Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gapjunctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gapjunctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Identification of Cells Expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in Gap Junctions of Rat Brain and Spinal Cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with “permissive” connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

Gap Junctions: Structure, Functions, and Regulation

The review presents data on the role of gap junctions in intercellular communication. The review includes information on history of the appearance of this problem. Data are presented on ultrastructure and function of gap junctions as well as on the mechanisms providing for their activity. A part of the review deals with the problem of regulation of intercellular communication realized by the gap junctions.     

Regulation of Lens Gap Junctions by Transforming Growth Factor Beta

Gap junction–mediated intercellular communication (GJIC) is essential for the proper function of many organs, including the lens. GJIC in lens epithelial cells is increased by FGF in a concentration-dependent process that has been linked to the intralenticular gradient of GJIC required for lens transparency. Unlike FGF, elevated levels of TGF-β are associated with lens dysfunction. We show that TGF–β1 or -2 up-regulates dye coupling in serum-free primary cultures of chick lens epithelial cells (dissociated cell-derived monolayer cultures [DCDMLs]) via a mechanism distinct from that utilized by other growth factors. Remarkably, the ability of TGF-β and of FGF to up-regulate GJIC is abolished if DCDMLs are simultaneously exposed to both factors despite undiminished cell–cell contact. This reduction in dye coupling is attributable to an inhibition of gap junction assembly. Connexin 45.6, 43, and 56–containing gap junctions are restored, and intercellular dye coupling is increased, if the activity of p38 kinase is blocked. Our data reveal a new type of cross-talk between the FGF and TGF-β pathways, as well as a novel role for TGF-β and p38 kinase in the regulation of GJIC. They also provide an explanation for how pathologically increased TGF-β signaling could contribute to cataract formation.     

Structure,Regulation and Function of Gap Junctions in Liver

Abstract

Gap junctions are a specialized group of cell-to-cell junctions that mediate direct intercellular communication between cells. They arise from the interaction of two hemichannels of adjacent cells, which in turn are composed of six connexin proteins. In liver, gap junctions are predominantly found in hepatocytes and play critical roles in virtually all phases of the hepatic life cycle, including cell growth, differentiation, liver-specific functionality and cell death. Liver gap junctions are directed through a broad variety of mechanisms ranging from epigenetic control of connexin expression to post-translational regulation of gap junction activity. This paper reviews established and novel aspects regarding the architecture, control and functional relevance of liver gap junctions.     

HeLa细胞中缝隙连接蛋白Cx26/Cx32的表达及其功能调控

目的 在HeLa宫颈癌细胞中研究不同浓度的多西环素对缝隙连接蛋白Cx26/Cx32表达及由其形成的缝隙连接通讯功能的影响.方法 采用Western印迹检测HeLa细胞中Cx26/Cx32的蛋白表达;荧光示踪实验用于检测HeLa细胞中由Cx26/Cx32形成的缝隙连接通讯功能.结果 Western印迹结果显示多西环素在0.01～1 μg/ml的范围内,随着剂量的增加,Cx26/Cx32蛋白表达水平增加;荧光示踪实验结果显示HeLa细胞之间的荧光传递随着多西环素增加也相应增强.结论 采用加入不同浓度多西环素的方法,可制备缝隙连接通讯功能强弱不同的细胞模型.     

Connexin 46 (Cx46) Gap Junctions Provide a Pathway for the Delivery of Glutathione to the Lens Nucleus

Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (P_Na/P_Cl ∼5 for Cx46 and ∼3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (P_Na/P_GSH ∼12 for Cx46 and ∼8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions.     

Regulation of Gap Junction Coupling Through the Neuronal Connexin Cx35 by Nitric Oxide and cGMP

Gap-junctional coupling among neurons is subject to regulation by a number of neurotransmitters including nitric oxide. We studied the mechanisms by which NO regulates coupling in cells expressing Cx35, a connexin expressed in neurons throughout the central nervous system. NO donors caused potent uncoupling of HeLa cells stably transfected with Cx35. This effect was mimicked by Bay 21-4272, an activator of guanylyl cyclase. A pharmacological analysis indicated that NO-induced uncoupling involved both PKG-dependent and PKG-independent pathways. PKA was involved in both pathways, suggesting that PKG-dependent uncoupling may be indirect. In vitro, PKG phosphorylated Cx35 at three sites: Ser110, Ser276, and Ser289. A mutational analysis indicated that phosphorylation on Ser110 and Ser276, sites previously shown also to be phosphorylated by PKA, had a significant influence on regulation. Ser289 phosphorylation had very limited effects. We conclude that NO can regulate coupling through Cx35 and that regulation is indirect in HeLa cells.     

Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus)

Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.     

外胚层细胞诱导后的间隙连接

用电镜观察了经受诱导作用之后胚胎细胞的冷冻蚀刻复型膜。和未经诱导作用的对照组比较,早期和中期神经胚的神经上皮细胞以及经过豚鼠骨髓粗提液(BME)——一种有效的异源中胚层诱导物——处理过的早期原肠胚外胚层,它们的间隙连接都处于活跃的动态状态。用图像分析仪测得的间隙连接连接子的排列密度,指出经受过诱导作用的三组分别和未经受诱导作用的对照组比较,计算求出P值,神经上皮两组和对照组的差别为非常显著,BME处理过的细胞和对照组的差别为显著。结合对照组与诱导后胚胎细胞间隙连接连接子的变化讨论了它们在信息传递上可能起的作用。     

LIN7 Mediates the Recruitment of IRSp53 to Tight Junctions

In this study, we examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein–protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin–Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain (ΔL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domains of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7 small hairpin RNA experiments) and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53Δ5) or fused to the L27 domain of LIN7 (L27-IRSp53Δ5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity. These data therefore indicate that LIN7–IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells.     

Increasing Gap Junctional Coupling: A Tool for Dissecting the Role of Gap Junctions

Much of our current knowledge about the physiological and pathophysiological role of gap junctions is based on experiments where coupling has been reduced by either chemical agents or genetic modification. This has brought evidence that gap junctions are important in many physiological processes. In a number of cases, gap junctions have been implicated in the initiation and progress of disease, and experimental uncoupling has been used to investigate the exact role of coupling. The inverse approach, i.e., to increase coupling, has become possible in recent years and represents a new way of testing the role of gap junctions. The aim of this review is to summarize the current knowledge obtained with agents that selectively increase gap junctional intercellular coupling. Two approaches will be reviewed: increasing coupling by the use of antiarrhythmic peptide and its synthetic analogs and by interfering with the gating of gap junctional channels.     

Slow Gating of Gap Junction Channels and Calmodulin

Certain COOH-terminus mutants of connexin32 (Cx32) were previously shown to form channels with unusual transjuctional voltage (V _j ) sensitivity when tested heterotypically in oocytes against Cx32 wild type. Junctional conductance (G _j ) slowly increased by severalfold or decreases to nearly zero with V _j positive or negative, respectively, at mutant side, and V _j positive at mutant side reversed CO₂-induced uncoupling. This suggested that the CO₂-sensitive gate might be a V _j -sensitive slow gate. Based on previous data for calmodulin (CaM) involvement in gap junction function, we have hypothesized that the slow gate could be a CaM-like pore plugging molecule (cork gating model). This study describes a similar behavior in heterotypic channels between Cx32 and each of four new Cx32 mutants modified in cytoplasmic-loop and/or COOH-terminus residues. The mutants are: ML/NN+3R/N, 3R/N, ML/NN and ML/EE; in these mutants, N or E replace M105 and L106, and N replace R215, R219 and R220. This study also reports that inhibition of CaM expression strongly reduces V _j and CO₂ sensitivities of two of the most effective mutants, suggesting a CaM role in slow and chemical gating. Received: 19 April 2000/Revised: 11 August 2000     

Structured Functional Domains of Myelin Basic Protein: Cross Talk between Actin Polymerization and Ca-Dependent Calmodulin Interaction

The 18.5-kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. Solution NMR spectroscopy and a hydrophobic moment analysis of MBP's amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-helices. These regions, located in the central, N- and C-terminal parts of the protein, have been shown to play a role in the interactions of MBP with cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca²⁺-activated calmodulin (Ca²⁺-CaM), and myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5-kDa murine MBP: (A22–K56), (S72–S107), and (S133–S159) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca²⁺-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca²⁺-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca²⁺-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca²⁺-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition fragments with multiple roles in both membrane- and protein-association.     

Correlations of Differentially Expressed Gap Junction Connexins Cx26, Cx30, Cx32, Cx43 and Cx46 with Breast Cancer Progression and Prognosis

Background and Aims

Connexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers.

Materials and Methods

Meta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models.

Results

The expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other’s prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively.

Conclusion

Differential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers.     

Modelling the Effect of Gap Junctions on Tissue-Level Cardiac Electrophysiology

When modelling tissue-level cardiac electrophysiology, a continuum approximation to the discrete cell-level equations, known as the bidomain equations, is often used to maintain computational tractability. Analysing the derivation of the bidomain equations allows us to investigate how microstructure, in particular gap junctions that electrically connect cells, affect tissue-level conductivity properties. Using a one-dimensional cable model, we derive a modified form of the bidomain equations that take gap junctions into account, and compare results of simulations using both the discrete and continuum models, finding that the underlying conduction velocity of the action potential ceases to match up between models when gap junctions are introduced at physiologically realistic coupling levels. We show that this effect is magnified by: (i) modelling gap junctions with reduced conductivity; (ii) increasing the conductance of the fast sodium channel; and (iii) an increase in myocyte length. From this, we conclude that the conduction velocity arising from the bidomain equations may not be an accurate representation of the underlying discrete system. In particular, the bidomain equations are less likely to be valid when modelling certain diseased states whose symptoms include a reduction in gap junction coupling or an increase in myocyte length.     

Neuroprotective Role of Astrocytic Gap Junctions in Ischemic Stroke

The role of astrocytic gap junctions in ischemia remains controversial. Several studies support that astrocytic gap junctions play a role in the spread of hypoxic injury, while other reports have demonstrated that blocking astrocytic gap junctions increases neuronal death. Using a stroke model on animals in which the astrocytic gap junction protein connexin43 (Cx43) was compromised, we explored the neuroprotective role of astrocytic gap junctions. A focal brain stroke was performed on heterozygous Cx43 null [Cx43(+/?)] mice, wild type [Cx43(+/+)] mice, astrocyte-directed Cx43 deficient [Cx43^{fl/ fl}/hGFAP-cre] mice (here designated as Cre(+) mice), and their corresponding controls [Cx43^fl/fl] (here designated as Cre(?) mice). Four days following stroke, ischemic lesions were measured for size and analyzed immunohistochemically. Stroke volume was significantly larger in Cx43(+/?) and Cre(+) mice compared to Cx43(+/+) and Cre(?) mice, respectively. Apoptosis as detected by TUNEL labeling and caspase-3 immunostaining was amplified in Cx43(+/?) and Cre(+) mice compared to their control groups. Furthermore, increased inflammation as characterized by the immunohistochemical staining of the microglial marker CD11b was observed in the Cre(+) mice penumbra. Astrocytic gap junctions may reduce apoptosis and inflammation in the penumbra following ischemic insult, suggesting that coupled astrocytes fulfill a neuroprotective role under ischemic stroke conditions.