Immunological properties of gap junction protein from mouse liver

Hepatic gap junctions were purified as plaques from BALB/c mice and separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Antisera were raised in rabbits and rats against gap junction plaques as well as protein bands of the following apparent molecular weights: 44K to 49K ("dimer" proteins), 26K, and 21K. Using an enzyme immunoassay, we found that the reactivities of the different antisera towards gap junction plaques decreased in the following order: anti-plaque antisera, anti-26K antisera, anti-"dimer" protein antisera, and anti-21K antisera. The gap junction protein bands separated by SDS-polyacrylamide gel electrophoresis were transferred by blotting onto nitrocellulose paper and the immunological cross-reactivities were compared: the anti-26K antisera reated with the dimer protein bands and the 26K band but did not cross-react with the 21K protein band. The rabbit anti-21K antiserum reacted weakly with the 21K protein. The missing immunological cross-reaction of the 26K and the 21K protein band can be most easily explained if both proteins were independent of each other. No inhibition of metabolic cooperation between fibroblastoid mouse 3T6 cells was observed in the presence of Fab fragments prepared from rabbit antiplaque antiserum or from rabbit anti 26K antiserum. When the total proteins of plasma membranes from mouse liver were separated by SDS-polyacrylamide electrophoresis, only the 26K protein reacted with rabbit anti 26K antiserum. This result opens the possibility for direct quantitation of gap junction protein in tissues and cell fractions.     

Visualization of gap junction mobility in living cells

In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.     

TLR2 regulates gap junction intercellular communication in airway cells

The innate immune response to inhaled bacteria, such as the opportunist Pseudomonas aeruginosa, is initiated by TLR2 displayed on the apical surface of airway epithelial cells. Activation of TLR2 is accompanied by an immediate Ca(2+) flux that is both necessary and sufficient to stimulate NF-kappaB and MAPK proinflammatory signaling to recruit and activate polymorphonuclear leukocytes in the airway. In human airway cells, gap junction channels were found to provide a regulated conduit for the movement of Ca(2+) from cell to cell. In response to TLR2 stimulation, by either lipid agonists or P. aeruginosa, gap junctions functioned to transiently amplify proinflammatory signaling by communicating Ca(2+) fluxes from stimulated to adjacent, nonstimulated cells thus increasing epithelial CXCL8 production. P. aeruginosa stimulation also induced tyrosine phosphorylation of connexin 43 and association with c-Src, events linked to the closure of these channels. By 4 h postbacterial stimulation, gap junction communication was decreased indicating an autoregulatory control of the connexins. Thus, gap junction channels comprised of connexin 43 and other connexins in airway cells provide a mechanism to coordinate and regulate the epithelial immune response even in the absence of signals from the immune system.     

Migrating cells retain gap junction plaque structure and function

Cell migration is an essential process in organ development, differentiation, and wound healing, and it has been hypothesized that gap junctions play a pivotal role in these cell processes. However, the changes in gap junctions and the capacity for cell communication as cells migrate are unclear. To monitor gap junction plaques during cell migration, adrenocortical cells were transfected with cDNA encoding for the connexin 43-green fluorescent protein. Time-lapse imaging was used to analyze cell movements and concurrent gap junction plaque dynamics. Immunocytochemistry was used to analyze gap junction morphology and distribution. Migration was initiated by wounding the cell monolayer and diffusional coupling was demonstrated by monitoring Lucifer yellow dye transfer and fluorescence recovery after photobleaching (FRAP) in cells at the wound edge and in cells located some distance from the wound edge. Gap junction plaques were retained at sites of contact while cells migrated in a "sheet-like" formation, even when cells dramatically changed their spatial relationship to one another. Consistent with this finding, cells at the leading edge retained their capacity to communicate with contacting cells. When cells detached from one another, gap junction plaques were internalized just prior to cell process detachment. Although gap junction plaque internalization clearly was a method of gap junction removal during cell separation, cells retained gap junction plaques and continued to communicate dye while migrating.     

Distribution and dynamics of gap junction channels revealed in living cells

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.     

Freeze-fracture analysis of gap junction disruption in rat ovarian granulosa cells

The effects of chemical dissociation on rat ovarian granulosa cell gap junctions has been studied using freeze-fracture electron microscopy. Sequential exposure of granulosa cells within follicles to solutions containing 6·8 mM EGTA [ethylene-bis-(β-aminoethyl ether)-N,N′-tetra acetic acid] and 0·5 M sucrose results in extensive cellular dissociation of the follicular epithelium. Freeze-fracture replicas made from fixed, control or EGTA-treated ovarian follicles exhibit extensive gap junctions between granulosa cells that are characterized by a range of packing order of constituent P-face particles or E-face pits. In contrast, exposure to 0·5 M sucrose containing 1·8 mM EGTA for as little as 1 min results in a consistently close packing of particles or pits which is accompanied by splitting of gap junctions between granulosa cells. The process of junction splitting was studied in detail in replicas prepared from follicles treated sequentially for various periods of time with EGTA and sucrose solutions. Initially, large gap junctions lose their regular shape and fragment into numerous tightly packed aggregates of P-face particles or E-face pits which are separated by unspecialized areas of plasma membrane. Subsequent to junction fragmentation, individual junction plaques separate at sites of cell contact and generate hemijunctions that border the intercellular space, Hemijunctions undergo particle dispersion of the P fracture face which results in an increased density of large intramembrane particles; no corresponding change in E-face pits is discernible at this stage. Morphometric analysis of replicas of tissue undergoing junction splitting indicates that junctional surface area decreases to 10–20% of control levels during this same treatment and so further supports the qualitative observations on junction fragmentation. Viabilities of granulosa cells obtained by these techniques also agree with the sequence observed in the morphometric analysis of the replicas. Finally, within 15 min after placing ovaries in isotonic, Ca²⁺-containing salt solutions, gap junction reformation occurs by aggregation of particles at sites of intercellular contact. These sites are distinguished by the appearance of short surface protrusions or indentations on their respective P and E fracture faces. The data suggest a mechanism for EGTA-sucrose mediated cellular dissociation in the follicular epithelium in which gap junctional particles are free to move in the plane of the plasma membrane and may be re-utilized to form gap junctions in the presence of extracellular calcium.     

Chloral hydrate decreases gap junction communication in rat liver epithelial cells

Gap junction communication (GJC) is involved in controlling cell proliferation and differentiation. Alterations in GJC are associated with carcinogenesis, but the mechanisms involved are unknown. Chloral hydrate (CH), a by-product of chlorine disinfection of water, is carcinogenic in mice, and we demonstrated that CH reduced GJC in a rat liver epithelial cell line (Clone 9). To examine the mechanism(s) by which CH inhibits GJC, Clone 9 cells treated with CH were examined using Western blot, real-time polymerase chain reaction, immunocytochemical, and dye-communication techniques. Treatment with CH (0.1–5 mM for 24 h) resulted in a dose-dependent inhibition of GJC as measured by Lucifer yellow dye transfer. Western blot analysis demonstrated expression of connexin (Cx) 43 and 26 in control cells and reduced expression of Cx 43 but not Cx 26 protein from 0.1 to 1 mM CH. CH treatment from 2.5 to 5 mM caused an apparent increase in expression of both connexins that was concomitant with a reduction in mRNA expression for both connexins. Similarly, with immunocytochemistry, a dose-dependent decrease in Cx 43 staining at sites of cell–cell contact was apparent in CH (0.5–5 mM)-treated cultures, whereas no Cx 26 staining was observed. Thus, Clone 9 cells contain two types of connexins but only one type of plasma membrane channel. Understanding of the regulation of connexin may shed light on mechanisms responsible for inhibition of GJC by chemical carcinogens.     

On gap junction structure

We have studied the stain distribution within rat liver gap junctions for specimens prepared by thin sectioning and negative staining. Pools of stain molecules exist in two specific locations with respect to the distinctive morphological units (connexons) of the junction. One pool of stain surrounds the connexons and is restricted to the extracellular space in the gap between the adjacent plasma membranes. The other pool of stain is located along in the central axis of each connexon, measures 1-2 nm in diameter and 4-5 nm in length, and is restricted to the gap region. On rare occasions, barely discernible linear densities seem to extend from this latter pool of stain and traverse the entire width of the junction. The data indicate the existence of a hydrophilic cavity along the central axis of te connexon which, in most instances, is restricted to the gap region. However, the precise depth to which this cavity may further extend along the connexon axis is still uncertain.     

Calcium-mediated changes in gap junction structure: evidence from the low angle X-ray pattern

Rat liver gap junctions were isolated in Ca2+-free media and analyzed in controlled environments by x-ray diffraction of partially oriented pellets. Different treatments of the same preparations were compared. The ordered hexagonal lattices gave rise to detail that was sensitive to low Ca2+ concentrations (0.05 mM), but not to Mg2+ (up to 0.16 mM) or pH (between 6.0 and 8.0). The major Ca2+-mediated responses were reductions in the intensity of the (1, 0) peak and in the off- equatorial contributions to the (2, 1) peak, and changes of scale equivalent to a decrease (approximately 2%) in lattice dimension, but an increase (approximately 4%) in the dimension perpendicular to the lattice. A simple structural interpretation of these findings is that Ca2+ induces the subunits of the channel-forming assembly, the connexon, to align more nearly parallel to the channel, thereby causing the connexon to become slightly longer and more radially compact. The rearrangement is of the same nature as one found under less physiological circumstances by electron microscopy (Unwin, P. N. T., and G. Zampighi, 1980, Nature (Lond.)., 283:545-549), and may be part of a coordinated mechanism by which the channel closes.     

A germline-specific gap junction protein required for survival of differentiating early germ cells

Germ cells require intimate associations and signals from the surrounding somatic cells throughout gametogenesis. The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Animals with a null mutation in zpg are viable but sterile and have tiny gonads. Adult zpg-null gonads contain small numbers of early germ cells, resembling stem cells or early spermatogonia or oogonia, but lack later stages of germ cell differentiation. In the male, Zpg protein localizes to the surface of spermatogonia, primarily on the sides adjacent to the somatic cyst cells. In the female, Zpg protein localizes to germ cell surfaces, both those adjacent to surrounding somatic cells and those adjacent to other germ cells. We propose that Zpg-containing gap junctional hemichannels in the germ cell plasma membrane may connect with hemichannels made of other innexin isoforms on adjacent somatic cells. Gap junctional intercellular communication via these channels may mediate passage of crucial small molecules or signals between germline and somatic support cells required for survival and differentiation of early germ cells in both sexes.     

Connexin45 gap junction channels in rat cerebral vascular smooth muscle cells

Cells in blood vessel walls express connexin (Cx)43, Cx40, and Cx37. We recently characterized gap junction channels in rat basilar artery smooth muscle cells and found features attributable not only to these three connexins but also to an unidentified connexin, including strong voltage dependence and single channel conductance of 30-40 pS. Here, we report data consistent with identification of Cx45. Immunofluorescence using anti-human Cx45 and anti-mouse Cx45 antibodies revealed labeling between alpha-actin-positive cells, and RT-PCR of mRNA from arteries after endothelial destruction yielded amplicons exhibiting 90-98% identity with mouse Cx45 and human Cx45. Dual-perforated patch clamping was performed after exposure to oligopeptides that interfere with docking of Cx43, Cx40, or Cx45. Cell pairs pretreated with blocking peptides for Cx43 and Cx40 exhibited strongly voltage-dependent transjunctional conductances [voltage at which voltage-dependent conductance declines by one-half (V1/2) = +/-18.9 mV] and small single channel conductances (31 pS), consistent with the presence of Cx45, whereas cell pairs pretreated with blocking peptide for Cx45 exhibit weaker voltage-dependent conductances (V1/2 = +/-37.9 mV), consistent with block of Cx45. Our data suggest that Cx45 is transcribed, expressed, and forms functional gap junction channels in rat cerebral arterial smooth muscle.     

Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells

Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca(2+), whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, V(m). These currents did not occur in non-transfected cells. The hemichannel currents were inhibited by raising extracellular Ca(2+) or by acidification with CO(2). The unitary conductance exhibited V(m) dependence (i.e., gamma(hc,main) increased/decreased with hyperpolarization/depolarization). Extrapolation to V(m) = 0 mV led to a gamma(hc,main) of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, P(o), was V(m)-dependent, declining at negative V(m) (P(o) < 0.11, V(m) < -50 mV), and increasing at positive V(m) (P(o) approximately 0.76, V(m) > 50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca(2+) level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in non-transfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca(2+)-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca(2+). Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca(2+) concentration is reduced.     

Sharing signals: connecting lung epithelial cells with gap junction channels

Gap junction channels enable the direct flow of signaling molecules and metabolites between cells. Alveolar epithelial cells show great variability in the expression of gap junction proteins (connexins) as a function of cell phenotype and cell state. Differential connexin expression and control by alveolar epithelial cells have the potential to enable these cells to regulate the extent of intercellular coupling in response to cell stress and to regulate surfactant secretion. However, defining the precise signals transmitted through gap junction channels and the cross talk between gap junctions and other signaling pathways has proven difficult. Insights from what is known about roles for gap junctions in other systems in the context of the connexin expression pattern by lung cells can be used to predict potential roles for gap junctional communication between alveolar epithelial cells.     

Characteristics of C6 glioma cells overexpressing a gap junction protein

1. C6 glioma cells transfected with connexin43 cDNA display a dramatic increase in the level of connexin43 mRNA and protein. 2. This overexpression of connexin43 is evident at the cellular level, as revealed with in situ hybridization and immunocytochemistry. Transfection with connexin43 cDNA also induced actin stress fibers in these glioma cells. 3. Although we observed up to a 50-fold increase in the level of connexin43 mRNA following transfection, virtually all of this mRNA was present in the polysomal fraction. 4. Overexpression of connexin43 mRNA did not appear to compete with other cellular mRNAs for access to the translational machinery. 5. It is likely that the reduced proliferation rate of the transfected cells, reported earlier, is due to enhanced connexin43 expression and intercellular coupling.     

Rat mast cells communicate with fibroblasts via gap junction intercellular communications

Usually mast cells (MCs) modulate other cellular activities through the release of their cytoplasmic granules. Recently, gap junctional intercellular communication (GJIC) between an established human MC cell line (HMC-1) co-cultured with human dermal fibroblasts in fibroblast populated collagen lattices (FPCLs), enhanced the rate and degree of FPCL contraction. However, HMC-1 cells were unable to generate GJIC with human neonatal fibroblasts in monolayer culture. Here freshly isolated rat peritoneal MCs are co-cultured with fibroblasts in collagen lattices and in monolayer culture in vitro and introduced into rat polyvinyl alcohol (PVA) sponge implants in vivo. Co-cultured MC-FPCL contracted faster and to a greater degree. Loading Calcein AM green fluorescent dye into red fluorescent Dil tagged MC generates MC-paratroopers. When MC-paratroopers form GJIC with fibroblasts, some green dye is passed into the fibroblast, while the MC-paratrooper retains both its red and green fluorescence. MC-paratroopers passed green fluorescent dye into both human and rat dermal fibroblasts in monolayer culture. In rats 7-day-old subcutaneous PVA sponge implants, which received an injection of MC-paratroopers, exhibited auto-fluorescent green fibroblasts, when harvested 24 h later. MC-paratroopers pretreated with a long-acting GJIC inhibitor prior to their introduction into PVA sponge implants, failed to pass dye into fibroblasts. It is proposed that GJIC between granulation tissue fibroblasts and MCs can modulate some aspects of wound repair and fibrosis.