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Published on: October 18, 2014
Gap junctional intercellular communication capacity by gap-FRAP technique: a comparative study.
Muriel Abbaci1, Muriel Barberi-Heyob, Jean-René Stines
1Faculté de Médecine, Nancy University, Vandoeuvre-les-Nancy, France.
Biotechnology Journal
|January 17, 2007
Summary
This study shows that the gap-FRAP technique effectively quantifies gap junction communication (GJIC) functionality. Dye choice impacts GJIC kinetics, highlighting limitations of CFDA for 3D studies.
Area of Science:
- Cell Biology
- Biophysics
Background:
- Gap junctions, formed by connexins like Cx43, are crucial for cell growth and differentiation.
- Phosphorylated Cx43 localization influences gap junctional intercellular communication (GJIC) capacity.
Purpose of the Study:
- To assess GJIC functionality in different cell types using the gap-FRAP technique.
- To compare the performance of CFDA and calcein AM tracers in quantifying GJIC.
- To investigate the relationship between phosphorylated Cx43 localization and GJIC capacity.
Main Methods:
- Utilized fluorescence recovery after photobleaching (FRAP) to measure GJIC.
- Employed two tracers: 5(6)-carboxyfluorescein diacetate (CFDA) and calcein acetoxymethylester (AM).
- Performed gap junction channel inhibition assays to confirm GJIC involvement.
Main Results:
- GJIC functionality correlated with phosphorylated Cx43 localization at cell-cell contacts.
- Tracer choice significantly impacted fluorescence kinetic profiles, not recovery percentages.
- CFDA exhibited cell line-dependent release, suggesting potential limitations.
Conclusions:
- The gap-FRAP approach is valuable for quantifying changes in gap junction functionality.
- Calcein AM may be more suitable than CFDA for certain kinetic analyses.
- CFDA's limitations for 3D applications warrant consideration.
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