scholarly journals Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43.

1995 ◽  
Vol 130 (4) ◽  
pp. 987-995 ◽  
Author(s):  
M Koval ◽  
S T Geist ◽  
E M Westphale ◽  
A E Kemendy ◽  
R Civitelli ◽  
...  
Keyword(s):  
Gap Junction ◽  
Lucifer Yellow ◽  
Direct Interaction ◽  
Full Length ◽  
Osteosarcoma Cell ◽  
Dye Transfer ◽  
Fluorescent Reporter ◽  
Intercellular Transfer ◽  
Carboxyl Terminal ◽  
Gap Junctional

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin.

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.

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.

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)

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 Gap Junctional Communication in the Early Xenopus Embryo

2000 ◽  
Vol 150 (4) ◽  
pp. 929-936 ◽  
Author(s):  
Yosef Landesman ◽  
Daniel A. Goodenough ◽  
David L. Paul
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Synergistic Effects ◽  
Xenopus Embryo ◽  
Dye Transfer ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Whole Mount ◽  
Gap Junctional ◽  
Whole Mounts

In the Xenopus embryo, blastomeres are joined by gap junctions that allow the movement of small molecules between neighboring cells. Previous studies using Lucifer yellow (LY) have reported asymmetries in the patterns of junctional communication suggesting involvement in dorso-ventral patterning. To explore that relationship, we systematically compared the transfer of LY and neurobiotin in embryos containing 16–128 cells. In all cases, the junction-permeable tracer was coinjected with a fluorescent dextran that cannot pass through gap junctions. Surprisingly, while LY appeared to transfer in whole-mount embryos, in no case did we observe junctional transfer of LY in fixed and sectioned embryos. The lack of correspondence between data obtained from whole-mounts and from sections results from two synergistic effects. First, uninjected blastomeres in whole-mounts reflect and scatter light originating from the intensely fluorescent injected cell, creating a diffuse background interpretable as dye transfer. Second, the heavier pigmentation in ventral blastomeres masks this scattered signal, giving the impression of an asymmetry in communication. Thus, inspection of whole-mount embryos is an unreliable method for the assessment of dye transfer between embryonic blastomeres. A rigorous and unambiguous demonstration of gap junctional intercellular communication demands both the coinjection of permeant and impermeant tracers followed by the examination of sectioned specimens. Whereas LY transfer was never observed, neurobiotin was consistently transferred in both ventral and dorsal aspects of the embryo, with no apparent asymmetry. Ventralization of embryos by UV irradiation and dorsalization by Xwnt-8 did not alter the patterns of communication. Thus, our results are not compatible with current models for a role of gap junctional communication in dorso-ventral patterning.

A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes

1998 ◽  
Vol 111 (1) ◽  
pp. 31-43 ◽  
Author(s):  
F. Cao ◽  
R. Eckert ◽  
C. Elfgang ◽  
J.M. Nitsche ◽  
S.A. Snyder ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Hela Cells ◽  
Xenopus Oocytes ◽  
Lucifer Yellow ◽  
Expression System ◽  
Electrical Conductance ◽  
Dye Transfer ◽  
Specific Permeability ◽  
Fluorescent Molecules

Gap junctions provide direct intercellular communication by linking adjacent cells with aqueous pores permeable to molecules up to 1 kDa in molecular mass and 8–14 A in diameter. The identification of over a dozen connexins in the mammalian gap junction family has stimulated interest in the functional significance of this diversity, including the possibility of selectivity for permeants as seen in other channel classes. Here we present a quantitative comparison of channel permeabilities of different connexins expressed in both HeLa transfectants (rat Cx26, rat Cx32 and mouse Cx45) and Xenopus oocytes (rat Cx26 and rat Cx32). In HeLa cells, we examined permeability to two fluorescent molecules: Lucifer Yellow (LY: anionic, MW 457) and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, cationic, MW 350). A comparison of the kinetics of fluorescent dye transfer showed Cx32, Cx26 and Cx45 to have progressively decreasing permeabilities to LY, but increasing permeabilities to DAPI. This pattern was inconsistent with selection based on physical size of the probe, nor could it be accounted for by the differences between clones in the electrical conductance of the monolayers. In Xenopus oocytes, where electrical and dye coupling could be assessed in the same cells, Cx32 coupled oocytes showed an estimated 6-fold greater permeability to LY than those coupled by Cx26, a comparable result to that seen in HeLa cells, where an approximately 9-fold difference was seen. The oocyte system also allowed an examination of Cx32/Cx26 heterotypic gap junction that proved to have a permeability intermediate between the two homotypic forms. Thus, independent of the expression system, it appears that connexins show differential permeabilities that cannot be predicted based on size considerations, but must depend on other features of the probe, such as charge.

scholarly journals Regulation of gap junction coupling in bovine ciliary epithelium

2010 ◽  
Vol 298 (4) ◽  
pp. C798-C806 ◽  
Author(s):  
Zhao Wang ◽  
Chi Wai Do ◽  
Virginijus Valiunas ◽  
Chi Ting Leung ◽  
Angela K. W. Cheng ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Quantitative Polymerase Chain Reaction ◽  
Donor Cell ◽  
Ciliary Epithelium ◽  
Dibutyryl Camp ◽  
Dye Transfer ◽  
Qrt Pcr ◽  
Junctional Communication ◽  
Gap Junctional

Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 ± 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (Rf) in recipient to donor cell was 0.47 ± 0.09 ( n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased Rf by ∼60% to 0.20 ± 0.07 ( n = 13, P < 0.02). Dibutyryl-cAMP (500 μM) also reduced LY dye transfer by ∼60%, reducing Rf from 0.41 ± 0.05 ( n = 15) to 0.17 ± 0.05 ( n = 20) after 10 min. Junctional currents were lowered by ∼50% ( n = 6) after 10-min perfusion with 500 μM dibutyryl-cAMP ( n = 6); thereafter, heptanol abolished the currents ( n = 5). Preincubation with the PKA inhibitor H-89 (2 μM) prevented cAMP-triggered current reduction ( n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication.

scholarly journals Gap junctional coupling and connexin immunoreactivity in rabbit retinal glia

2006 ◽  
Vol 23 (1) ◽  
pp. 1-10 ◽  
Author(s):  
KATHLEEN R. ZAHS ◽  
PAUL W. CEELEN
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Lucifer Yellow ◽  
Müller Cells ◽  
Müller Cell ◽  
Muller Cells ◽  
Junctional Coupling ◽  
Muller Cell ◽  
Gap Junctional ◽  
Gap Junctional Coupling

Gap junctions provide a pathway for the direct intercellular exchange of ions and small signaling molecules. Gap junctional coupling between retinal astrocytes and between astrocytes and Müller cells, the principal glia of vertebrate retinas, has been previously demonstrated by the intercellular transfer of gap-junction permeant tracers. However, functional gap junctions have yet to be demonstrated between mammalian Müller cells. In the present study, when the gap-junction permeant tracers Neurobiotin and Lucifer yellow were injected into a Müller cellviaa patch pipette, the tracers transferred to at least one additional cell in more than half of the cases examined. Simultaneous whole-cell recordings from pairs of Müller cells in the isolated rabbit retina revealed electrical coupling between closely neighboring cells, confirming the presence of functional gap junctions between rabbit Müller cells. The limited degree of this coupling suggests that Müller cell–Müller cell gap junctions may coordinate the functions of small ensembles of these glial cells. Immunohistochemistry and immunoblotting were used to identify the connexins in rabbit retinal glia. Connexin30 (Cx30) and connexin43 (Cx43) immunoreactivities were associated with astrocytes in the medullary ray region of the retinas of both pigmented and albino rabbits. Connexin43 was also found in Müller cells, but antibody recognition differed between astrocytic and Müller cell connexin43.

Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery

2001 ◽  
Vol 280 (6) ◽  
pp. H2441-H2450 ◽  
Author(s):  
Andrew T. Chaytor ◽  
Patricia E. M. Martin ◽  
David H. Edwards ◽  
Tudor M. Griffith
Keyword(s):  
Gap Junctions ◽  
Hepatic Artery ◽  
Lucifer Yellow ◽  
Dye Transfer ◽  
Cell Coupling ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
A7r5 Cells ◽  
Gap Junctional

Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26,43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 μM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 μM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.

Cell-to-cell diffusion of fluorescent dyes in paired ventricular cells

1987 ◽  
Vol 252 (1) ◽  
pp. H223-H232 ◽  
Author(s):  
I. Imanaga ◽  
M. Kameyama ◽  
H. Irisawa
Keyword(s):  
Gap Junction ◽  
Rhodamine B ◽  
Fluorescent Dyes ◽  
Lucifer Yellow ◽  
Fluorescein Sodium ◽  
Guinea Pig Heart ◽  
Lucifer Yellow Ch ◽  
Ventricular Cells ◽  
Molecular Sizes ◽  
Gap Junctional

The intracellular and cell-to-cell diffusion of fluorescent dyes of various molecular sizes were studied in enzymatically isolated paired ventricular cells of the guinea pig heart. Fluorescein sodium (mol wt 332), 6-carboxyfluorescein (mol wt 376), Lucifer yellow CH (mol wt 457), lissamine rhodamine B-200 (mol wt 559), and tetraglycine-conjugated lissamine rhodamine B-200 (mol wt 859) were all diffused into the single ventricular cell through the patch-clamp pipette. All these dyes were able to diffuse through the gap junction of the paired cells. The diffusion coefficient of 6-carboxyfluorescein in the cytoplasm was 5.8 X 10(-6) cm2/s, Lucifer yellow CH was 3.0 X 10(-6) cm2/s, and lissamine rhodamine B-200 was 8.6 X 10(-7) cm2/s. Permeability of the gap-junctional membrane was 6.8 X 10(-4) cm/s for 6-carboxyfluorescein, 2.8 X 10(-4) cm/s for Lucifer yellow CH and 7.4 X 10(-5) cm/s for lissamine rhodamine B-200. Thus lissamine rhodamine B-200 diffused in the cytoplasm and through the gap junction approximately 10 times slower than 6-carboxyfluorescein. Tetraglycine-conjugated lissamine rhodamine B-200 (mol wt 859) passed through the gap junction, suggesting that the critical pore size of the ventricular gap junction may be somewhat larger than that of the conjugated tetraglycine rhodamine.

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