Junctions In Cultures Research Articles

Striatins as plaque molecules of zonulae adhaerentes in simple epithelia, of tessellate junctions in stratified epithelia, of cardiac composite junctions and of various size classes of lateral adherens junctions in cultures of epithelia- and carcinoma-derived cells

Proteins of the striatin family (striatins 1–4; sizes ranging from 90 to 110 kDa on SDS-polyacrylamide gel electrophoresis) are highly homologous in their amino acid sequences but can differ in their cell-type-specific gene expression patterns and biological functions. In various cell types, we have found one, two or three polypeptides of this evolutionarily old and nearly ubiquitous family of proteins known to serve as scaffold proteins for diverse protein complexes. Light and electron microscopic immunolocalization methods have revealed striatins in mammalian cell-cell adherens junctions (AJs). In simple epithelia, we have localized striatins as constitutive components of the plaques of the subapical zonulae adhaerentes of cells, including intestinal, glandular, ductal and urothelial cells and hepatocytes. Striatins colocalize with E-cadherin or E–N-cadherin heterodimers and with the plaque proteins α- and β-catenin, p120 and p0071. In some epithelia and carcinomas and in cultured cells derived therefrom, striatins are also seen in lateral AJs. In stratified epithelia and in corresponding squamous cell carcinomas, striatins can be found in plaques of some forms of tessellate junctions. Moreover, striatins are major plaque proteins of composite junctions (CJs; areae compositae) in the intercalated disks connecting cardiomyocytes, colocalizing with other CJ molecules, including plectin and ankyrin-G. We discuss the “multimodulator” scaffold roles of striatins in the initiation and regulation of the formation of various complex particles and structures. We propose that striatins are included in the diagnostic candidate list of proteins that, in the CJs of human hearts, can occur in mutated forms in the pathogeneses of hereditary cardiomyopathies, as seen in some types of genetically determined heart damage in boxer dogs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-014-2053-z) contains supplementary material, which is available to authorized users.

Bone marrow stromal cells as an inducer for cardiomyocyte differentiation from mouse embryonic stem cells

Bone marrow stromal cells (BMSCs) secrete soluble factors and display varied cell-biological functions. To confirm the ability and efficiency of BMSCs to induce embryonic stem cells (ESCs) into cardiomyocytes, mouse embryoid bodies (EBs) were co-cultured with rat BMSCs. After about 10 days, areas of rhythmically contracting cells in more solid aggregates became evident with bundle-like structures formed along borders between EB outgrowth and BMSC layer. ESC-derived cardiomyocytes exhibited sarcomeric striations when stained with troponin I (Trop I), organized in separated bundles. Besides, the staining for connexin 43 was detected in cell-cell junctions, which demonstrated that ESC-derived cardiomyocytes were coupled by gap junction in culture. The related genes of cardiomyocytes were found in these beating and no-beating EBs co-cultured with BMSCs. In addition, an improved efficiency of cardiomyocyte differentiation from ESC-BMSC co-culture was found in the serum-free medium: 5-fold up-regulation in the number of beating area compared with the serum medium. Effective cardiac differentiation was also recognized in transfer filter assay and in condition medium obtained from BMSC culture. A clear increase in the expression of cardiac genes and TropI protein confirmed further cardiac differentiation by BMP4 and Retinoic Acid (RA) treatment. These results demonstrate that BMSCs can induce cardiomyocyte differentiation from ESCs through soluble factors and enhance it with BMP4 or RA treatment. Serum-free ESC-BMSC co-culture represents a defined in vitro model for identifying the cardiomyocyte-inducing activity from BMSCs and, in addition, a straightforward experimental system for assessing clinical applications.

Intercellular calcium signaling between astrocytes and oligodendrocytes via gap junctions in culture

To understand further how oligodendrocytes regulate brain function, the mechanism of communication between oligodendrocytes and other cell types needs to be explored. An important mode of communication between various cell types in the nervous system involves gap junctions. Astroglial cells are extensively connected through gap junctions forming the glial syncytium. Although the presence of gap junctions between oligodendrocytes and astrocytes have been well documented, evidence for gap junction-mediated calcium transfer between these two glial populations is still missing. To measure functional coupling between astrocytes and oligodendrocytes and to test whether this coupling is mediated by gap junctions we used laser photostimulation and monitored Ca2+ propagation in cultures from transgenic animals in which oligodendrocytes express enhanced green fluorescent protein (eGFP). We show that waves of Ca2+ spread from astrocytes to oligodendrocytes and that these waves are blocked by the broad-spectrum gap junction blocker carbenoxolone, but not the neuron-specific gap junction blocker quinine. We also show that the spread of Ca2+ waves between astrocytes and oligodendrocytes is bi-directional. Thus, increase of Ca2+ concentration in astrocytes triggered by surrounding neuronal activity may feed back onto different neuronal populations through oligodendrocytes.

PlGF-1 and VEGFR-1 Pathway Regulation of the External Epithelial Hemato-Ocular Barrier

VEGF is considered as an important factor in the pathogenesis of macular edema. VEGF induces the rupture of the blood retinal barrier and may also influence the retinal pigment epithelial (RPE) outer retinal barrier. The aim of this work was to analyze the influence of the VEGF receptor pathways in the modulation of the RPE barrier breakdown in vitro and in vivo. The ARPE19 human junctions in culture are modulated by VEGF through VEGFR-1 but not through VEGFR-2. PlGF-1, that is a pure agonist of VEGFR-1, is produced in ARPE-19 cells under hypoxic conditions and mimics VEGF effects on the external retinal barrier as measured by TER and inulin flux. In vivo, the intravitreous injection of PlGF-1 induces a rupture of the external retinal barrier together with a retinal edema. This effect is reversible within 4 days. VEGF-E, that is a pure agonist of VEGFR-2, does not induce any acute effect on the RPE barrier. These results demonstrate that PlGF-1 can reproduce alterations of the RPE barrier occurring during diabetic retinopathy.

Controlling Retinal Pigment Epithelium Injury after Experimental Detachment of the Retina

Damage induced by detachment of the neural retina and the retinal pigment epithelium (RPE) can be reduced by dark adaptation. The authors evaluated the influence of the duration of dark adaptation, time of day, and modification of the melatonin-dopamine pathway on acute RPE lesions induced by mechanical detachment. BALB/c mice were studied at different times of day and different periods of dark adaptation. Some mice were treated with melatonin or sulpiride, a D2 dopamine receptor antagonist. Enucleated eyes and different saline solutions were used in experiments ex vivo. Retinal detachments in vivo were made by subretinal injections of hyaluronic acid. RPE cell damage was quantitatively evaluated with a dye exclusion procedure, and their viability was tested by preservation of tight junctions in culture. Lectin histochemistry was used to examine the interphotoreceptor matrix (IPM). Significant propidium iodide (PI) incorporation in RPE cells was detected after ex vivo separation during daytime, but it was very low when detachment took place at night after 24 to 48 hours of dark adaptation. PI exclusion was achieved during daytime after a single hour of dark adaptation when mice were pretreated with melatonin or sulpiride. Reduction of RPE cell damage was accompanied by decreased lectin binding to cone sheaths. A combination of time of day and length of dark adaptation decreased damage induced by detachment of the retina ex vivo and in vivo. Melatonin or sulpiride could replace these environmental factors. Therefore, melatonin and dopamine pathways might be involved in the control of IPM properties and retina/RPE interactions.

Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells.

Cardiomyocytes derived from human embryonic stem (hES) cells could be useful in restoring heart function after myocardial infarction or in heart failure. Here, we induced cardiomyocyte differentiation of hES cells by a novel method and compared their electrophysiological properties and coupling with those of primary human fetal cardiomyocytes. hES cells were cocultured with visceral-endoderm (VE)-like cells from the mouse. This initiated differentiation to beating muscle. Sarcomeric marker proteins, chronotropic responses, and ion channel expression and function were typical of cardiomyocytes. Electrophysiology demonstrated that most cells resembled human fetal ventricular cells. Real-time intracellular calcium measurements, Lucifer yellow injection, and connexin 43 expression demonstrated that fetal and hES-derived cardiomyocytes are coupled by gap junctions in culture. Inhibition of electrical responses by verapamil demonstrated the presence of functional alpha1c-calcium ion channels. This is the first demonstration of induction of cardiomyocyte differentiation in hES cells that do not undergo spontaneous cardiogenesis. It provides a model for the study of human cardiomyocytes in culture and could be a step forward in the development of cardiomyocyte transplantation therapies.

Activity-Dependent Expression of NT-3 in Muscle Cells in Culture: Implications in the Development of Neuromuscular Junctions

Although activity-dependent expression of neurotrophins has been studied extensively in the CNS, its physiological role during synapse development is not well established. At the developing neuromuscular junction in culture, exogenous application of the neurotrophin BDNF or NT-3 has been shown to acutely potentiate synaptic transmission and chronically promote synapse maturation. Using the same cell culture model, we have investigated activity-dependent neurotrophin expression in muscle cells and its role in developing neuromuscular synapses. Membrane depolarization, elicited by either depolarizing agents or repetitive electric stimulation, rapidly and specifically increased the levels of NT-3 mRNA in developing Xenopus laevis muscle cells in culture. NT-3 gene expression also was enhanced by acetylcholine (ACh), the neurotransmitter that causes muscle membrane depolarization. The effects of depolarization were mediated by increasing intracellular calcium concentration. Moreover, factor(s) induced by membrane depolarization appeared to enhance synaptic transmission at the developing neuromuscular junction. The frequency of spontaneous synaptic currents (SSCs) recorded from neuromuscular synapses was increased significantly after treatment with conditioned medium from depolarized muscle cultures. The amplitude, rise time, and decay time of SSCs were not affected, indicating a presynaptic action of the conditioned medium. The effects of the conditioned medium were blocked, partially, by the NT-3 scavenger TrkC-IgG, suggesting that the potentiation of synaptic efficacy was attributable, at least in part, to elevated NT-3 as a consequence of muscle depolarization. Thus, activity-dependent expression of muscle NT-3 may contribute to the development of the neuromuscular synapse.

Na(+)-dependent glucose transporter SGLT1 is localized in the apical plasma membrane upon completion of tight junction formation in MDCK cells.

SGLT1, an isoform of Na(+)-dependent glucose transporters, is localized at the apical plasma membrane in the epithelial cells of the small intestine and the kidney. In the present study we examined its location in SGLT1 cDNA-transfected MDCK cells, which form an epithelial sheet connected by tight junctions in culture. Formation of tight junctions was monitored by staining for occludin, an integral tight junction protein. In the cells demarcated by an uninterrupted occludin meshwork, SGLT1 was specifically localized at the apical plasma membrane, showing that SGLT1 has a signal to accomplish this restricted localization. In the cells with little or no occludin accumulation in the tight junction, however, SGLT1 was present along the entire aspect of the plasma membrane. Similar distribution of SGLT1 was observed in the cells as long as the occludin meshwork remained incomplete. These observations suggest that apical localization of SGLT1 occurs upon the completion of the uninterrupted meshwork of tight junctions.

Changes in Cellular Distribution of Connexins 32 and 26 during Formation of Gap Junctions in Primary Cultures of Rat Hepatocytes

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sectionsin vivo.Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.

Erratic Deposition of Agrin during the Formation of Xenopus Neuromuscular Junctions in Culture

In order to disclose the mechanisms that regulate synapse development we compared the distributions of agrin, acetylcholine receptors (AChR) and a basal lamina heparan sulfate proteoglycan (HSPG) in sections and cultures prepared from Xenopus laevis and Ambystoma mexicanum embryos. While agrin, AChR and HSPG may accumulate almost synchronously at synapses in vivo, agrin deposition usually lagged well behind the other synaptic markers during development in culture, and was not detectable at many differentiated junctions. Agrin deposition at nerve-muscle contacts in culture also appeared to require the presence of other synaptic components. A similarly variable deposition occurred on noninnervated myocytes, where agrin again collected near sites of HSPG and AChR accumulation on some cells. Profuse agrin accretion occurred consistently, however, within the extracellular matrices of surrounding epithelial cells derived from both myotomes and neural tubes. In cocultures of Ambystoma neurons and Xenopus myocytes Ambystoma agrin collected at some chimeric neuromuscular junctions, but also accumulated on noninnervated myocytes and in the extracellular matrices of salamander neuroendothelial cells. Based upon these observations we conclude that (a) focal agrin deposition is not required for synaptic differentiation on Xenopus myocytes and (b) agrin may be one of several muscle basal lamina components that stem mainly from the secreted products of nearby epithelial cells.

Properties of an identified dopamine-containing neurone in culture from the snail Helisoma.

The giant neurone in the left pedal ganglion of the snail Helisoma trivolvis is homologous with the giant dopaminergic neurone of Planorbis corneus, because the neurones have a very similar location and morphology, and react similarly with glyoxylic acid to produce an intense blue fluorescence, indicating the presence of dopamine. Each of these neurones is therefore referred to as a giant dopaminergic neurone, or GDN. Conditions for the extension of neurites and formation of chemical junctions in culture have been determined for the H. trivolvis GDN, and compared with other neurones from this species. The pattern of neurites that extended from the neurone was indistinguishable from that of another identified aminergic neurone, the large serotonergic neurone (LSN), but differed markedly from many other central neurones. However, the type of substrate also greatly affected the pattern of the neurites observed. Some of the electrical properties of the GDN in culture differed from those recorded in situ: peak spike amplitude was increased, spike half-width reduced and the firing pattern of the neurone was altered. However, the resting membrane potential was very similar. The GDN formed chemical and electrical junctions in culture. The chemical junctions formed were of the same type as those found in situ. They formed rapidly, within 18 h after plating, but were not stable and were lost within 48 h, to be replaced by a non-rectifying electrical junction. A chemical junction may form in either direction between the GDN and the LSN, but only rarely did such junctions allow transmission in both directions, as observed in situ. Experiments in which neurones were plated out at different times suggested that the direction of formation of the chemical junction was not dependent on the degree or state of neurite extension.

Quantal and non-quantal ACh release at developing Xenopus neuromuscular junctions in culture.

1. Single acetylcholine receptor (AChR) channel openings, detected by the whole-cell patch clamp technique, were used to monitor quantal and non-quantal ACh release at synapses in 1- and 2-day-old co-cultures of Xenopus embryonic motoneurons and muscle cells. motoneuron growth cones in ways that presumably reflect muscle-nerve inductive influences and the development of neurotransmitter release mechanisms. 2. Miniature endplate currents (MEPCs) occurred at a mean frequency of approximately 0.6 s-1 with a skewed distribution and mean amplitude of about twenty channel openings. In addition, occasional brief episodes of rapid deviations in the baseline were observed in some cells, with mean amplitudes of 4-8 pA and durations of a few hundred milliseconds. However, these episodes did not closely resemble summated openings of AChR channels. Moreover, where tested, these episodes were not blocked by curare; and comparable episodes were seen in an uninnervated myocyte. Thus they appear not to reflect ACh release from the nerve terminal. 3. Single-channel openings that might have been responses to non-quantal release of ACh were observed at rates of 0.9-12.3 min-1 (mean 3.0 min-1), only 1-5 times the rate of spontaneous AChR channel openings in uninnervated myocytes (mean 1.4 min-1). 4. We conclude that there is no significant non-quantal ACh leak from the presynaptic contacts in these immature synapses under these culture conditions. This is in disagreement with other, less direct, experimental reports, but consistent with findings in mature frog motor nerve terminals.

Increases in pericellular proteolysis at developing neuromuscular junctions in culture

To determine whether localized changes in pericellular proteolysis contribute to synapse formation, we examined the degradative actions of developing Xenopus laevis nerve and muscle cells on films of extracellular matrix proteins adsorbed to the glass surface of a tissue culture chamber. Skeletal myocytes, growing neurites, and fibroblasts all removed fluorescent fibronectin and laminin from the culture substratum at regions of close cell-surface contact. In addition, however, motor neurites also displayed a particularly enhanced rate of gelatin elimination at developing neuromuscular junctions. It has already been shown (a) that there is a similar remodeling of organized muscle basal lamina proteoglycan accumulations along the path of nerve-muscle contact and (b) that this is the earliest detectable biochemical change specific to developing neuromuscular junctions. Our observations thus suggest that the establishment of motoneuron-muscle contact leads to a further activation of pericellular proteinases along both the pre- and the postsynaptic surfaces of the developing junction. We therefore consider whether site-specific proteinase-activation cascades could contribute to the inductive signals that direct synaptic differentiation.

Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule.

PECAM-1 is a 130-120-kD integral membrane glycoprotein found on the surface of platelets, at endothelial intercellular junctions in culture, and on cells of myeloid lineage. Previous studies have shown that it is a member of the immunoglobulin gene superfamily and that antibodies against the bovine form of this protein (endoCAM) can inhibit endothelial cell-cell interactions. These data suggest that PECAM-1 may function as a vascular cell adhesion molecule. The function of this molecule has been further evaluated by transfecting cells with a full-length PECAM-1 cDNA. Transfected COS-7, mouse 3T3 and L cells expressed a 130-120-kD glycoprotein on their cell surface that reacted with anti-PECAM-1 polyclonal and monoclonal antibodies. COS-7 and 3T3 cell transfectants formed cell-cell junctions that were highly enriched in PECAM-1, reminiscent of its distribution at endothelial cell-cell borders. In contrast, this protein remained diffusely distributed within the plasma membrane of PECAM-1 transfected cells that were in contact with mock transfectants. Mouse L cells stably transfected with PECAM-1 demonstrated calcium-dependent aggregation that was inhibited by anti-PECAM antibodies. These results demonstrate that PECAM-1 mediates cell-cell adhesion and support the idea that it may be involved in some of the interactive events taking place during thrombosis, wound healing, and angiogenesis.

Cultured myometrial cells establish communicating gap junctions

Myometrial cells were isolated and cultured from term rat uterus. The myometrial origin of the cultures was verified by antibody staining of cellular desmin and α-smooth muscle actin. The presence of functional gap junctions was indicated by transfer of radiolabeled nucleotide and microinjected Lucifer yellow dye. The cultured cells expressed mRNA recognized by a connexin43 gap junction cDNA probe. To our knowledge, this is the first report that isolated myometrial cells form gap junctions in culture.

Presynaptic calcium currents evoking quantal transmission from avian ciliary ganglion neurons

Using whole-cell patch clamp techniques, we simultaneously recorded presynaptic Ca++ current and excitatory postsynaptic currents (EPSCs) from avian neuromuscular junctions in culture. Quantal synaptic transmission was proportional to evoked presynaptic Ca++ current except with large stimuli, which evoked bursts of quanta, reflecting a shift to synchronized release. Synaptic delay, measured from the onset of presynaptic depolarization to the appearance of the first postsynaptic quantal response, was often greater than 100 msec for weak depolarizations but declined as stimulus intensity was increased. Quantal events evoked by Ca++ tail currents had a mean synaptic delay of 1.67 msec. The single type of presynaptic Ca++ current observed displayed an inactivation time constant of greater than 100 msec and tail currents well fit by a single exponential function.

Structure of gap junctions in cultures of normal and neoplastic bladder epithelial cells.

Normal rat urinary bladder epithelial cells contain small subunit (PF-1) and large subunit (PF-2) gap junctions, whereas carcinoma cells only contain PF-1 gap junctions. The absence of PF-2 gap junctions, which are composed of larger connections with slightly larger ionic channels, may contribute to altered metabolic coupling between urinary bladder carcinoma cells.

Effects of glycolipids on in vitro development of neuromuscular junction.

THE nervous system has a unique distribution of glycolipids1, which changes markedly during growth of the animals2 and in various inherited metabolic disorders3. No physiological roles for glycolipids in neural function are known, however. But, in nonneural cells glycolipids of the cell membrane are essential for cell recognition and interaction4. For example, some gangliosides act as receptors for various bacterial toxins5 and globoside has been identified as the human P blood type antigen6. When administered in the medium, ganglioside can be incorporated into cell membranes and function as toxin receptors5,7. Ganglioside added to the medium competes with membrane receptors by binding to the toxins5. We reported previously8,9 that the effects of some drugs and sera on the formation of neuromuscular junction in culture could be evaluated by recording the endplate potential (e.p.p.) from muscle cells combined with the spinal cord. We describe here the effect of glycolipids on the development of neuromuscular junction in culture of skeletal muscle and the spinal cord. Globoside had biphasic effects, an inhibitory effect at the higher concentration and a facilitatory one at the lower concentration or on brief exposure at the beginning of the cultivation. Our evidence suggests that glycolipids are involved in formation of the neuromuscular junction.

Development of neuromuscular transmission in culture with a variety of neurons and in the presence of cholinergic substances and tetrodotoxin

Summary The formation of neuromuscular junctions was investigated electrophysiologically in co-cultures of skeletal muscle cells and a variety of neurons obtained from chick embryo and rat. Neurons were plated as dissociated cells or tissue explants. One to 10 days after plating neurons, end-plate potentials (EPPs) were recorded from embryonic chick myotubes co-cultured with embryonic chick spinal cord, medulla oblongata and ciliary ganglion. The EPPs occurred spontaneously with frequency of 0.2–60/sec in 39–83% of myotube. Nerve stimulation also induced EPPs. No selectivity in junction formation on pectoral or thigh muscle was found among cervical, brachial and lumbar regions of the spinal cord. EPPs were observed in cultures of not only chick muscle-chick spinal cord but also rat muscle-rat cord, rat muscle-chick cord and chick muscle-rat cord. On the other hand EPPs were not observed in co-cultures of chick myotubes with chick cerebellum, cerebrum, superior cervical ganglia or dorsal root ganglia. Muscle cells and spinal cord explants of chick embryo were cultured in the presence of high concentrations of acetylcholine (ACh), carbachol, d -tubocurarine, gallamine, atropine, dibenamine or tetrodotoxin. When these substances were removed after 1–5 days of culture, spontaneous EPPs were observed similarly. It is concluded that neither functional ACh receptors nor nerve action potentials were necessary for the development of the neuromuscular junction in culture.

Ultrastructural Aspects and Functional Implications of Some Specialized Junctions in Cultures in Vitro of Nervous Tissue

Ultrastructural Aspects and Functional Implications of Some Specialized Junctions in Cultures in Vitro of Nervous Tissue