Expression of a dominant negative inhibitor of intercellular communication in the early Xenopus embryo causes delamination and extrusion of cells

Development ◽  
1995 ◽  
Vol 121 (2) ◽  
pp. 371-381
Author(s):  
D.L. Paul ◽  
K. Yu ◽  
R. Bruzzone ◽  
R.L. Gimlich ◽  
D.A. Goodenough
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Positional Information ◽  
Embryonic Cell ◽  
Dominant Negative ◽  
Dye Transfer ◽  
Cell Stage ◽  
The Right

A chimeric construct, termed 3243H7, composed of fused portions of the rat gap junction proteins connexin32 (Cx32) and connexin43 (Cx43) has been shown to have selective dominant inhibitory activity when tested in the Xenopus oocyte pair system. Co-injection of mRNA coding for 3243H7 together with mRNAs coding for Cx32 or Cx43 completely blocked the development of channel conductances, while the construct was ineffective at blocking intercellular channel assembly when coinjected with rat connexin37 (Cx37). Injection of 3243H7 into the right anterodorsal blastomere of 8-cell-stage Xenopus embryos resulted in disadhesion and delamination of the resultant clone of cells evident by embryonic stage 8; a substantial number, although not all, of the progeny of the injected cell were eliminated from the embryo by stage 12. A second construct, 3243H8, differing from 3243H7 in the relative position of the middle splice, had no dominant negative activity in the oocyte pair assay, nor any detectable effects on Xenopus development, even when injected at four-fold higher concentrations. The 3243H7-induced embryonic defects could be rescued by coinjection of Cx37 with 3243H7. A blastomere reaggregation assay was used to demonstrate that a depression of dye-transfer could be detected in 3243H7-injected cells as early as stage 7; Lucifer yellow injections into single cells also demonstrated that injection of 3243H7 resulted in a block of intercellular communication. These experiments indicate that maintenance of embryonic cell adhesion with concomitant positional information requires gap junction-mediated intercellular communication.

scholarly journals Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly.

1994 ◽  
Vol 127 (6) ◽  
pp. 1895-1905 ◽  
Author(s):  
P D Lampe
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Freeze Fracture ◽  
Dye Transfer ◽  
Novikoff Hepatoma ◽  
Junctional Communication ◽  
Junction Protein

The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level.

Intercellular communication in the early embryo of the ascidian Ciona intestinalis

Development ◽  
1988 ◽  
Vol 102 (1) ◽  
pp. 55-63 ◽  
Author(s):  
F. Serras ◽  
C. Baud ◽  
M. Moreau ◽  
P. Guerrier ◽  
J.A.M. Van den Biggelaar
Keyword(s):  
Intercellular Communication ◽  
Lucifer Yellow ◽  
Ciona Intestinalis ◽  
Early Embryo ◽  
Cell Stage ◽  
Early Embryos ◽  
Junctional Conductance ◽  
Voltage Dependent ◽  
Cytoplasmic Bridges ◽  
Series Of Experiments

We have studied the intercellular communication pathways in early embryos of the ascidian Ciona intestinalis. In two different series of experiments, we injected iontophoretically the dyes Lucifer Yellow and Fluorescein Complexon, and we analysed the spread of fluorescence to the neighbouring cells. We found that before the 32-cell stage no dye spread occurs between nonsister cells, whereas sister cells are dye-coupled, possibly via cytoplasmic bridges. After the 32-cell stage, dye spread occurs throughout the embryo. However, electrophysiological experiments showed that nonsister cells are ionically coupled before the 32-cell stage. We also found that at the 4-cell stage junctional conductance between nonsister cells is voltage dependent, which suggests that conductance is mediated by gap junctions in a way similar to that observed in other embryos.

scholarly journals Modulation of gap junction mediated intercellular communication in TM3 Leydig cells

2003 ◽  
Vol 177 (2) ◽  
pp. 327-335 ◽  
Author(s):  
RC Goldenberg ◽  
FS Fortes ◽  
JM Cristancho ◽  
MM Morales ◽  
CR Franci ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Leydig Cells ◽  
Connexin 43 ◽  
Lucifer Yellow ◽  
Surface Membrane ◽  
Hormone Secretion ◽  
Functional Coupling ◽  
Western Blots

Long-term modulation of intercellular communication via gap junctions was investigated in TM3 Leydig cells, under low and high confluence states, and upon treatment of the cells for different times with activators of protein kinase A (PKA) and protein kinase C (PKC). Cells in low confluence were readily coupled, as determined by transfer of the dye Lucifer Yellow; on reaching confluence, the cells uncoupled. Western blots and RT-PCR revealed that connexin 43 (Cx43) was abundantly expressed in TM3 Leydig cells and its expression was decreased after the cells achieved confluence. Stimulation of PKA or PKC induced a decrease in cell-cell communication. Staurosporin, an inhibitor of protein kinases, increased coupling and was able to prevent and reverse the uncoupling actions of dibutyryl cAMP and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Under modulation by confluence, Cx43 was localized to the appositional membranes when cells were coupled and was mainly in the cytoplasm when they were uncoupled. In addition, cAMP and TPA reduced the surface membrane labeling for Cx43, whereas staurosporin increased it. These data show a strong correlation between functional coupling and the membrane distribution of Cx43, implying that this connexin has an important role in intercellular communication between TM3 cells. Furthermore, increased testosterone secretion in response to luteinizing hormone was accompanied by a decrease in intercellular communication, suggesting that gap junction mediated coupling may be a modulator of hormone secretion in TM3 cells.

Transgenic Disruption of Gap Junctional Intercellular Communication Enhances Early but Not Late Stage Hepatocarcinogenesis in the Rat

2005 ◽  
Vol 33 (6) ◽  
pp. 695-701 ◽  
Author(s):  
Naomi Hokaiwado ◽  
Makoto Asamoto ◽  
Kumiko Ogawa ◽  
Tomoyuki Shirai
Keyword(s):  
Intercellular Communication ◽  
Early Stage ◽  
Intraperitoneal Administration ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Connexin 32 ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Transgenic Rats ◽  
Gap Junctional

Much experimental evidence supports the conclusion that loss of gap junctional intercellular communication (GJIC) contributes to carcinogenesis. Transgenic rats featuring a dominant negative mutant of the connexin 32 gene under albumin promoter control (Cx32ΔTg-High and Cx32ΔTg-Low lines, respectively with high and low copy numbers of the transgene) have disrupted GJIC, as demonstrated by scrape dye-transfer assay in vivo as previous report by Asamoto et al. (2004) . In the present study, we investigated the susceptibility of these transgenic rats to a single intraperitoneal administration of diethylnitrosamine (DEN), and found a significant increase in preneoplastic glutathione S-transferase placental form (GST-P) positive lesions in the livers of Cx32ΔTg-High but not Cx32ΔTg-Low rats. However, incidences of adenomas and hepatocellular carcinomas were not elevated at the end of the experiment (52 weeks). In addition, we investigated the promotional effect of phenobarbital (PB) on Cx32ΔTg-High rats pretreated with DEN and found enhanced formation of GST-P positive lesions, in contrast to the lack of promoting effects reported for Cx32 deficient mice. The results indicate that although both high and low expression of the dominant negative connexin 32 mutant gene in our rats is able to inhibit gap junctional capacity, only high expression is effective at enhancing susceptibility to early stage DEN-induced liver carcinogenesis.

scholarly journals High-throughput measurement of gap junctional intercellular communication

2014 ◽  
Vol 306 (12) ◽  
pp. H1708-H1713 ◽  
Author(s):  
Jun Liu ◽  
Vinayakumar Siragam ◽  
Jun Chen ◽  
Michael D. Fridman ◽  
Robert M. Hamilton ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Cell Lines ◽  
High Throughput ◽  
Intercellular Communication ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Cell Injection ◽  
Operation Speed ◽  
Gap Junctional

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.

Mechanism of a novel missense mutation, p.V174M, of the human connexin31 (GJB3) in causing nonsyndromic hearing loss

2014 ◽  
Vol 92 (4) ◽  
pp. 251-257 ◽  
Author(s):  
Tung-Cheng Li ◽  
Yu-Hsiang Kuan ◽  
Tzu-Yu Ko ◽  
Chuan Li ◽  
Jiann-Jou Yang
Keyword(s):  
Hearing Loss ◽  
Gap Junction ◽  
Missense Mutation ◽  
Functional Change ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Dominant Negative Effect ◽  
Nonsyndromic Hearing Loss ◽  
Dye Transfer ◽  
Gap Junction Channel

Hearing loss is the most common sensory disorder, worldwide. In a recent study, we have identified a missense mutation, p.V174M, in the connexin 31 encoded by the GJB3 gene, in a patient with nonsyndromic hearing loss. However, the functional change in the CX31V174M mutant remains unknown. This study compared the intracellular distribution and assembly of the mutant CX31V174M with that of the wild-type (WT) CX31 in HeLa cells, and it examined the effect that the mutant protein had on those cells. A fluorescent localization assay of WT CX31 showed the typical punctuate pattern of a gap junction channel between the neighboring expression cells. Conversely, the p.V174M missense mutation resulted in the accumulation of the mutant protein in the lysosomes rather than in the cytoplasmic membrane. Moreover, dye transfer experiments have also demonstrated that the CX31V174M mutant did not form functional gap junction channels, probably due to the incorrect assembly or the altered properties of the CX31 channels. In addition, we found that CX31V174M-transfection can cause cell death by MTT assay. CX31V174M co-expressed with either CX31WT or CX26WT studies, suggested the impairment of the ability of CX26WT proteins to intracellular trafficking and targeting to the plasma membrane, but did not influence the trafficking of CX31WT. Based on these findings, we suggest that the CX31V174M mutant may have an effect on the formation and function of the gap junction, and CX31V174M has a trans-dominant negative effect on the function of wild types CX26. These results provide a novel molecular explanation for the role that GJB3 plays in hearing loss.

scholarly journals Asymmetric patterns of gap junctional communication in developing chicken skin

Development ◽  
1993 ◽  
Vol 119 (1) ◽  
pp. 85-96 ◽  
Author(s):  
F. Serras ◽  
S. Fraser ◽  
C.M. Chuong
Keyword(s):  
Lucifer Yellow ◽  
Single Cells ◽  
Dye Coupling ◽  
Dye Transfer ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Chicken Skin ◽  
Preferential Pathways ◽  
Gap Junctional ◽  
Feather Development

To study the pattern of gap junctional communication in chicken skin and feather development, we injected Lucifer Yellow into single cells and monitored the transfer of the fluorescent dye through gap junctions. Dye coupling is present between cells of the epithelium as well as between cells of the mesoderm. However, dye transfer did not occur equally in all directions and showed several consistent patterns and asymmetries, including: (1) no dye coupling between mesoderm and epithelium, (2) partial restriction of dye coupling at the feather bud/interbud boundary during early feather bud development, (3) preferential distribution of Lucifer Yellow along the anteroposterior axis of the feather placode and (4) absence of dye coupling in some epithelial cells. These results suggest the presence of preferential pathways of communication that may play a role in the patterning of chicken skin.

Block of intercellular communication: interaction of intracellular H+ and Ca2+

1987 ◽  
Vol 253 (4) ◽  
pp. C607-C612 ◽  
Author(s):  
J. M. Burt
Keyword(s):  
Intercellular Communication ◽  
Lucifer Yellow ◽  
Contractile Activity ◽  
Neonatal Rat ◽  
Dye Coupling ◽  
Myocardial Cells ◽  
Dye Transfer ◽  
Intracellular Acidosis ◽  
Communication Interaction ◽  
Junctional Permeability

The influence of elevated intracellular levels of H+ and Ca2+ on intercellular communication between cultured neonatal rat myocardial cells was examined by quantifying the percent of primary neighboring cells to which intracellularly injected Lucifer yellow had spread within 10 s of injection. Partial acidosis was induced by incubation in and then removal of NH4Cl. Intracellular Ca2+ was raised through the use of treatments that are standard in studies of heart muscle: reduction of the Na+ gradient, addition of caffeine, and combinations of these interventions. Under control conditions and during application of NH4Cl, cells exhibited spontaneous electrical and contractile activity and were well coupled (dye detectable in 100% of primary neighbors). Sustained intracellular acidosis without simultaneous elevation of intracellular Ca2+ (NH4Cl exposure followed by zero Na+, zero Ca2+) reduced the incidence of dye transfer to 90%. Elevation of intracellular Ca2+ (exposure to zero Na+, Ca2+-containing solution, with or without 10 mM caffeine) had no effect on coupling. These same interventions, when employed together, reduced the incidence of dye coupling to 18%. The results are consistent with a synergism of action of Ca2+ and H+ in the regulation of junctional permeability.

Increased gap junction assembly between cultured cells upon cholesterol supplementation

1990 ◽  
Vol 96 (2) ◽  
pp. 231-238
Author(s):  
R. Meyer ◽  
B. Malewicz ◽  
W.J. Baumann ◽  
R.G. Johnson
Keyword(s):  
Gap Junction ◽  
Actinomycin D ◽  
Cultured Cells ◽  
Lucifer Yellow ◽  
Freeze Fracture ◽  
Cholesterol Content ◽  
Dye Transfer ◽  
Novikoff Hepatoma ◽  
Fourfold Increase ◽  
Cholesterol Supplementation

Novikoff hepatoma cells provide an excellent model system for the study of gap junction assembly, a process that could be influenced by lipids and other factors at numerous points. Since it is possible to alter the cellular levels of cholesterol in these cells, it was added to the cells in serum-supplemented medium and changes in gap junction assembly were evaluated. Cells were dissociated and reaggregated following exposure to a range of cholesterol concentrations for 24 h. A five- to sixfold increase in the number of aggregated gap junction particles and a 50% increase in cellular cholesterol content were observed with 20 microM added cholesterol. A 1-h exposure to added cholesterol, during cell reaggregation, resulted in a fourfold increase in the number of aggregated gap junction particles, demonstrating that the effect was rapid. The number of aggregated gap junction particles and formation plaque areas were used as measures of junction assembly and assayed by quantitative freeze-fracture and electron microscopy. Junctional permeabilities were evaluated by means of dye transfer times following the intracellular microinjection of Lucifer Yellow. Increased dye transfer was observed between cholesterol-treated cells, which suggested that the increase in assembly was accompanied by an increase in junction permeability. Cells were treated with cycloheximide (100 micrograms ml-1) and actinomycin D (10 micrograms ml-1) to determine whether protein and RNA syntheses were involved in the enhanced gap junction assembly. Cycloheximide but not actinomycin D blocked the increased junction assembly observed with added cholesterol. These results suggested that protein synthesis, but not RNA synthesis, is necessary for the increased gap junction formation observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The correlation between patterns of dye transfer junctions and future developmental fate in Xenopus: u.v. irradiation and lithium treatment

Development ◽  
1989 ◽  
Vol 105 (4) ◽  
pp. 747-752 ◽  
Author(s):  
D.J. Nagajski ◽  
S.C. Guthrie ◽  
C.C. Ford ◽  
A.E. Warner
Keyword(s):  
Lucifer Yellow ◽  
Lithium Treatment ◽  
Cell Communication ◽  
Embryonic Axis ◽  
Axis Formation ◽  
Dye Transfer ◽  
Cell Stage ◽  
The Future ◽  
Vegetal Pole ◽  
Cell Embryo

The correlation between cell-to-cell communication junctions at the 32-cell stage and the subsequent embryonic axis has been examined in Xenopus laevis Disturbances of embryonic axis formation were u.v. irradiation at the vegetal pole before 0.6 in the which generates embryos with dorsal axial embryos were treated with 100mM-lithium chloride 32-cell stage, which generates embryos with ventral The cell-to-cell transfer of Lucifer Yellow was used junctional permeability. Injections were made into cells, lying in tiers 1 and 2 of the 32-cell embryo, relative to the future dorsoventral axis of the embryo on the basis of differences in pigmentation. The Yellow transfer in the future dorsal half of the compared with that in the future ventral half for u.v.-irradiated and Li-treated embryos. Injected subsequently scored for axial developmenf for transfer frequencies. In control embryos at the 32- Yellow transfer was both more frequent and more dorsal regions than in future ventral regions, as In embryos that had been u.v. irradiated before 0.6 in cycle, Lucifer transfer was the same in both light and the animal hemisphere and at the low level ventral regions in normal embryos. These embryos reductions in dorsal axial structures. Embryos the first cell cycle, when u.v. irradiation no longer cytoplasmic movements initiated at fertilization, dorsoventral difference in Lucifer Yellow transfer and normal dorsoventral polarity. Embryos exposed to

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