Probing the function of connexin channels in primary tissues

Connexin channels provide for a widespread mechanism of cell-to-cell cross-talk within primary tissues, which is mediated by intercellular exchanges of cytoplasmic ions and molecules. Experimental and clinical studies have recently provided evidence that these exchanges are most likely to play multiple roles, which are critical for the proper development and function of primary tissues. There is also increasing evidence that major clinical disorders may result when the formation and function of connexin channels are altered. Still, the physiological functions that the cell-to-cell communication mediated by connexin channels subserve in most primary tissues are still uncertain. Here, I review two approaches that may aid in identifying these specific functions.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

Reversible Interruption of Gap Junctional Communication by Testosterone Propionate in Cultured Sertoli Cells and Cardiac Myocytes

A direct cell-to-cell exchange of ions and molecules occurs through specialized membrane channels built by the interaction of two half channels, termed connexons, contributed by each of the two adjacent cells. The electrical and diffusional couplings have been investigated by monitoring respectively the cell-to-cell conductance and the fluorescence recovery after photobleaching, in Sertoli and cardiac cells of young rat. In both cell types, a rapid impairment of the intercellular coupling has been observed in the presence of testosterone propionate. This interruption of the cell-to-cell communication through gap junction channels was dose-dependent, observed in the concentration range 1 to 25 μm and was progressively reversed after withdrawing the testosterone ester. Pretreatment with cyproterone acetate, an antiandrogen which blocks the nuclear testosterone receptor by binding, did not prevent the uncoupling action of the androgen ester. This observation, together with the rapid time course of the uncoupling and recoupling, and the rather high effective concentration (micromolar) of the steroid compound, suggests a nongenomic mechanism of action. The uncoupling concentrations were very similar to those of other steroid compounds known to interrupt gap junctional communication. The uncoupling could result from a direct interaction of the steroid with the proteolipidic structure of the membrane, that might alter the conformation of the gap junction channels and their functional state. Received: 10 April 1995/Revised: 27 October 1995     

Gap junction-mediated intercellular communication between dendritic cells (DCs) is required for effective activation of DCs

Gap junctions, formed by members of the connexin (Cx) family, are intercellular channels allowing direct exchange of signaling molecules. Gap junction-mediated intercellular communication (GJIC) is a widespread mechanism for homeostasis in organs. GJIC in the immune system is not yet fully understood. Although dendritic cells (DC) reportedly form cell-to-cell contact between DCs in nonlymphoid and lymphoid organs, GJIC between DCs remains unknown. In this study we examined whether DCs form GJIC. XS52 and bone marrow-derived DCs (BMDCs) were tested for GJIC by counting intercellular transfer of Lucifer Yellow microinjected into a cell. Either DC became effectively dye-coupled when activated with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma. LPS- plus IFN-gamma-induced dye-coupling was mediated by DC-derived TNF-alpha. In addition, CpG plus IFN-gamma induced dye-coupling in BMDCs, which was also mediated by DC-derived TNF-alpha. LPS- plus IFN-gamma-induced activation of DCs (assessed by CD40 expression) was observed when there was cell-to-cell contact and was significantly blocked by heptanol, a gap junction blocker. These results indicate that cell-to-cell contact and GJIC are required for effective DC activation. In addition, heptanol significantly inhibited the LPS- plus IFN-gamma-induced up-regulation of the other costimulatory (i.e., CD80 and CD86) and MHC class II molecules expressed by BMDCs, and it significantly reduced their allostimulatory capacity. Among Cx members, Cx43 was up-regulated in dye-coupled BMDCs, and Cx mimetic peptide, a blocker of Cx-mediated GJIC, significantly inhibited the dye-coupling and activation, suggesting the involvement of Cx43. Thus, our study provides the first evidence for GJIC between DCs, which is required for effective DC activation.     

Opening of single gap junction channels during formation of electrical coupling between embryonic muscle cells

Gap junctions, which are low-resistance intercellular pathways, may contribute to normal embryogenesis by allowing cell-to-cell passage of as yet unidentified regulatory or inductive signals. But little is known about the properties of newly formed single junctional channels which are the basis of the communicating junctions. Reported here are the first direct measurements of current passing through single junctional channels as they form. Individual pairs of embryonic Xenopus muscle cells in culture were manipulated into contact, allowing control of the onset time and area of cellular contact, and current was recorded with the patch clamp technique. The opening of single channels which pass current between the two cells at a conductance of about 100 pS was observed within minutes of cell-cell contact. The channels opened one-at-a-time, and once formed, remained open for long periods of time, with infrequent brief closures. This suggests that formation of electrical coupling between two cells proceeds by addition of single conducting junctional channels one channel-at-a-time.     

Transport of macromolecules through plasmodesmata and the phloem

Cell-to-cell communication is a pivotal process in the determination of cell fate during development and physiological adaptation in response to environmental stimuli. The intercellular trafficking of proteins and RNAs has emerged as a novel mechanism of cell-to-cell signaling in plants. As a strategy for efficient intercellular communication, plants have evolved plant-specific symplasmic communication networks via plasmodesmata (PD) and the phloem. PD are symplasmic channels connecting the cytoplasm of neighboring cells and are responsible for the local exchange of metabolites and signaling molecules. The phloem is the sieve-tube system that allows rapid, long-distance translocation of molecules. Together, PD and phloem conduits have been shown to allow the transport of proteins and RNAs in non-selective or/and selective modes. This review describes the current understanding of macromolecule trafficking through PD and the phloem.     

Cell-to-cell movement and assembly of a plant closterovirus: roles for the capsid proteins and Hsp70 homolog.

Diverse animal and plant viruses are able to translocate their virions between neighboring cells via intercellular connections. In this work, we analyze the virion assembly and cell-to-cell movement of a plant closterovirus and reveal a strong correlation between these two processes. The filamentous virions of a closterovirus possess a long body formed by the major capsid protein (CP) and a short tail formed by the minor capsid protein (CPm). Genetic and biochemical analyses show that the functions of these virion components are distinct. A virion body is required primarily for genome protection, whereas a tail represents a specialized device for cell-to-cell movement. Furthermore, tail assembly is mediated by the viral Hsp70 homolog (Hsp70h) that becomes an integral part of the virion. Inactivation of the ATPase domain of Hsp70h results in assembly of tailless virions that are incapable of translocation. A dual role for the viral molecular chaperone Hsp70h in virion assembly and transport, combined with the previous finding of this protein in intercellular channels, allowed us to propose a model of closteroviral movement from cell to cell.     

Tunneling nanotubes: a new route for the exchange of components between animal cells

Recently, highly sensitive nanotubular structures mediating membrane continuity between mammalian cells have been discovered. With respect to their peculiar architecture, these membrane channels were termed tunneling nanotubes (TNTs). TNTs could form de novo between animal cells leading to the generation of complex cellular networks. They have been shown to facilitate the intercellular transfer of organelles as well as, on a limited scale, of membrane components and cytoplasmic molecules. It has been proposed that TNTs represent a novel and general biological principle of cell-to-cell communication and it becomes increasingly apparent that they fulfill important functions in the physiological processes of multicellular organisms.     

Ultrastructural Aspects and Possible Origin of Cytoplasmic Channels Providing Intercellular Connection in Vegetative Tissues of Anthers1

Cytoplasmic channel represents a huge intercellular connection other than plasmodesma. They are proposed to play an essential role in controlling cell-to-cell trafficking of macromolecules. The present study ultrastructurally examined the occurrence, structure, and formation of this intercellular path in somatic tissues of wheat, tobacco, and onion anthers as well as their differences from those present in anther generative tissue. It was shown that cytoplasmic channels existed not only in the pollen mother cells, but also in both epidermis and vascular parenchyma of the anthers. In somatic tissues, they appeared as plasmallema-lined tubes 400–750 nm wide filled with nuclear or cytoplasmic material, the latter including cytoplasmic matrix, ribosomes, and filamentous structures. Their biogenesis seems to result from the thinning of the local portions of cell wall containing multiple plasmodesmata and the fusion of plasmodesmata in such regions induced by the wall-digesting enzymes released by nearby located vesicles. In contrast to the channels existing in the pollen mother cells of tobacco, the cytoplasmic channels in the epidermal or vascular parenchyma cells of wheat, onion, and tobacco anthers usually do not appear in groups, but are single. Perhaps this is the way for nuclear material to migrate from cell to cell via a single channel and then form a single chromatin spherical body in the adjoining cell. An individual cell could not accept the nuclear material from another cell while extruding its own to the third cell. Cell-to-cell migration of organelles through the cytoplasmic channels was not shown in the somatic tissues.     

Mobile Macromolecules in Plant Development

Plant cells transmit developmental signals and distribute nutrients via dynamic intercellular channels termed plasmodesmata (PD). Multidisciplinary inquiries have provided evidence that plasmodesmatal regulation is critical to fundamental plant functions, such as development, host–pathogen interactions, and systemic RNA silencing. This review focuses on macromolecules that transport cell-to-cell through PD and describes their implications on plant development.     

Cell organization of the rat pars tuberalis. Evidence for open communication between pars tuberalis cells, cerebrospinal fluid and tanycytes

The pars tuberalis (PT) is the only pituitary region in close contact with the medial-basal hypothalamus and bathed by cerebrospinal fluid (CSF). Although PT has long been recognized as an endocrine gland, certain aspects of its structure remain obscure. The present investigation has been designed to gain information concerning (1) the cellular organization of PT, (2) the PT/median eminence spatial relationship and (3) the exposure of various cell compartments of PT to CSF. Non-endocrine cells (S100-reactive) appear as the organizer of the PT architecture. The apical poles of these cells line large cistern-like cavities and the processes of these cells establish a close spatial relationship with PT-specific secretory cells, portal capillaries and tanycytes. The cisterns are also endowed with clusters of ciliated cells and with a highly electron-dense and PAS-reactive content. The unique spatial organization of endocrine and non-endocrine cells of the PT supports a functional relationship between both cell populations. PT endocrine cells display a hallmark of PT-specific cells, namely, the paranuclear spot, which is a complex structure involving the Golgi apparatus, a large pool of immature secretory granules and a centriole from which originates a single 9+0 cilium projecting to the intercellular channels. Horseradish peroxidase (HRP) injected into the CSF readily reaches the intercellular channels of PT and the inner channel of the single cilium and is incorporated by the endocytic machinery of the secretory cells. The PT endocrine cells, through their single 9+0 cilium, may act as sensors of the CSF. HRP also reaches the lumen of the cisterns, indicating that this PT compartment is also exposed to CSF. PT endocrine cells establish direct cell-to-cell contacts with hypothalamic β₁ tanycytes, suggesting a second means of brain-PT communication.     

Cell-to-cell transport of macromolecules during early plant development

Plant cells exchange developmental signals, distribute nutrients and ribonucleoprotein complexes through dynamic intercellular channels termed plasmodesmata (PD). Multidisciplinary investigations over the last decade have provided evidence that plasmodesmatal regulation is critical to various basic plant functions, such as development, host-pathogen interactions, and systemic RNA-silencing. This review highlights the cell-to-cell transport of micro- and macromolecules via PD during embryo and seedling growth.     

Aluminum-induced 1-->3-beta-D-glucan inhibits cell-to-cell trafficking of molecules through plasmodesmata. A new mechanism of aluminum toxicity in plants

Symplastic intercellular transport in plants is achieved by plasmodesmata (PD). These cytoplasmic channels are well known to interconnect plant cells to facilitate intercellular movement of water, nutrients, and signaling molecules including hormones. However, it is not known whether Al may affect this cell-to-cell transport process, which is a critical feature for roots as organs of nutrient/water uptake. We have microinjected the dye lucifer yellow carbohydrazide into peripheral root cells of an Al-sensitive wheat (Triticum aestivum cv Scout 66) either before or after Al treatment and followed the cell-to-cell dye-coupling through PD. Here we show that the Al-induced root growth inhibition is closely associated with the Al-induced blockage of cell-to-cell dye coupling. Immunofluorescence combined with immuno-electron microscopic techniques using monoclonal antibodies against 1-->3-beta-D-glucan (callose) revealed circumstantial evidence that Al-induced callose deposition at PD may responsible for this blockage of symplastic transport. Use of 2-deoxy-D-glucose, a callose synthesis inhibitor, allowed us to demonstrate that a reduction in callose particles correlated well with the improved dye-coupling and reduced root growth inhibition. While assessing the tissue specificity of this Al effect, comparable responses were obtained from the dye-coupling pattern in tobacco (Nicotiana tabacum) mesophyll cells. Analyses of the Al-induced expression of PD-associated proteins, such as calreticulin and unconventional myosin VIII, showed enhanced fluorescence and co-localizations with callose deposits. These results suggest that Al-signal mediated localized alterations to calcium homeostasis may drive callose formation and PD closure. Our data demonstrate that extracellular Al-induced callose deposition at PD could effectively block symplastic transport and communication in higher plants.     

INTERCELLULAR COMMUNICATION IN RAPIDLY PROLIFERATING AND DIFFERENTIATED C6 GLIOMA CELLS IN CULTURE

Glial cells in the brain are known to provide structural and functional supports to neurons. To sustain such a supportive role, they have developed cell-to-cell communicating gap junctional channels. The authors studied the effect of dbcAMP on gap junctional channels mediated communication in C6 cells, a rat glioma cell line. Quantitative assessment of coupled cells under microscope after microinjection of a fluorescent dye was taken as a measure of junctional permeability. An enhanced coupling between cells was observed following dbcAMP treatment and this elevated coupling was found to be dependent on the duration of exposure of cells to dbcAMP. The studies have focused on a subtle shift in the spatial organization of the functional channels to the processes of dbcAMP induced differentiated cells from the cell cytoplasms and membranes of dbcAMP untreated cells. Immunofluorescence study with affinity purified antibody against gap junction further confirmed the spatial distribution of gap junctional protein(s) in the processes and also showed an increase in the density of the protein at the intercellular spaces in dbcAMP induced differentiated C6 glioma cells.     

Gap Junctional Channels Regulate Acid Secretion in the Mammalian Gastric Gland

Gap junction channels are regarded as a primary pathway for intercellular message transfer, including calcium wave propagation. Our study identified two gap junctional proteins, connexin26 and connexin32, in rat gastric glands by RT-PCR, Western blot analysis, and immunofluorescence. We demonstrated a potential physiological role of the gap junctional channels in the acid secretory process using the calcium indicator fluo-3, and microinjection of Lucifer Yellow. Application of gastrin (10⁻⁷ m) to the basolateral membrane resulted in the induction of uniphasic calcium signals in adjacent parietal cells. In addition, single parietal cell microinjections in intact glands with the cell-impermeant dye Lucifer Yellow resulted in a transfer of dye from the injected cell to the adjacent parietal cell following gastrin stimulation, demonstrating gastrin-induced cell-to-cell communication. Both calcium wave propagation and Lucifer Yellow transfer were blocked by the gap junction inhibitor 18α-glycyrrhetinic acid. Our studies demonstrate that functional gap junction channels in gastric glands provide an effective means for rapid cell-to-cell communication and allow for the rapid onset of acid secretion. Received: 4 December 2000/Revised: 5 June 2001     

Connexin channels modulation in pathophysiology and treatment of immune and inflammatory disorders

Connexin-mediated intercellular communication mechanisms include bidirectional cell-to-cell coupling by gap junctions and release/influx of molecules by hemichannels. These intercellular communications have relevant roles in numerous immune system activities. Here, we review the current knowledge about the function of connexin channels, mainly those formed by connexin-43, on immunity and inflammation. Focusing on those evidence that support the design and development of therapeutic tools to modulate connexin expression and/or channel activities with treatment potential for infections, wounds, cancer, and other inflammatory conditions.     

Subcellular localization determines the availability of non-targeted proteins to plasmodesmatal transport

BACKGROUND: Individual plant cells are encased in a cell wall. To enable cell-to-cell communication, plants have evolved channels, termed plasmodesmata, to span thick walls and interconnect the cytoplasm between adjacent cells. How macromolecules pass through these channels is now beginning to be understood. RESULTS: Using two green fluorescent protein (GFP) reporters and a non-invasive transfection system, we assayed for intercellular macromolecular traffic in leaf epidermal cells. Plasmodesmata were found in different states of dilation. We could distinguish two forms of protein movement across plasmodesmata, non-targeted and targeted. Although leaves have generally been considered closed to non-specific transport of macromolecules, we found that 23% of the cells had plasmodesmatal channels in a dilated state, allowing GFP that was not targeted to plasmodesmata to move into neighboring cells. GFP fusions that were targeted to the cytoskeleton or to the endoplasmic reticulum did not move between cells, whereas those that were localized to the cytoplasm or nucleus diffused to neighboring cells in a size-dependent manner. Superimposed upon this non-specific exchange, proteins that were targeted to the plasmodesmata could transit efficiently between 62% of transfected cells. CONCLUSIONS: A significant population of leaf cells contain plasmodesmata in a dilated state, allowing macromolecular transport between cells. Protein movement potential is regulated by subcellular address and size. These parameters of protein movement illustrate how gradients of signaling macromolecules could be formed and regulated, and suggest that non-cell-autonomous development in plants may be more significant than previously assumed.     

Targeting and modification of prokaryotic cell–cell junctions by tobacco mosaic virus cell‐to‐cell movement protein

The movement protein (MP) of tobacco mosaic virus (TMV) facilitates the cell-to-cell spread of infection by altering the structure and function of plasmodesmata, the intercellular communication channels in plants. Because the protein was shown to interfere with intercellular communication when expressed in the cyanobacterium Anabaena sp. strain PCC 7120, whether the ability of the protein to target and to modify intercellular communication channels in plants is conserved in this prokaryote was investigated. It was found that the MP localizes to the cell junctions and induces the formation of filamentous structures that traverse the septa. It is proposed that the protein interacts with host components that are similar between plants and Anabaena and that may be evolutionarily related. The observations in Anabaena suggest that the MP modifies plasmodesmata by forming a filamentous aggregate within the pore.     

Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48 h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.