Confocal Laser and Electron Microscopic Investigation of Gap Junctions in Anaplastic Astrocytomas.

Connexin 43 (Cx43) is a multifunction protein that forms gap junction channels and hemichannels and is suggested to play an essential role in oxygen-glucose deprivation, induced via neuroinflammation during astrocytoma expansion into healthy tissue. To prove this assumption we studied connexin 43 localisation and ultrastructure of gap junctions in samples of malignant brain tumour (anaplastic astrocytomas grade III). For confocal laser microscopy, vibratome sections of tumour fragments were incubated in a mixture of primary antibodies to connexin 43 and glial fibrillary acidic protein (GFAP), then in a mixture of secondary antibodies conjugated with a fluorescent label. After the immunofluorescence study, sections were washed in phosphate buffer, additionally postfixed with 1% OsO4 solution, dehydrated and embedded in epoxy resin by a plane-parallel method. Ultra-thin sections obtained from these samples were contrasted with uranyl acetate and lead citrate and viewed under a Jem 1011 electron microscope. Confocal laser examination detected a positive reaction to Cx43 in the form of point fluorescence. These points were of various sizes. Most of them were localised around or at the intersection of small processes containing GFAP. Electron microscopy of the tumour samples containing the most significant number of Cx43 revealed single and closely spaced gap junctions with a typical ultrastructure on the processes and bodies of tumour cells. Sequential analysis in the fields of view revealed 62 gap junctions in the area of 100 µm2. Numerous gap junctions in anaplastic astrocytomas revealed in our study may indicate electrotonic and metabolic transmission between glioma cells, possibly promoting its progression.

Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects.

The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.

Cardiac Connexin-43 Hemichannels and Pannexin1 Channels: Provocative Antiarrhythmic Targets.

Cardiac connexin-43 (Cx43) creates gap junction channels (GJCs) at intercellular contacts and hemi-channels (HCs) at the peri-junctional plasma membrane and sarcolemmal caveolae/rafts compartments. GJCs are fundamental for the direct cardiac cell-to-cell transmission of electrical and molecular signals which ensures synchronous myocardial contraction. The HCs and structurally similar pannexin1 (Panx1) channels are active in stressful conditions. These channels are essential for paracrine and autocrine communication through the release of ions and signaling molecules to the extracellular environment, or for uptake from it. The HCs and Panx1 channel-opening profoundly affects intracellular ionic homeostasis and redox status and facilitates via purinergic signaling pro-inflammatory and pro-fibrotic processes. These conditions promote cardiac arrhythmogenesis due to the impairment of the GJCs and selective ion channel function. Crosstalk between GJCs and HCs/Panx1 channels could be crucial in the development of arrhythmogenic substrates, including fibrosis. Despite the knowledge gap in the regulation of these channels, current evidence indicates that HCs and Panx1 channel activation can enhance the risk of cardiac arrhythmias. It is extremely challenging to target HCs and Panx1 channels by inhibitory agents to hamper development of cardiac rhythm disorders. Progress in this field may contribute to novel therapeutic approaches for patients prone to develop atrial or ventricular fibrillation.

Localization of connexin 43 gap junctions and hemichannels in tanycytes of adult mice.

Tanycytes are specialized glial cells lining the lateral walls and the floor of the third ventricle behind the optic chiasm. In addition to functioning as barrier cells, they also have an important role in the regulation of neuroendocrine axes and energy homeostasis. To determine whether tanycytes communicate with each other via Connexin 43 (Cx43) gap junctions, individual tanycytes were loaded with Lucifer yellow (LY) through a patch pipette. In all cases, LY filled a larger group of tanycytes as well as blood vessels adjacent to tanycyte processes. The Cx43-blocker, carbenoxolone, inhibited spreading of LY. The greatest density of Cx43-immunoreactive spots was observed in the cell membrane of α-tanycyte cell bodies. Cx43-immunoreactivity was also present in the membrane of ß-tanycyte cell bodies, but in lower density. Processes of both types of tanycytes also contained Cx43-immunoreactivity. At the ultrastructural level, Cx43-immunoreactivity was present in the cell membrane of all types of tanycytes including their ventricular surface, but gap junctions were more frequent among α-tanycytes. Cx43-immunoreactivity was also observed in the cell membrane between contacting tanycyte endfeet processes, and between tanycyte endfeet process and axon varicosities in the external zone of the median eminence and capillaries in the arcuate nucleus and median eminence. These results suggest that gap junctions are present not only among tanycytes, but also between tanycytes and the axons of hypophysiotropic neurons. Cx43 hemichannels may also facilitate the transport between tanycytes and extracellular fluids, including the cerebrospinal fluid, extracellular space of the median eminence and bloodstream.

Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood.

Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.

Structure and permeability of ion-channels by integrated AFM and waveguide TIRF microscopy.

Membrane ion channels regulate key cellular functions and their activity is dependent on their 3D structure. Atomic force microscopy (AFM) images 3D structure of membrane channels placed on a solid substrate. Solid substrate prevents molecular transport through ion channels thus hindering any direct structure-function relationship analysis. Here we designed a ~70 nm nanopore to suspend a membrane, allowing fluidic access to both sides. We used these nanopores with AFM and total internal reflection fluorescence microscopy (TIRFM) for high resolution imaging and molecular transport measurement. Significantly, membranes over the nanopore were stable for repeated AFM imaging. We studied structure-activity relationship of gap junction hemichannels reconstituted in lipid bilayers. Individual hemichannels in the membrane overlying the nanopore were resolved and transport of hemichannel-permeant LY dye was visualized when the hemichannel was opened by lowering calcium in the medium. This integrated technique will allow direct structure-permeability relationship of many ion channels and receptors.

Light- and transmission-electron-microscopic investigations on distribution of CD44, connexin 43 and actin cytoskeleton during the foreign body reaction to a nanoparticular hydroxyapatite in mini-pigs.

Foreign body giant cells (FBGCs) are formed by fusion of mononucleated macrophages during the foreign body response to a nanoparticulate hydroxyapatite (HA) implanted in defects of mini-pig femura. The molecular mechanisms underlying the formation of FBGCs are still largely obscure. Here we propose connexin 43 (cx43) and CD44 as candidate molecules involved in the fusion process. Immunohistochemistry and ultrastructural immunogold labeling indicated that cx43 is present within the ruffled border of FBGCs and is the main component of gap junctions formed between fusing macrophages. CD44 was strongly expressed during clustering and fusion of mononucleated macrophages. FBGCs adhering apically at the implanted HA showed CD44 reactivity only along the basolateral aspects of the plasma membranes, while podosome formation was observed within the sealing zone and ruffled border. Taken together, these findings demonstrate that cx43 and CD44 are part of the fusion machinery responsible for the formation of FBGCs. Furthermore, the results of microfilament and cx43 labeling suggest a functional role for podosomes and hemi-channels in biomaterial degradation.

Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria.

Cardiomyocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ in their respiratory and calcium retention capacity. Connexin 43 (Cx43) is located at the inner membrane of SSM, and Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). The function of Cx43-formed channels is regulated in part by phosphorylation at residues in the carboxy terminus of Cx43. The aim of the present study was (1) to investigate whether Cx43 is also present in IFM, and (2) to characterize its spatial orientation in the inner mitochondrial membrane (IMM). Confirming previous findings, ADP-stimulated respiration was greater in IFM than in SSM from rat ventricles. In preparations from rats and mice not contaminated with sarcolemmal proteins, Cx43 was exclusively detected in SSM, but not in IFM by Western blot analysis (n = 6). SSM were exposed to different proteinase K concentrations to cleave peptide bonds, and Western blot analysis was performed for ATP synthase alpha (IMM, subunit in the matrix), uncoupling protein 3 (UCP3, IMM, intermembrane space epitope), and manganese superoxide dismutase (MnSOD, matrix). At a proteinase K concentration of 50 microg/ml, immunoreactivities of all the analyzed proteins were completely lost. The use of 5 microg/ml proteinase K resulted in similarly reduced immunoreactivities for Cx43 (19.4 +/- 5.8% of untreated mitochondria, n = 6) and UCP3 (23.0 +/- 4%, n = 7), whereas the immunoreactivities of ATP synthase alpha (49.1 +/- 6.4%, n = 7) and MnSOD (79.9 +/- 17.4%, n = 6) were better preserved, suggesting that the carboxy terminus of Cx43 is directed towards the intermembrane space. The results were confirmed in digitonin-treated mitochondria. Taken together, Cx43 is exclusively localized in SSM, with its carboxy terminus directed towards the intermembrane space. Since loss of mitochondrial Cx43 abolishes IP's cardioprotection, SSM and IFM apparently differ in their function in the signal transduction of IP.

Down regulation of immuno-detectable cardiac connexin-43 in BALB/c mice following acute fasting.

Acute starvation effects for connexin-43 protein expression, in the heart, had not been previously explored. Hence we examined acute fasting on the myocardial immuno-histochemical expression of connexin-43 in 3 groups of 8-week old female BALB/c mice. Groups consisted of control mice (n=5), fasting for 24 h (N=5) and 48 h (N=3). Under light microscopy all control fed cases revealed the presence of some immuno-detectable staining for connexin-43 that is either present or weakly observed in some or all of the regions of interest, that include the cross-sectional left ventricular sub-endocardium, mid-myocardium and papillary muscle. Whereas mice that underwent 24 or 48 h of acute starvation, connexin-43 expression was either difficult to detect visually (N=3) or was completely absent (N=5) at 40x magnification using a light microscope. In starved mice with no membrane staining for connexin-43 we observed an increase in the intracellular accumulation of cytoplasmic connexin-43 expression.

Bootstrap resampling for voxel-wise variance analysis of three-dimensional density maps derived by image analysis of two-dimensional crystals.

Difference density maps are commonly used in structural biology for identifying conformational changes in macromolecular complexes. For interpretation of the results, it is essential to estimate the variance or standard deviation of the difference density and the distribution of errors in space. In order to compare three-dimensional density maps of gap junction channels with and without the C-terminal regulatory domain, we developed a bootstrap resampling method for estimation of the voxel-wise standard deviation. The bootstrap approach has been successfully used for estimating the sampling distribution from a limited data set and for estimating the statistical properties of the derived quantities [Efron, B., 1979. Bootstrap methods: another look at the jackknife. Ann. Stat. 7, 1-26]. In our application, the standard deviation map can be estimated by bootstrapping the images. Our results show that, apart from the symmetry axes and small regions bordering the lumen of the extracellular vestibule, difference maps normalized by the mean of the standard deviation map can be used as a good approximation of the t-test map of the gap junction crystals.

Fibrosis of collagen I and remodeling of connexin 43 in atrial myocardium of patients with atrial fibrillation.

BACKGROUND: Fibrosis in atrial myocardium is a common phenomenon for patients with atrial fibrillation (AF). Remodeling of connexins was found accompanying with AF. The aim of the study is to investigate whether it is by causing the remodeling of connexin 43 (Cx43) that the fibrosis of atrial muscle plays an important role during the initiation and maintenance of AF. METHODS: Samples of right atrial appendage were taken from 24 patients with rheumatic valvular disease during surgery. Fibrosis and remodeling of Cx43 was examined by microscopy and ultramicroscopy technique and analyzed by image analyzer. The collagen volume fraction of type I (CVF-I) and the volume fraction of Cx43 (Cx43VF) were studied between AF and sinus rhythm (SR) groups. RESULTS: (1) Microscopic examination demonstrated that CVF-I significantly increased and Cx43VF decreased in patients with AF compared to those with SR. (2) The CVF-I was negatively correlated with the Cx43VF. CONCLUSION: The results suggest that fibrosis and remodeling of Cx43 are involved in the pathophysiologic mechanism of human AF. Fibrosis of atrial muscle may play an important role in the process of AF by means of interfering with remodeling of connexins.

High-pressure freezing and freeze substitution of rat myocardium for immunogold labeling of connexin 43.

The value of high-pressure freezing (HPF) and freeze substitution (FS) for immunoelectron microscopy (immuno-EM) of the heart was investigated in bioptic specimens taken from isolated hearts of 0-, 5-, and 14-day-old rats at baseline and at 15, 30, 45, and 60 min after induction of ischemia. The target antigen chosen here was the gap junction protein connexin 43 (Cx43). After HPF and FS, immunogold labeling was applied for detection of Cx43. Gold particles associated with gap junction areas, free plasma membrane, and annular gap junctions (AGJs) were counted and distributions compared by contingency table analysis. HPF and FS resulted in excellent preservation of antigenicity for Cx43. The mostly good preservation of the ultrastructure was limited by mechanical damage at the border and by ice crystal formation in the center of the tissue blocks. In normal myocardium of newborns, gold particles associated with free plasma membrane were frequently observed, with AGJs only seldom. In older rats, the opposite relation was found. During ischemia, no distribution changes occurred in newborn or 14-day-old rats. In 5-day-old rats, however, ischemia induced a shift of Cx43 from gap junction plaques to AGJs. In conclusion, HPF and FS are an ideal alternative to chemical fixation for immuno-EM as the excellent preservation of antigenicity is combined with a well-preserved ultrastructure. The results indicate that the process of degradation of gap junctions via AGJs gradually increases during postnatal rat heart development, a process that may be accelerated by ischemia in an early developmental state.

Effects of two-step freezing on the ultra-structural components of murine osteoblast cultures.

Understanding the ultra-structural response of cells to the cryopreservation process is important for designing cryopreservation strategies for cells and tissues. Cell-cell interaction and cell-scaffold interactions alter cryopreservation response and, in turn, the cellular structures involved in adhesion and intercellular contact are possible targets of cryopreservation-induced damage. Immuno-fluorescence was used to assess the status of the actin filaments (F-actin), focal adhesions (vinculin) and gap junctions (connexin-43) of murine osteoblasts attached to hydroxyapatite (HA) discs and plastic coverslips for a two-step freezing process. The freezing process de-polymerized and distorted the actin filaments of dead cells, while those of live cells experienced little change. Vinculin and connexin-43 structures were rarely seen in dead cells, while a portion of vinculin (8.14+/-2.27 percent) and connexin-43 (21.7+/-4.7 percent) structures remained in live cells. These results suggest that focal adhesions and gap junctions may support cell robustness during cryopreservation. The present study contributes to our knowledge of the damage mechanisms associated with attached cells during a freezing process.

Upregulation of connexin43 gap junctions between neointimal smooth muscle cells.

Increased expression of connexin43 gap junctions in smooth muscle cells (SMC) is implicated in the response to primary arterial injury and in the early stages of human coronary atherosclerosis, but the relevance of these findings to restenosis is unknown. Here we investigated the expression of connexin43 gap junctions in restenotic aortas of cholesterol-fed double injured rabbits. Immunofluorescence confocal microscopy was used to evaluate temporal and spatial expression patterns and to characterize the major expressing cell type. Parallel studies were conducted by electron microscopy, in situ hybridization and Northern blot analysis. Connexin43 gap junctions- and connexin43 mRNA-expressing cells were abundant in the media of non-injured control aorta. Following primary injury and 6 weeks cholesterol diet, connexin43 gap junctions were found distributed throughout the primary intimal layer; although medial expression was reduced, the overall mRNA expression level remained similar to that of non-injured controls. After secondary injury, no major change in distribution pattern of connexin43 gap junctions occurred up to day 10, when marked neointimal labeling was observed. This overall pattern persisted, though with some diminution, at later stages. On the mRNA level total connexin43 mRNA expression declined to about 40% of control values within 4 days after secondary injury (P < 0.05), but subsequently increased four-fold, attaining levels double that of non-injured controls in the 10-day group (P < 0.005 versus control and 4 days). At later stages mRNA expression levels returned to values similar to those of non-injured controls. At all stages, connexin43 gap junctions were localized to the SMC, not to macrophages. We conclude that the enhanced gap junction formation may contribute to the coordination of the response of SMC after secondary injury, particularly in the early phase of restenosis.

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.

Expression, two-dimensional crystallization, and electron cryo-crystallography of recombinant gap junction membrane channels.

We used electron cryo-microscopy and image analysis to examine frozen-hydrated, two-dimensional (2D) crystals of a recombinant, 30-kDa C-terminal truncation mutant of the cardiac gap junction channel formed by 43-kDa alpha(1) connexin. To our knowledge this is the first example of a structural analysis of a membrane protein that has been accomplished using microgram amounts of starting material. The recombinant alpha(1) connexin was expressed in a stably transfected line of baby hamster kidney cells and spontaneously assembled gap junction plaques. Detergent treatment with Tween 20 and 1,2-diheptanoyl-sn-phosphocholine resulted in well-ordered 2D crystals. A three-dimensional density (3D) map with an in-plane resolution of approximately 7.5 A revealed that each hexameric connexon was formed by 24 closely packed rods of density, consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. In the extracellular gap the aqueous channel was bounded by a continuous wall of protein that formed a tight electrical and chemical seal to exclude exchange of substances with the extracellular milieu.

Electron cryo-crystallography of a recombinant cardiac gap junction channel.

Gap junctions in the heart play an important functional role by electrically coupling cells, thereby organizing the pattern of current flow to allow co-ordinated muscle contraction. Cardiac gap junctions are therefore intimately involved in normal conduction as well as the genesis of potentially lethal arrhythmias. We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated 2D crystals of a recombinant, C-terminal truncated form of connexin 43 (Cx43; alpha 1), the principal cardiac gap junction protein. The projection map at 7 A resolution revealed that each 30 kDa connexin subunit has a transmembrane alpha-helix that lines the aqueous pore and a second alpha-helix in close contact with the membrane lipids. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by approximately 30 degrees. We are now recording images of tilted, frozen-hydrated 2D crystals, and a preliminary 3D map has been computed at an in-plane resolution of approximately 7.5 A and a vertical resolution of approximately 25 A. As predicted by our model, the two apposing connexons that form the channel are staggered with respect to each other for certain connexin molecular boundaries within the hexamer. Within the membrane interior each connexin subunit displays four rods of density, which are consistent with an alpha-helical conformation for the four transmembrane domains. Preliminary studies of BHK hamster cells that express the truncated Cx43 designated alpha 1 Cx263T demonstrate that oleamide, a sleep inducing lipid, blocks in vivo dye transfer, suggesting that oleamide causes closure of alpha 1 Cx263T channels. The comparison of the 3D structures in the presence and absence of oleamide may provide an opportunity to explore the conformational changes that are associated with oleamide-induced blockage of dye transfer. The structural details revealed by our analysis will be essential for delineating the molecular basis for intercellular current flow in the heart, as well as the general molecular design and functional properties of this important class of channel proteins.

Structure of cardiac gap junction intercellular channels.

Gap junction proteins, termed connexins, constitute a multigene family of polytopic membrane channel proteins that have four hydrophobic transmembrane domains with the N- and C-termini located on the cytoplasmic membrane face. The principal gap junction protein in the heart, alpha 1 connexin (also designated Cx43), mediates action potential propagation between cells in order to synchronize cardiac contraction. alpha 1 connexin channels are concentrated in gap junction plaques located in the intercalated disks. The intercellular channel is formed by the docking of two hemi-channels, termed connexons, formed by a ring of six 43-kDa alpha 1 connexin subunits. Each subunit is asymmetric with an axial ratio of 4-5:1 with approximately 20 A extending into the extracellular gap approximately 50 A spanning the lipid bilayer and approximately 50 A extending into the cytoplasmic space. We have recently grown two-dimensional crystals of a recombinant C-terminal truncation mutant of alpha 1 connexin (designated alpha 1Cx263T) that are ordered to better than 7 A resolution. Projection density maps derived by electron cryocrystallography revealed that the intercellular channel is lined by six alpha-helices, and there is a second ring of six alpha-helices at the interface with the membrane lipids. These rings of alpha-helices are staggered by 30 degrees, which predicts that the two connexons in the channel are staggered by 30 degrees such that each connexin subunit in one connexon interacts with two subunits in the apposed connexon. Such a quaternary arrangement may confer stability in the docking of the connexons to form a tight electrical seal for intercellular current flow during cardiac conduction.