Quantitative Determination of Gap Junction Intercellular Communication by Scrape Loading and Image Analysis

Opsahl, Hanne; Rivedal, Edgar

doi:10.3109/15419060009109019

Cited by 61 publications

(46 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Different strategies have been used to evaluate gap junction functionality, including metabolic cooperation, radioactive nucleotide transfer, fluorescent dye microinjection, scrape-loading, dual whole cell patch-clamp electrophysi-ology, standard eletrophysiology, fluorescence recovery after photobleaching, local activation of a molecular fluorescent probe, and the so-called ''parachute assay,'' in which cells labeled with a gap junction permeant indicator are plated atop unlabeled cells (9)(10)(11)(12)(13)(14).…”

Section: Resultsmentioning

confidence: 99%

Flow cytometry analysis of gap junction‐mediated cell–cell communication: Advantages and pitfalls

et al. 2006

View full text Add to dashboard Cite

Background: Since the first morphological description of the gap junctions use electron microscopy, a considerable number of techniques has been introduced to evaluate gap junction channel functionality, many of which use dye transfer techniques, such as dye injection and fluorescent dye transfer, analyzed by flow cytometry. Methods: To analyze dye transfer, generally one population of cells is incubated with calcein-AM (0.5 lM) for 30 min at 37°C, and the other population was incubated with the lipophilic dye DiIC 18 (3) (10 lM) for 1 h at 37°C; after incubation, these cells were washed five times with PBS and cocultured for different times, and then the dye transfer was analyzed by flow cytometry.

show abstract

Section: Resultsmentioning

confidence: 99%

Flow cytometry analysis of gap junction‐mediated cell–cell communication: Advantages and pitfalls

et al. 2006

View full text Add to dashboard Cite

show abstract

“…6A). To specifically assess gap junction function and intracellular communication between connecting NMVMs, we then performed dye microinjection studies as well as scrape loading assays (Boswell et al, 2009;elFouly et al, 1987;Li et al, 2011;Opsahl and Rivedal, 2000) ( Fig. 6B,C) .…”

Section: Vcl Reduction Causes a Decrease In Gap Junctional Communicationmentioning

confidence: 99%

“…They were then rinsed three times with PBS, and incubated for an additional 20 min in the culture medium saved prior to dye loading. Dye transfer was then assessed by image acquisition 25 min after dye loading, and analyzed as previously described (Boswell et al, 2009;el-Fouly et al, 1987;Li et al, 2011;Opsahl and Rivedal, 2000).…”

Section: Cytotoxicity Assaymentioning

confidence: 99%

Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin 43 containing gap junctions in cardiac Myocytes

Zemljic-Harpf

Godoy

Platoshyn

et al. 2014

Journal of Cell Science

View full text Add to dashboard Cite

Vinculin (Vcl) links actin filaments to integrin-and cadherin-based cellular junctions. Zonula occludens-1 (ZO-1, also known as TJP1) binds connexin-43 (Cx43, also known as GJA1), cadherin and actin. Vcl and ZO-1 anchor the actin cytoskeleton to the sarcolemma. Given that loss of Vcl from cardiomyocytes causes maldistribution of Cx43 and predisposes cardiomyocyte-specific Vcl-knockout mice with preserved heart function to arrhythmia and sudden death, we hypothesized that Vcl and ZO-1 interact and that loss of this interaction destabilizes gap junctions. We found that Vcl, Cx43 and ZO-1 colocalized at the intercalated disc. Loss of cardiomyocyte Vcl caused parallel loss of ZO-1 from intercalated dics. Vcl coimmunoprecipitated Cx43 and ZO-1, and directly bound ZO-1 in yeast two-hybrid studies. Excision of the Vcl gene in neonatal mouse cardiomyocytes caused a reduction in the amount of Vcl mRNA transcript and protein expression leading to (1) decreased protein expression of Cx43, ZO-1, talin, and b1D-integrin, (2) reduced PI3K activation, (3) increased activation of Akt, Erk1 and Erk2, and (4) cardiomyocyte necrosis. In summary, this is the first study showing a direct interaction between Vcl and ZO-1 and illustrates how Vcl plays a crucial role in stabilizing gap junctions and myocyte integrity.

show abstract

“…Quantitative scrape loading was performed as previously described (Leithe et al, 2003;Opsahl and Rivedal, 2000). Digital monochrome images were acquired using a COHU 4912 CCD camera (COHU, San Diego, CA) and a Scion LG-3 frame grabber card (Scion Corporation, Frederick, MD).…”

Section: Determination Of Gap Junctional Communication By Quantitativmentioning

confidence: 99%

Ubiquitylation of the gap junction protein connexin-43 signals its trafficking from early endosomes to lysosomes in a process mediated by Hrs and Tsg101

Leithe

Kjenseth

Sirnes

et al. 2009

Journal of Cell Science

Self Cite

128

View full text Add to dashboard Cite

Gap junctions are dynamic plasma membrane domains, and their protein constituents, the connexins, have a high turnover rate in most tissue types. However, the molecular mechanisms involved in degradation of gap junctions have remained largely unknown. Here, we show that ubiquitin is strongly relocalized to connexin-43 (Cx43; also known as Gja1) gap junction plaques in response to activation of protein kinase C. Cx43 remained ubiquitylated during its transition to a Triton X-100-soluble state and along its trafficking to early endosomes. Following internalization, Cx43 partly colocalized with the ubiquitin-binding proteins Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate; also known as Hgs) and Tsg101 (tumor susceptibility gene 101). Depletion of Hrs or Tsg101 by small interfering RNA abrogated trafficking of Cx43 from early endosomes to lysosomes. Under these conditions, Cx43 was able to undergo dephosphorylation and deubiquitylation, locate to the plasma membrane and form functional gap junctions. Simultaneous depletion of Hrs and Tsg101 caused accumulation of a phosphorylated and ubiquitylated subpopulation of Cx43 in early endosomes and in hybrid organelles between partly degraded annular gap junctions and endosomes. Collectively, these data reveal a central role of early endosomes in sorting of ubiquitylated Cx43, and identify Hrs and Tsg101 as crucial regulators of trafficking of Cx43 to lysosomes.

show abstract

Quantitative Determination of Gap Junction Intercellular Communication by Scrape Loading and Image Analysis

Cited by 61 publications

References 19 publications

Flow cytometry analysis of gap junction‐mediated cell–cell communication: Advantages and pitfalls

Flow cytometry analysis of gap junction‐mediated cell–cell communication: Advantages and pitfalls

Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin 43 containing gap junctions in cardiac Myocytes

Ubiquitylation of the gap junction protein connexin-43 signals its trafficking from early endosomes to lysosomes in a process mediated by Hrs and Tsg101

Contact Info

Product

Resources

About