Gap junctional intercellular communication capacity by gap‐FRAP technique: A comparative study

Abbaci, Muriel; Barberi‐Heyob, Muriel; Stines, Jean René; Blondel, Walter; Dumas, Dominique; Guillemin, François; Didelon, Jacques

doi:10.1002/biot.200600092

Cited by 63 publications

(55 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Other confocal laser microscopy techniques, such as fluorescent recovery after photobleaching (FRAP), have also been used to analyse gap junction communications (Abbaci et al 2007), but are limited to investigation of the target cell and its immediate neighbour. By contrast, FLIP provides the potential to analyse multiple cell-cell communications in isolation.…”

Section: Discussionmentioning

confidence: 99%

Gap junction permeability between tenocytes within tendon fascicles is suppressed by tensile loading

Maeda

Wang

et al. 2011

Biomech Model Mechanobiol

View full text Add to dashboard Cite

Gap junction communication is an essential component in the mechanosensitive response of tenocytes. However, little is known about direct mechanoregulation of gap junction turnover and permeability. The present study tests the hypothesis that mechanical loading alters gap junction communication between tenocyte within tendon fascicles. Viable tenocytes within rat tail tendon fasicles were labelled with calcein-AM and subjected to a fluorescent loss induced by photobleaching (FLIP) protocol. A designated target cell within a row of tenocytes was continuously photobleached at 100% laser power whilst recording the fluorescent intensity of neighbouring cells. A mathematical compartment model was developed to estimate the intercellular communication between tenocytes based upon the experimental FLIP data. This produced a permeability parameter, k, which quantifies the degree of functioning gap functions between cells as confirmed by the complete inhibition of FLIP by the inhibitor 18α-glycyrrhentic acid. The application of 1N static tensile load for 10 min had no effect on gap junction communication. However, when loading was increased to 1 h, there was a statistically significant reduction in gap junction permeability. This coincided with suppression of connexin 43 protein expression in loaded samples as determined by confocal immunofluorescence. However, there was an upregulation of connexin 43 mRNA. These findings demonstrate that tenocytes remodel their gap junctions in response to alterations in mechanical loading with a complex mechanosensitive mechanism of breakdown and remodelling. This is therefore the first study to show that tenocyte gap junctions are not only important in transmitting mechanically activated signals but that mechanical loading directly regulates gap junction permeability.

show abstract

Section: Discussionmentioning

confidence: 99%

Gap junction permeability between tenocytes within tendon fascicles is suppressed by tensile loading

Maeda

Wang

et al. 2011

Biomech Model Mechanobiol

View full text Add to dashboard Cite

show abstract

“…Fluorescence recovery after photobleaching (FRAP) was used to evaluate functional coupling between different cell types similar to previously described studies (1,45). In this method, fluorescence in a photobleached cell is recovered by passage of the dye from surrounding cells through functional gap junctions, and the speed and level of the recovery reveal the degree of cell coupling.…”

Section: Fluorescence Recovery After Photobleachingmentioning

confidence: 99%

Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level

McSpadden¹,

Kirkton²,

Bursac³

2009

American Journal of Physiology-Cell Physiology

View full text Add to dashboard Cite

Understanding how electrotonic loading of cardiomyocytes by unexcitable cells alters cardiac impulse conduction may be highly relevant to fibrotic heart disease. In this study, we optically mapped electrical propagation in confluent, aligned neonatal rat cardiac monolayers electrotonically loaded with cardiac fibroblasts, control human embryonic kidney (HEK-293) cells, or HEK-293 cells genetically engineered to overexpress the gap junction proteins connexin-43 or connexin-45. Gap junction expression and function were assessed by immunostaining, immunoblotting, and fluorescence recovery after photobleaching and were correlated with the optically mapped propagation of action potentials. We found that neonatal rat ventricular fibroblasts negative for the myofibroblast marker smooth muscle alpha-actin expressed connexin-45 rather than connexin-43 or connexin-40, weakly coupled to cardiomyocytes, and, without significant depolarization of cardiac resting potential, slowed cardiac conduction to 75% of control only at high (>60%) coverage densities, similar to loading effects found from HEK-293 cells expressing similar levels of connexin-45. In contrast, HEK-293 cells with connexin-43 expression similar to that of cardiomyocytes significantly decreased cardiac conduction velocity and maximum capture rate to as low as 22% and 25% of control values, respectively, while increasing cardiac action potential duration to 212% of control and cardiac resting potential from -71.6 +/- 4.9 mV in controls to -65.0 +/- 3.8 mV. For all unexcitable cell types and coverage densities, velocity anisotropy ratio remained unchanged. Despite the induced conduction slowing, none of the loading cell types increased the proportion of spontaneously active monolayers. These results signify connexin isoform and expression level as important contributors to potential electrical interactions between unexcitable cells and myocytes in cardiac tissue.

show abstract

“…3B, graph (Fritzsche and Charras, 2015). FRAP has been used to characterise protein movement (de Beco et al, 2009;Goehring et al, 2010;Renz and Langowski, 2008), protein-protein interaction (Dunham et al, 2004), nuclear export (Wagner et al, 2004), signal transduction (Khait et al, 2016), focal adhesion turn-over (Kumar et al, 2016), cell-cell junction dynamics (Verma et al, 2012;Yamada et al, 2005) or gap junction function via 'gap-FRAP' (Abbaci et al, 2007;Farnsworth et al, 2014). Fluorescence loss in photobleaching (FLIP) Loss of fluorescence in regions adjacent to the bleached region is recorded (Fig.…”

Section: Box 1 Subcellular Imaging Techniquesmentioning

confidence: 99%

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

Nobis

Warren

Lucas

et al. 2018

Journal of Cell Science

View full text Add to dashboard Cite

Molecular mobility, localisation and spatiotemporal activity are at the core of cell biological processes and deregulation of these dynamic events can underpin disease development and progression. Recent advances in intravital imaging techniques in mice are providing new avenues to study real-time molecular behaviour in intact tissues within a live organism and to gain exciting insights into the intricate regulation of live cell biology at the microscale level. The monitoring of fluorescently labelled proteins and agents can be combined with autofluorescent properties of the microenvironment to provide a comprehensive snapshot of in vivo cell biology. In this Review, we summarise recent intravital microscopy approaches in mice, in processes ranging from normal development and homeostasis to disease progression and treatment in cancer, where we emphasise the utility of intravital imaging to observe dynamic and transient events in vivo. We also highlight the recent integration of advanced subcellular imaging techniques into the intravital imaging pipeline, which can provide in-depth biological information beyond the singlecell level. We conclude with an outlook of ongoing developments in intravital microscopy towards imaging in humans, as well as provide an overview of the challenges the intravital imaging community currently faces and outline potential ways for overcoming these hurdles.

show abstract

Gap junctional intercellular communication capacity by gap‐FRAP technique: A comparative study

Cited by 63 publications

References 62 publications

Gap junction permeability between tenocytes within tendon fascicles is suppressed by tensile loading

Gap junction permeability between tenocytes within tendon fascicles is suppressed by tensile loading

Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

Contact Info

Product

Resources

About