Can gap junctions deliver?

In vivo delivery of small interfering RNAs (siRNAs) to target cells via the extracellular space has been hampered by dilution effects and immune responses. Gap junction-mediated transfer between cells avoids the extracellular space and its associated limitations. Because of these advantages cell based delivery via gap junctions has emerged as a viable alternative for siRNA or miRNA delivery. Here we discuss the advantages and disadvantages of extracellular delivery and cell to cell delivery via gap junction channels composed of connexins. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Gap junctions: fates worse than death?

The reasons for the molecular heterogeneity of connexin channels in vivo remain unclear. Functional replacement of one connexin gene with another has now revealed unexpected phenotypes and shows that cellular homeostasis depends not simply on cell-cell communication but also on the correct types of connexin.     

Cardiac telocytes — their junctions and functional implications

Telocytes (TCs) form a cardiac network of interstitial cells. Our previous studies have shown that TCs are involved in heterocellular contacts with cardiomyocytes and cardiac stem/progenitor cells. In addition, TCs frequently establish 'stromal synapses' with several types of immunoreactive cells in various organs ( www.telocytes.com ). Using electron microscopy (EM) and electron microscope tomography (ET), we further investigated the interstitial cell network of TCs and found that TCs form 'atypical' junctions with virtually all types of cells in the human heart. EM and ET showed different junction types connecting TCs in a network (puncta adhaerentia minima, processus adhaerentes and manubria adhaerentia). The connections between TCs and cardiomyocytes are 'dot' junctions with nanocontacts or asymmetric junctions. Junctions between stem cells and TCs are either 'stromal synapses' or adhaerens junctions. An unexpected finding was that TCs have direct cell-cell (nano)contacts with Schwann cells, endothelial cells and pericytes. Therefore, ultrastructural analysis proved that the cardiac TC network could integrate the overall 'information' from vascular system (endothelial cells and pericytes), nervous system (Schwann cells), immune system (macrophages, mast cells), interstitium (fibroblasts, extracellular matrix), stem cells/progenitors and working cardiomyocytes. Generally, heterocellular contacts occur by means of minute junctions (point contacts, nanocontacts and planar contacts) and the mean intermembrane distance is within the macromolecular interaction range (10-30 nm). In conclusion, TCs make a network in the myocardial interstitium, which is involved in the long-distance intercellular signaling coordination. This integrated interstitial system appears to be composed of large homotropic zones (TC-TC junctions) and limited (distinct) heterotropic zones (heterocellular junctions of TCs).     

Regulating cell–cell junctions from A to Z

Epithelial sheets often present a “cobblestone” appearance, but the mechanisms underlying the dynamics of this arrangement are unclear. In this issue, Choi et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506115) show that afadin and ZO-1 regulate tension and maintain zonula adherens architecture in response to changes in contractility.The textbook view of epithelial cells is that once such cells adopt a close, hexagonal packing, their “honeycomb” or “cobblestone” arrangement is static. This fixed appearance is misleading, as these cells are more like players in a rugby scrum, locked in a tussle in which the forces exerted by each of the players on the others maintains their seemingly static arrangement, but by a very dynamic force balance. How such balance is maintained in epithelia is a subject of substantial interest. A crucial role is played by F-actin and nonmuscle myosin II isoforms, which are deployed in contractile networks that transiently attach to cell–cell junctions to generate tensile forces along cell–cell boundaries (Lecuit and Yap, 2015). Contractile arrays of actomyosin are regulated by the monomeric G protein Rho, its upstream regulators, including Rho guanine nucleotide exchange factors (Quiros and Nusrat, 2014), and its effectors ROCK/Rho kinase and Shroom3 (Nishimura and Takeichi, 2008), but also by tension-mediated feedback between the myosin network and the junction (Lecuit and Yap, 2015). Cell–cell adhesion, including cadherin-dependent adhesion, also plays a crucial role in this process. As cells engage with one another via interactions of the extracellular domains of their cadherin complexes, they transduce forces to the actomyosin cytoskeleton through catenins. β-Catenin binds to the cytoplasmic domain of classical cadherins and recruits α-catenin, which binds F-actin.Given the dynamic nature of epithelia, the attachment of contractile actomyosin networks to junctions are also subject to regulation. One aspect of epithelial architecture that has received relatively little attention is that a typical epithelial monolayer (Fig. 1 A) displays two main types of cell–cell interfaces: bilateral junctions (BCJs), in which two cells establish a relatively long stretch of contact, and cellular vertices, which represent a confluence of three or more cell edges to form tricellular junctions (TCJs) or multicellular junctions. TCJs are not well understood, but are known to contain unique molecular components (Furuse et al., 2014; Flores-Benitez and Knust, 2015). In this issue, Choi et al. show that the multivalent scaffolding proteins afadin and ZO-1/2 regulate the spacing of and tension along lateral contacts in cultured cells, thereby shedding light on how contractile arrays containing bilateral and tri- or multicellular contact points are regulated in epithelia.Open in a separate windowFigure 1.ZO proteins and afadin regulate junctional tension and organization in cultured cells. (A) Untreated MDCK cells have sinuous cell boundaries, whereas ZO KD cells show extremely straight boundaries. When ZO proteins and afadin are knocked down, cells adopt contact zones of irregular length with other cells, sometimes clustering into foci (asterisks). Images courtesy of Mark Peifer (Choi et al., 2016). (B) A model for actomyosin organization at adherens junctions (adapted from Choi et al., 2016). Contractile actomyosin arrays run parallel to bicellular junctions and are anchored by side-on attachments (pink circles). At TCJs, end-on binding of actin, likely stabilized by afadin, anchors actomyosin filaments. In ZO KD cells, contractile elements and cadherin complexes collapse toward TCJs, and myosin minifilaments adopt a regularly spaced arrangement.Afadin and ZO-1/2 are far from new players at junctions. Afadin binds α-catenin, actin, and other cytoskeletal and junctional proteins and associates with the transmembrane protein nectin, which appears to form an alternative adhesion system at adherens junctions (Mandai et al., 2013). The zonula occludens proteins ZO-1 and ZO-2 are tight junction proteins that bind claudins and are required for tight junction formation (Itoh et al., 1999; Balda and Matter, 2008). In addition, ZO proteins also bind to α-catenin (Itoh et al., 1997), are involved in establishing the zonula adherens (ZA; Ikenouchi et al., 2007), and potentiate cadherin-dependent adhesion in Caenorhabditis elegans (Lockwood et al., 2008) and Drosophila melanogaster (Choi et al., 2011). Knockdown of ZO-1 and ZO-2 (ZO KD) in MDCK cells has previously been shown (Fanning et al., 2012) to lead to dramatic alterations of the ZA: F-actin and myosin IIs assemble into striking apical arrays at the ZA, spaced at regular intervals. In addition, the normally sinuous boundaries between cells give way to very straight borders (Fig. 1 A).Using superresolution microscopy, diffraction-limited junctional laser ablation, cell morphometry, kinetic analysis, and a whole-monolayer approach to contractility, Choi et al. (2016) now extend this story. To test whether contractility is increased after ZO KD, the authors first measured the recoil after laser ablation of ZO KD cells; an increase in recoil velocity indicated that the straight junctional boundaries between ZO-depleted cells are under tension. Imaging analysis of BCJs showed that the increase in contractility in ZO KD cells is associated with a strikingly dynamic behavior of the BCJs. Individual BCJs were found to undergo periods of shortening and elongation, whereas neighboring BCJs underwent compensatory, opposite changes in length. These changes in contractility have effects on the entire tissue sheet as well: whereas control cell sheets remained flat when detached from the substratum, ZO KD cells contracted into a cup-like shape. This constriction was blocked by the myosin inhibitor blebbistatin. Overall, these experiments indicated that ZO proteins regulate myosin assembly and contractility across the cellular sheet.To dissect the protein network mediating increased contractility in ZO KD cells, Choi et al. (2016) examined the role of ROCK and found that ROCK inhibitors abolished the straight BCJs, which became curvilinear. Additionally, Shroom3, which is known to recruit ROCK (Nishimura and Takeichi, 2008), was cytoplasmic in control cells but junctional in ZO KD cells. Transient Shroom3 overexpression led to ROCK recruitment to the ZA and drove formation of an actomyosin network similar to that in ZO KD cells. Conversely, Shroom3 knockdown resulted in loss of the actomyosin arrays in ZO KD cells. Collectively, these data indicated that Shroom3 is an effector of increased apical contractility in ZO KD cells.The researchers used ZO KD cells to test how tissue integrity is maintained despite elevated contractibility and how junctions are remodeled to maintain integrity when increased tension is present. Afadin is a good candidate: the Drosophila homologue of afadin, Canoe, plays roles in convergent extension and collective cell migration; in its absence, actomyosin networks at the apex of constricting epithelial cells in the embryo contract in a catastrophic, uncontrolled fashion (Sawyer et al., 2009), suggesting a potential role for afadin in the maintenance of tissue integrity during morphogenetic movements. Choi et al. (2016) therefore turned their attention to afadin. ZO KD cells have significantly more afadin at their adherens junctions and TCJs, a pattern reminiscent of the normal distribution of Canoe in Drosophila (Sawyer et al., 2009). Knocking down afadin by shRNA in ZO KD cells led to further defects in cell–cell boundary maintenance. In addition to the taut appearance of bicellular borders, cell boundary length became much more irregular, with occasional foci of highly contracted cells (Fig. 1 A). Velocimetry analysis and live-cell imaging indicated that loss of both ZO proteins and afadin led to large-scale cell movements within the monolayer not seen after ZO KD alone.New imaging techniques used by Choi et al. (2016) revealed further details about the changes in actomyosin arrays in ZO KD cells. Superresolution imaging of myosin light chain kinase staining via structured illumination showed that myosin II assembles into arrays of myosin minifilaments spaced ∼415 nm apart along bicellular contacts. Superresolution and transmission electron microscopy also revealed reorganization of F-actin and E-cadherin at TCJs in ZO KD cells. Lateral F-actin bundles appeared to terminate end-on at TCJs at sites where E-cadherin was present. ZO KD therefore induces assembly of a remarkably ordered actomyosin array along BCJs, and these arrays appear to be separate contractile units that anchor end-on at the ZA. Moreover, based on staining for vinculin and a specific epitope in αE-catenin that serve as markers for regions under high tension (Yonemura et al., 2010), the end-on attachments of actin cables to the ZA at TCJs experience significant tensile stress. Strikingly, although vinculin and αE-catenin accumulation at TCJs was relatively uniform after ZO KD, their distribution was more heterogeneous after ZO/afadin KD. Differences in staining paralleled differences in cell border length and correlated with the level of tension measured at BCJs after laser cutting, suggesting that afadin contributes to the ability of cells to distribute forces at TCJ/multicellular junctions throughout the monolayer. Lastly, the researchers investigated whether internal cues downstream of ZO KD are sufficient for myosin recruitment or whether such recruitment depends on mechanical cues exerted by neighboring cells. They designed an assay mixing small islands of wild-type cells surrounded by ZO KD cells (or vice versa) and found that the development of the contractile array at the ZA depends on the contractility of neighboring cells; however, afadin recruitment to the ZA was less dependent on the sustained contractility of neighboring cells. Taking these data together, Choi et al. (2016) propose that cells respond to elevated contractility by increasing junctional afadin; because combined ZO/afadin knockdown dramatically alters cell shape and barrier function in response to elevated contractility, afadin acts as a robust scaffold that maintains ZA architecture most crucially at TCJs.Although many aspects of the model proposed by Choi et al. (2016) remain to be tested, their data suggest new features regarding the detailed assembly of actomyosin contractile arrays in confluent cells (Fig. 1 B). In control cells, actomyosin arrays presumably extend parallel to individual BCJs. Choi et al. (2016) propose that these actomyosin bundles act as separate contractile units that terminate near TCJs, allowing the generation of tension along BCJs. In ZO KD cells, excessive assembly of actomyosin filaments, perhaps exacerbated by the tendency of F-actin/myosin minifilament arrays to self-assemble, somehow leads to regularly spaced actomyosin arrays, and perhaps collapse of cadherin complexes and other components toward TCJs. There is a precedent for such lateral collapse of cadherin-dependent attachments: it is a prominent feature of cadherin complexes at sites of high tension in the epidermis of the C. elegans embryo (Choi et al., 2015). If the new model of Choi et al. (2016) is correct, then the foci seen in ZO KD/afadin KD cells may be similar to what happens in a game of tug of war when one team stops pulling. If some end-on attachments (assisted by afadin) fail, filaments might be expected to collapse along BCJs as the other, still tethered end of a set of filaments contracts toward the remaining attachment at the opposite cell vertex.Several other interesting questions remain. First, what is the relationship of the striking, regularly spaced bipolar myosin II minifilaments that form in ZO KD cells to myosin arrays in normal cells? It is clear that untreated cells have junctional actomyosin networks, but not with this strict periodicity. One possibility is that this spacing is simply an epiphenomenon; when not appropriately anchored along junctions, actomyosin networks may self-organize as they are known to do in other systems, such as in the contractile ring and in migrating cells (Srivastava et al., 2015; Fenix et al., 2016). More optimistically, the spacing may represent an intensified version of processes that operate in normal cells at bicellular and multicellular contact sites. If so, components of the model of Choi et al. (2016) will require further investigation. For example, the organization of F-actin along BCJs remains unclear, as are the proteins that mediate the side-on binding envisioned in this model. It is also uncertain whether proteins assist bundling of filaments and what role dynamic growth and shrinkage of actin filaments plays in end-on binding. In some contexts, junctions are capable of seeding polymerization of F-actin (Brieher and Yap, 2013), and it may be that actin dynamics are important in the processes studied here.A second question has to do with the community events within monolayers that Choi et al. (2016) describe. The neighbor effects on ZA morphology that they document are intriguing, as are the long-range accelerated movements of cells lacking both ZO proteins and afadin. Collective properties of monolayers are only beginning to be explored; connecting these properties with subcellullar events is an exciting future challenge. Whatever the answers to these new questions, the work of Choi et al. (2016) refines our understanding of the roles of key scaffolding proteins in organizing and anchoring junctions in epithelia.     

Intercellular junctions: downstream and upstream of Ras?

Most human tumors are of epithelial origin, and these tumors gradually lose their epithelial character in a process termed the epithelial-mesenchymal transition. Approximately 40% of human tumors have activating mutations in one of the three RAS genes. Given these statistics, it is critically important to understand the role of Ras signaling in the epithelial-mesenchymal transition. This review considers the mechanisms and effectors through which Ras may regulate intercellular junction formation in epithelial cells. Conversely, intercellular junction proteins themselves may play a role in regulating Ras activation and signaling.     

Organization of multiprotein complexes at cell–cell junctions

The formation of stable cell-cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell-cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell-cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell-cell contact formation and maintenance.     

The cell–cell junctions of mammalian testes: I. The adhering junctions of the seminiferous epithelium represent special differentiation structures

The seminiferous tubules and the excurrent ducts of the mammalian testis are physiologically separated from the mesenchymal tissues and the blood and lymph system by a special structural barrier to paracellular translocations of molecules and particles: the “blood–testis barrier”, formed by junctions connecting Sertoli cells with each other and with spermatogonial cells. In combined biochemical as well as light and electron microscopical studies we systematically determine the molecules located in the adhering junctions of adult mammalian (human, bovine, porcine, murine, i.e., rat and mouse) testis. We show that the seminiferous epithelium does not contain desmosomes, or “desmosome-like” junctions, nor any of the desmosome-specific marker molecules and that the adhering junctions of tubules and ductules are fundamentally different. While the ductules contain classical epithelial cell layers with E-cadherin-based adherens junctions (AJs) and typical desmosomes, the Sertoli cells of the tubules lack desmosomes and “desmosome-like” junctions but are connected by morphologically different forms of AJs. These junctions are based on N-cadherin anchored in cytoplasmic plaques, which in some subforms appear thick and dense but in other subforms contain only scarce and loosely arranged plaque structures formed by α- and β-catenin, proteins p120, p0071 and plakoglobin, together with a member of the striatin family and also, in rodents, the proteins ZO-1 and myozap. These N-cadherin-based AJs also include two novel types of junctions: the “areae adhaerentes”, i.e., variously-sized, often very large cell-cell contacts and small sieve-plate-like AJs perforated by cytoplasm-to-cytoplasm channels of 5–7 nm internal diameter (“cribelliform junctions”). We emphasize the unique character of this epithelium that totally lacks major epithelial marker molecules and structures such as keratin filaments and desmosomal elements as well as EpCAM- and PERP-containing junctions. We also discuss the nature, development and possible functions of these junctions.     

Holliday junctions in the eukaryotic nucleus: resolution in sight?

The Holliday junction is a key recombination intermediate whose resolution generates crossovers. Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism. Holliday junction resolvases in the eukaryotic nucleus have long eluded identification. The endonucleases Mus81/Mms4-Eme1 and XPF-MEI-9/MUS312 are structurally related to the archaeal resolvase Hjc and were found to be involved in crossover formation in budding yeast and flies, respectively. Although these endonucleases might represent one class of eukaryotic resolvases, their substrate preference opens up the possibility that junctions other than classical Holliday junctions might contribute to crossovers. Holliday junction resolution to non-crossover products can also be achieved topologically, for example, by the action of RecQ-like DNA helicases combined with topoisomerase III.     

Mucosal junctions: open doors to HPV and HIV infections?

Throughout adult life, new developmental commitment of adult stem cells causes reversible epithelial replacements in various mucosal surfaces, including the uterine cervix and the anal canal. Located at the squamocolumnar junctions, these metaplastic conversions are associated with chronic inflammation and deregulated expression of soluble and cell-membrane factors important for antiviral immune response. In this paper, we propose that these histological and immunological features increase the susceptibility of these metaplastic microenvironments to human papillomavirus and human immunodeficiency virus infections. Identification of the anatomical sites and cell populations within the anogenital tract, which is the site primary infected by these viruses, is crucial for the understanding of the pathogenesis of viral disease and development of antiviral strategies.     

Development of sertoli cell junctions in vitro—A freeze-fracture study

Summary Seminiferous tubules of 1-day-old rats were maintained in organ culture for up to 40 days. Five classes of intercellular junctions between Sertoli cells were observed by the freeze-fracture method as the tissue aged: (a) typical gap junctions; (b) focal tight junctions; (c) macular tight junctions; (d) meandering tight junctions; and (e) extensive tight junctions. The relative proportions of these types of Sertoli cell junctions were quantitated as the organ cultures progressed. The junctional structures observed and classified in organ culture were identical to those seen in vivo, but the timing of their appearance and/or disappearance, as well as their relative proportions, was different from that observed in the developing animal. Extensive tight junctions, with numerous parallel strands, were observed in the 40-day cultures; however, their oblique orientation with respect to the myoid layer was in contrast to the parallel orientation observed in vivo. This study was supported by Grant 801D185 (Dr. Posalaky) from the Medical Education and Research Foundation, St. Paul-Ramsey Hospital.     

Connexon rearrangement in cardiac gap junctions: Evidence for cytoskeletal control?

Summary Using ultrarapid-freezing techniques and freezefracture electron microscopy, we report here a close association between cardiac gap junctions and specialized membrane domains containing regularly-spaced furrows. These specialized furrowed domains are observed only during periods of gap junction re-organisation (i.e., connexon redistribution) and may reflect the presence of underlying cytoskeletal elements controlling the position of connexons in the membrane.     

Gap junctions in IL-1β-mediated cell survival response to strain

Mechanical stimuli play important roles in proliferation and differentiation of connective tissue cells, and development and homeostatic maintenance of tissues. However, excessive mechanical loading to a tissue can injure cells and disrupt the matrix, as occurs in tendinopathy. Tendinopathy is a common clinical problem in athletes and in many occupational settings due to overuse of the tendon. Moreover, interleukin (IL)-1β is generally considered to be a "bad" cytokine, activating NF-κb and cell death and inducing matrix metalloproteinase (MMPs 1, 2, 3) expression and matrix destruction. However, activated NF-κB can also drive a cell survival pathway. We have reported that cyclic strain induced tenocyte death in three-dimensional (3D) cultures, and IL-1β could promote cell survival under strain. Therefore, it was hypothesized that 1) cyclic strain could induce cell death in tenocytes as observed in pathologic tendons in vivo; 2) a gene expression profile indicative of tendinopathy could be identified; and 3) low-dose IL-1β could protect cells from strain-induced, tendinopathy-like changes. Human tenocytes were cultured in 3D type I collagen hydrogels and subjected to 3.5% elongation at 1 Hz for 1 h/day for up to 5 days with or without IL-1β. Real-time RT-PCR data showed that cyclic strain regulated the expression of tendinopathy marker genes in a manner similar to that found in pathological tendons from patients and that addition of IL-1β reversed the gene expression changes to control levels. Results of further studies showed that IL-1β may modulate cell survival through upregulating the expression of connexin 43, which is involved in the modulation of cell death/survival in a variety of cells and tissues. The elucidation of the mechanisms underlying strain-induced cell death and recovery from strain injury will facilitate our understanding of the pathogenesis of tendinopathy and may lead to the discovery of new molecular targets for early diagnosis and treatment of tendinopathy.     

Cell-cell junctions: α-catenin and E-cadherin help fence in Yap1

Metazoan cells translate adhesive events with neighbors into anti-proliferative signals in the nucleus. The cadherin-catenin adhesion complex has long been suspected of playing a key role in this process, and three recent papers suggest that it does so by modulating subcellular localization of the Hippo pathway component Yap1.     

Are there specialized junctions in the pars maculata of the distal tubule?

Summary In the present study the tight junctions at the macula densa were compared to those of the adjacent straight and convoluted segments of the distal tubule using freeze fracturing and thin sectioning techniques. Only insignificant differences were found in the number of strands and the apico-basal depth of the tight junctions in the three distal tubular segments of rat, dog and tree shrew. In experiments with horseradish peroxidase on mice and tree shrews, the tracer did not penetrate the apical junctions in any of the distal tubular segments. Our findings do not support the concept of considerably higher permeability of the tight junctions at the macula densa, as previously reported. Gap junctions were never observed in the distal nephron. The present results suggest that the glomerulo-tubular feedback is more likely to be mediated by transcellular resorption of solutes than by passive diffusion through a leaky paracellular shunt pathway.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Can 1000 reviews be wrong? Actin, alpha-Catenin, and adherens junctions

Coupling between cell adhesion and the actin cytoskeleton is thought to require a stable link between the cadherin-catenin complex and actin that is mediated by alpha-catenin. In this issue of Cell, the Weis and Nelson groups call this static model into question, showing that alpha-catenin can directly regulate actin dynamics (Drees et al., 2005 and Yamada et al., 2005).