scholarly journals Confirmation of Connexin45 Underlying Weak Gap Junctional Intercellular Coupling in HeLa Cells

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1389 ◽  
Author(s):  
Eun Ju Choi ◽  
Nicolás Palacios-Prado ◽  
Juan C. Sáez ◽  
Jinu Lee
Keyword(s):  
Hela Cells ◽  
Fluorescent Protein ◽  
Cell Model ◽  
Lucifer Yellow ◽  
Yellow Fluorescent Protein ◽  
Pcr Analysis ◽  
Gap Junctional Intercellular Communication ◽  
Null Cell ◽  
Cx43 Expression ◽  
Gap Junctional

Gap junctions (GJs) are intercellular channels that connect adjacent cells electrically and metabolically. The iodide-yellow fluorescent protein (I-YFP) gap junctional intercellular communication (GJIC) assay is a recently developed method with high sensitivity. HeLa cells have been widely used as GJ-deficient cells for GJ-related research. Herein, we present evidence showing that HeLa cells have functional GJs comprising connexin (Cx) 45 using the I-YFP GJ assay and CRISPR/Cas9 system. We conducted the I-YFP GJIC assay in HeLa cells, which revealed a weak level of GJIC that could not be detected by the Lucifer yellow scrape-loading assay. The mRNA expression of GJB5 (Cx31.1), GJA1 (Cx43), and GJC1 (Cx45) was detected in HeLa cells by RT-PCR analysis. Knocking out GJC1 (Cx45) abolished GJIC, as analyzed by the I-YFP assay and dual whole-cell patch-clamp assay. These results suggest that HeLa cells express Cx45-based GJs and that the I-YFP GJIC assay can be used for cells with weak GJIC, such as Cx45-expressing HeLa cells. Further, GJC1 (Cx45)-knockout HeLa cells are more suitable as a GJ-null cell model for transfection experiments than wild-type HeLa cells. This experimental design was successfully applied to knock out Cx43 expression and GJIC in A549 lung cancer cells and can thus be used to identify major Cxs in other cell types and to establish GJ assay systems for different Cxs.

scholarly journals Cx43 Present at the Leading Edge Membrane Governs Promigratory Effects of Osteoblast-Conditioned Medium on Human Prostate Cancer Cells in the Context of Bone Metastasis

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 3013
Author(s):  
Jonathan Boucher ◽  
Annie-Claire Balandre ◽  
Marjolaine Debant ◽  
Justine Vix ◽  
Thomas Harnois ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Leading Edge ◽  
Bone Microenvironment ◽  
Lncap Cells ◽  
Gap Junctional Intercellular Communication ◽  
Specific Expression ◽  
Cx43 Expression ◽  
Tissue Samples ◽  
Carboxy Terminal ◽  
Gap Junctional

Among the different interacting molecules implicated in bone metastases, connexin43 (Cx43) may increase sensitivity of prostate cancer (PCa) cells to bone microenvironment, as suggested by our in silico and human tissue samples analyses that revealed increased level of Cx43 expression with PCa progression and a Cx43 specific expression in bone secondary sites. The goal of the present study was to understand how Cx43 influences PCa cells sensitivity and aggressiveness to bone microenvironment. By means of Cx43-overexpressing PCa cell lines, we revealed a Cx43-dependent promigratory effect of osteoblastic conditioned media (ObCM). This effect on directional migration relied on the presence of Cx43 at the plasma membrane and not on gap junctional intercellular communication and hemichannel functions. ObCM stimulation induced Rac1 activation and Cx43 interaction with cortactin in protrusions of migrating PCa cells. Finally, by transfecting two different truncated forms of Cx43 in LNCaP cells, we determined that the carboxy terminal (CT) part of Cx43 is crucial for the responsiveness of PCa cells to ObCM. Our study demonstrates that Cx43 level and its membrane localization modulate the phenotypic response of PCa cells to osteoblastic microenvironment and that its CT domain plays a pivotal role.

scholarly journals Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment

2005 ◽  
Vol 288 (4) ◽  
pp. C795-C804 ◽  
Author(s):  
Lucia Formigli ◽  
Fabio Francini ◽  
Alessia Tani ◽  
Roberta Squecco ◽  
Daniele Nosi ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Myoblast Differentiation ◽  
Cell Contact ◽  
Cx43 Expression ◽  
Gap Junctional Communication ◽  
Skeletal Myoblasts ◽  
Junctional Communication ◽  
Direct Cell ◽  
Gap Junctional

The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C2C12 cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca2+ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca2+, in the modulation of myoblast differentiation is discussed.

scholarly journals Phylogenetic and structural analysis of annexins in pea (Pisum sativum L.) and their role in legume-rhizobial symbiosis development

2021 ◽  
Vol 25 (5) ◽  
pp. 502-513
Author(s):  
O. A. Pavlova ◽  
I. V. Leppyanen ◽  
D. V. Kustova ◽  
A. D. Bovin ◽  
E. A. Dolgikh
Keyword(s):  
Fluorescent Protein ◽  
Calcium Influx ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Plant Cells ◽  
Pcr Analysis ◽  
35S Promoter ◽  
Early Stages ◽  
Proteomic Approach ◽  
Rhizobial Symbiosis

Annexins as Ca2+/phospholipid-binding proteins are involved in the control of many biological processes essential for plant growth and development. In a previous study, we had shown, using a proteomic approach, that the synthesis of two annexins is induced in pea roots in response to rhizobial inoculation. In this study, phylogenetic analysis identified these annexins as PsAnn4 and PsAnn8 based on their homology with annexins from other legumes. The modeling approach allowed us to estimate the structural features of these annexins that might influence their functional activity. To verify the functions of these annexins, we performed comparative proteomic analysis, experiments with calcium influx inhibitors, and localization of labeled proteins. Essential down-regulation of PsAnn4 synthesis in a non-nodulating pea mutant P56 (sym10) suggests an involvement of this annexin in the rhizobial symbiosis. Quantitative RT-PCR analysis showed that PsAnn4 was upregulated at the early stages of symbiosis development, starting from 1–3 days after inoculation to up to 5 days after inoculation, while experiments with the Ca2+ channel blocker LaCl3 revealed its negative influence on this expression. To follow the PsAnn4 protein localization in plant cells, it was fused to the fluorophores such as red fluorescent protein (RFP) and yellow fluorescent protein (YFP) and expressed under the transcriptional regulation of the 35S promoter in Nicotiana benthamiana leaves by infiltration with Agrobacterium tumefaciens. The localization of PsAnn4 in the cell wall or plasma membrane of plant cells may indicate its participation in membrane modification or ion transport. Our results suggest that PsAnn4 may play an important role during the early stages of pea-rhizobial symbiosis development.

scholarly journals PDGF-AA promotes cell-to-cell communication in osteocytes through PI3K/Akt signaling pathway

2021 ◽  
Author(s):  
Yang Liu ◽  
Mengmeng Duan ◽  
Daimo Guo ◽  
Shiyi Kan ◽  
L i Zhang ◽  
...  
Keyword(s):  
Intercellular Communication ◽  
Bone Cells ◽  
Bone Matrix ◽  
Cell Communication ◽  
Akt Signaling ◽  
Gap Junctional Intercellular Communication ◽  
Cx43 Expression ◽  
Mineralized Bone Matrix ◽  
Cell Processes ◽  
Gap Junctional

Abstract Osteocytes are the main sensitive cells in bone remodeling due to their potent functional cell processes from the mineralized bone matrix to the bone surface and the bone marrow. Neighboring osteocytes communicate with each other by these cell processes to achieve molecular exchange through gap junction channels. Platelet-derived growth factor-AA (PDGF-AA) has been reported to enhance bone tissue remodeling by promoting cell proliferation, migration, and autocrine secretion in osteoid cell linage. However, the effect of PDGF-AA on intercellular communication between osteocytes is still unclear. In the present study, we elucidated that PDGF-AA could enhance the formation of dendritic processes of osteocytes and the gap junctional intercellular communication by promoting the expression of connexin43 (Cx43). This modulation process was mainly dependent on the activation of phosphorylation of Akt protein by phosphatidylinositol 3-kinase (PI3K)/Akt (also known as protein kinase B, PKB) signaling. Inhibition of PI3K/Akt signaling decreased the Cx43 expression induced by PDGF-AA. These results establish a bridge between PDGF-AA and cell–cell communication in osteocytes, which could help us understand the molecular exchange between bone cells and fracture healing.

Abstract WP135: Engineered Exosomes With Elevated Mir-27a Improve Neurological Outcome in Ischemic Mice

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Yi Zhang ◽  
Zhongwu Liu ◽  
Michael Chopp ◽  
Chao Li ◽  
Xinli Wang ◽  
...  
Keyword(s):  
Neurological Outcome ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
In Vitro Study ◽  
Axonal Growth ◽  
Stroke Recovery ◽  
Pcr Analysis ◽  
Inhibitory Protein

Background: Augmentation of axonal growth enhances improvement of neurological function after stroke. Our previous in vitro study demonstrated that miR-27a promotes axonal growth of embryonic neurons. The present in vivo study investigated whether exosomes carrying elevated miR-27a (27a-exos) enhance axonal growth and neurological outcome after stroke. Methods and Results: The 27a-exos were isolated from medium of mesenchymal stromal cells (MSCs) transfected with lenti-miR-27 vector. In order to investigate axonal growth and underlying mechanisms, Emx1-Thy1-YFP mice with selective expression of yellow fluorescent protein (YFP) in pyramidal neurons were subjected to the permanent middle cerebral artery occlusion (MCAO). Quantitative RT-PCR analysis showed that compared with exosomes derived from empty-vector transfected MSCs (empty-exos), 27a-exos had 3.7 fold increased miR-27a. 27a-exos (3x10 11 particles) were injected intravenously into mice at 24h after MCAO. Compared with control mice, the empty-exos (n=7) significantly (p<0.05) reduced the foot-fault (5±0.1 vs 11±0.5 in control) and the times to remove adhesive paper (seconds, 47±3 vs 70±4). However, compared with empty-exos, 27a-exos (n=7) further significantly (p<0.05) reduced foot-fault (3.0±0.2) and times to remove the paper (38±1.7). Histopathological and in situ hybridization analysis showed that 27a-exos localized to neurons and significantly (p<0.05) increased miR-27a in Emx1-YFP+ neurons (4±0.7 vs 1 in empty-exos), and augmented (p<0.05) the density of YFP+/pNFH+ axons (7±2 vs 1 in empty-exos). Moreover, 27a-exos substantially decreased the levels of the inhibitory protein Sema6a in the YFP+ axons in peri-infarct areas. Conclusion: Our data suggest that tailored 27a-exos augment the therapeutic effect of exosomes on stroke recovery and that suppression of axonal inhibitory proteins such as Sema6a may contribute to the 27a-exo-enhanced axonal outgrowth to promote neurological recovery post stroke.

Connexin 43 expression in the testis of the frog Rana esculenta

Zygote ◽  
2006 ◽  
Vol 14 (4) ◽  
pp. 349-357 ◽  
Author(s):  
G. Izzo ◽  
M. d'Istria ◽  
D. Ferrara ◽  
I. Serino ◽  
F. Aniello ◽  
...  
Keyword(s):  
Leydig Cells ◽  
Connexin 43 ◽  
Maximum Level ◽  
Rana Esculenta ◽  
Pcr Analysis ◽  
Cx43 Expression ◽  
Temporal And Spatial ◽  
Mammalian Testis ◽  
Gap Junctional

SummaryTesticular cell-to-cell interactions play a key role in the regulation of spermatogenesis. In the testis, cell contacts are mediated through several mechanisms, including paracrine and direct contacts depending on gap junctional pathways. Gap junctions require connexin (Cx) channels and connexin-43 (Cx43) represent the most abundant Cx found in mammalian testis. Little is known about Cx expression in non-mammalian testis. Here we report the partial cloning of a Cx43 transcript of 381 bp from Rana esculenta testis. We also demonstrate that, in the frog testis, Cx43 transcript and protein show a parallel temporal and spatial pattern of expression throughout the reproductive annual cycle, with higher levels from September to January (when spermatogenesis is at a maximum level). In situ hybridization, carried out on testis collected in October, indicated that Leydig cells (LC) and Sertoli cells express Cx43 transcript, while the hybridization signal was less intense in germ cells. To obtain more information on Cx43 expression in the frog testis, we have used ethane-dimethane sulphonate (EDS), a toxin that specifically destroys LC. RT-PCR analysis shows a progressive decrease in Cx43 expression in EDS-treated testis from day 1 to day 4 after the injection, associated with LC destruction. Moreover, Cx43 expression returns to normal on day 28, when a new population of LC reappear in the interstitium, indicating that Cx43 is mainly expressed by LC. Taken together our data provide evidence that Cx43 is present in the frog testis with an important role in spermatogenesis.

scholarly journals Prostaglandin E2 Enhances Gap Junctional Intercellular Communication in Clonal Epithelial Cells

2021 ◽  
Vol 22 (11) ◽  
pp. 5813
Author(s):  
Alejandro Ogazon del Toro ◽  
Lidia Jimenez ◽  
Mauricio Serrano Rubi ◽  
Aida Castillo ◽  
Lorena Hinojosa ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Prostaglandin E2 ◽  
Protein Kinase A ◽  
Intercellular Communication ◽  
Mdck Cells ◽  
Lucifer Yellow ◽  
Gap Junctional Intercellular Communication ◽  
Gap Junctional ◽  
Kinase A

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.

scholarly journals Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems

2020 ◽  
Vol 21 (17) ◽  
pp. 6420
Author(s):  
Ivana Pibiri ◽  
Raffaella Melfi ◽  
Marco Tutone ◽  
Aldo Di Leonardo ◽  
Andrea Pace ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Fluorescent Protein ◽  
Cell Model ◽  
Yellow Fluorescent Protein ◽  
Premature Termination Codon ◽  
Severe Form ◽  
Model Systems ◽  
Functional Protein ◽  
Nonsense Mutations ◽  
Translational Readthrough

Cystic fibrosis (CF) patients develop a severe form of the disease when the cystic fibrosis transmembrane conductance regulator (CFTR) gene is affected by nonsense mutations. Nonsense mutations are responsible for the presence of a premature termination codon (PTC) in the mRNA, creating a lack of functional protein. In this context, translational readthrough-inducing drugs (TRIDs) represent a promising approach to correct the basic defect caused by PTCs. By using computational optimization and biological screening, we identified three new small molecules showing high readthrough activity. The activity of these compounds has been verified by evaluating CFTR expression and functionality after treatment with the selected molecules in cells expressing nonsense–CFTR–mRNA. Additionally, the channel functionality was measured by the halide sensitive yellow fluorescent protein (YFP) quenching assay. All three of the new TRIDs displayed high readthrough activity and low toxicity and can be considered for further evaluation as a therapeutic approach toward the second major cause of CF.

scholarly journals Grb2 Regulates Internalization of EGF Receptors through Clathrin-coated Pits

2003 ◽  
Vol 14 (3) ◽  
pp. 858-870 ◽  
Author(s):  
Xuejun Jiang ◽  
Fangtian Huang ◽  
Andriy Marusyk ◽  
Alexander Sorkin
Keyword(s):  
Growth Factor ◽  
Hela Cells ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Receptor Internalization ◽  
Sequence Motifs ◽  
Coated Pits

The molecular mechanisms of clathrin-dependent internalization of epidermal growth factor receptor (EGFR) are not well understood and, in particular, the sequence motifs that mediate EGFR interactions with coated pits have not been mapped. We generated a panel of EGFR mutants and stably expressed these mutants in porcine aortic endothelial (PAE) cells. Interestingly, mutations of tyrosine phosphorylation sites 1068 and 1086 that interact with growth-factor-receptor-binding protein Grb2 completely abolished receptor internalization in PAE cells. Quantitative analysis of colocalization of EGF-rhodamine conjugate and coated pits labeled with yellow-fluorescent-protein–tagged β2 subunit of clathrin adaptor complex AP-2 revealed that EGFR mutants lacking Grb2 binding sites do not efficiently enter coated pits. The depletion of Grb2 from PAE as well as HeLa cells expressing endogenous EGFRs by RNA interference substantially reduced the rate of EGFR internalization through clathrin-dependent pathway, thus providing the direct evidence for the important role of Grb2 in this process. Overexpression of Grb2 mutants, in which the SH3 domains were either deleted or inactivated by point mutations, significantly inhibited EGFR internalization in both PAE and HeLa cells. These findings indicate that Grb2, in addition to its key function in signaling through Ras, has a major regulatory role at the initial steps of EGFR internalization through clathrin-coated pits. Furthermore, the EGFR mutant lacking Grb2 binding sites did not efficiently recruit c-Cbl and was not polyubiquitinated. The data are consistent with the model whereby Grb2 participates in EGFR internalization through the recruitment of Cbl to the receptor, thus allowing proper ubiquitylation of EGFR and/or associated proteins at the plasma membrane.

scholarly journals Expression of connexin 43 and gap junctional intercellular communication in the cumulus–oocyte complex in sheep

Reproduction ◽  
2005 ◽  
Vol 129 (2) ◽  
pp. 191-200 ◽  
Author(s):  
Disha Pant ◽  
Lawrence P Reynolds ◽  
Justin S Luther ◽  
Pawel P Borowicz ◽  
Tande M Stenbak ◽  
...  
Keyword(s):  
Estrous Cycle ◽  
Intercellular Communication ◽  
Connexin 43 ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Gap Junctional Intercellular Communication ◽  
Cx43 Expression ◽  
Cumulus Oocyte Complex ◽  
Gap Junctional

To evaluate the effects of FSH, LH and/or cAMP on expression of connexin 43 (Cx43) in the ovine cumulus-oocyte complex (COC) and gap junctional intercellular communication (GJIC) of cumulus cells, two experiments were carried out. In experiment 1, Cx43 was immunodetected in the COC, before or after maturation, obtained from non-treated or FSH-treated ewes. The expression of Cx43 in the COC was greater (P < 0.01) on day 16 than on day 15 of the estrous cycle. In vivo FSH treatment decreased (P < 0.02) Cx43 expression on day 16 but not on day 15 of the estrous cycle. In experiment 2, intact COCs or isolated cumulus cells obtained from small and large follicles from FSH-treated ewes were cultured with or without FSH, LH or cAMP agonist and evaluated for GJIC by laser cytometry. For large follicles, the basal rate of GJIC was greater (P < 0.01) for cumulus cells in intact COCs than for isolated cumulus cells. FSH increased (P < 0.04) GJIC in cumulus cells in intact COCs and tended to increase (P < 0.1) GJIC in isolated cumulus cells from small follicles but decreased (P < 0.01) GJIC in cumulus cells in intact COCs from large follicles. LH also increased (P < 0.01) GJIC in isolated cumulus cells from small follicles but decreased GJIC in intact COCs (P < 0.01) and isolated cumulus cells (P < 0.02) from large follicles. cAMP increased (P < 0.01) the GJIC in both intact COCs and cumulus cells from small and large follicles. These results indicate that day of estrous cycle, stage of maturation and duration of FSH treatment affect expression of Cx43 in ovine COCs. In intact COCs, GJIC in cumulus cells was enhanced, probably due to the presence of the oocyte. In addition, the effects of FSH and LH, but not cAMP, on GJIC of cumulus cells depended on the stage of follicular development and on the presence of the oocyte.

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