyme from the peak of AER height through its decline suggests that bulk transfer of morphogens between these two layers is not necessary for mouse limb development. © 1993 Wiley-Liss, Inc.
In the heart, individual cardiac muscle cells are linked by gap junctions. These junctions form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all the cells of the myocardium, ensuring their synchronous contraction. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera were raised to three synthetic peptides, each matching different cytoplasmically exposed portions of the sequence of connexin43, the major gap-junctional protein reported in the heart. The specificity of each antiserum for the peptide to which it was raised was established by dot blotting. New methods were developed for isolating enriched fractions of gap junctions from whole heart and from dissociated adult myocytes, in which detergent-treatment and raising the temperature (potentially damaging steps in previously described techniques) are avoided. Analysis of these fractions by SDS-polyacrylamide gel electrophoresis revealed major bands at 43 kDa (matching the molecular mass of connexin43) and at 70 kDa. Western blot experiments using our antisera indicated that both the 43-kDa and the 70-kDa bands represent cardiac gap-junctional proteins. Pre-embedding immunogold labelling of isolated gap junctions and post-embedding immunogold labelling of Lowicryl-embedded whole tissue demonstrated the specific binding of the antibodies to ultrastructurally defined gap junctions. One antiserum (raised to residues 131–142) was found to be particularly effective for cytochemical labelling. Using this antiserum for immunofluorescence labelling in combination with confocal scanning laser microscopy enabled highly sensitive detection and three-dimensional mapping of gap junctions through thick slices of cardiac tissue. By means of the serial optical sectioning ability of the confocal microscope, images of the entire gap junction population of complete en face-viewed disks were reconstructed. These reconstructions reveal the presence of large junctions arranged as a peripheral ring around the disk, with smaller junctions in an interior zone: an arrangement that may facilitate efficient intercellular transfer of current. By applying our immunolabelling techniques to tissue from hearts removed from transplant patients with advanced ischaemic heart disease, we have demonstrated that gap junction distribution between myocytes at the border zone of healed infarcts is markedly disordered. This abnormality may contribute to the genesis of reentrant arrhythmias in ischaemic heart disease.
Intracellular pH was measured in normal 8-cell stage mouse embryos and in embryos from a cross between DDK females and C3H males. DDK/C3H embryos display the DDK syndrome and spontaneously begin to decompact toward the late 16-cell stage. Ultimately, 90% fail to form blastocysts. Normal embryos have a resting intracellular pH close to neutrality. In DDK/C3H embryos a substantial proportion (46%) has an intracellular pH below 6.7. An equivalent proportion of DDK/C3H embryos was found previously to show slow communication through gap junctions at the 8-cell stage. This is probably a consequence of low intracellular pH. In normal embryos the weak acid, butyric acid, decreased intracellular pH and slowed the transfer of Lucifer Yellow through gap junctions. Normal embryos treated with butyrate for between 1 and 6 hr beginning at the 8-cell stage and cultured for 24 hr, reproduced the DDK/C3H phenotype. After 48 hr some butyrate treated embryos recovered, while others remained as decompacted morulae. Treatment of control and DDK/C3H 8-cell stage embryos with dibutyryl cyclic AMP or forskolin, which will increase intracellular cyclic AMP, speeded gap junctional communication. Forskolin treatment prevented expression of the DDK syndrome in DDK/C3H embryos, although the rescue was transient and the syndrome returned when forskolin was removed. The finding that the DDK syndrome is manifested as low intracellular pH may provide clues to the molecular basis of the defect.
The membrane topology and quaternary structure of rat cardiac gap junction ion channels containing α1, connexin (i.e. Cx43) have been examined using anti-peptide antibodies directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold electron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic “loop” domain of at least Mr = 5100 (residues (101 to 142) is readily accessible to peptide antibody labeling. The native Mr = 43,000 protein can be protease-cleaved on the cytoplasmic side of the membrane, resulting in an Mr ≈ 30,000 membrane-bound fragment. Western immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252–271. Immunoelectron microscopy demonstrated that the Mr ≈ 13,000 carboxy-terminal peptide(s) is released after protease cleavage and does not remain attached to the Mr ≈ 30,000 membrane-bound fragment via non-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of the α1 polypeptide. Therefore, the Mr ≈ 13,000 carboxy-terminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexons indicates conservation in the molecular design of the gap junction channels formed by α or β connexins.
We chemically synthesized 20 peptides corresponding to 75% of the HA1 molecule of the influenza virus. Antibodies to the majority (18) of these peptides were capable of reacting with the hemagglutinin molecule. These 18 peptides are not confined to the known antigenic determinants of the hemagglutinin molecule, but rather are scattered throughout its three-dimensional structure. In contrast, antibody raised to intact hemagglutinin did not react with any of the 20 peptides. Taken together these results suggest that the immunogenicity of an intact protein molecule is not the sum of the immunogenicity of its pieces.