Gap junctional intercellular communication capacity by gap-FRAP technique: a comparative study.
Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Connexins 43 (Cx43) are the most widely expressed gap junction proteins. Cellular localization of phosphorylated Cx43 has been implicated in the capacity of gap junctional intercellular communication (GJIC). To follow the functionality of GJIC of different cell types, in monolayer cultures, characterized by different patterns of phosphorylated Cx43, we used a fluorescence recovery after photobleaching (FRAP) technique, and compared two tracers, 5(6)-carboxyfluorescein diacetate (CFDA) and calcein acetoxymethylester (AM). The GJIC capacity was quantified by estimating fluorescence redistribution parameters. The functionality of GJIC was in relation with the staining localization of phosphorylated Cx43 to the cell-cell contact areas, corresponding to gap junctions between contacting cells. GJIC involvement in fluorescence restitution after photobleaching was checked by a gap junction channel inhibition assay. We demonstrated that the choice of the dye did not significantly influence the fluorescence recovery percentages despite a cell line-dependent CFDA release, whereas it had an important impact on fluorescence kinetic profiles. This study reinforces the interest of the gap-FRAP approach to quantify modifications in the functionality of gap junctions and, above all, argues about the limits of CFDA for 3-D future approaches.
Abbaci
Muriel
M
Faculté de Médecine, Nancy University, Vandoeuvre-les-Nancy, France.
Barberi-Heyob
Muriel
M
Stines
Jean-René
JR
Blondel
Walter
W
Dumas
Dominique
D
Guillemin
François
F
Didelon
Jacques
J
D002450:Cell Communication; D002460:Cell Line; D018031:Connexin 43 / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D017629:Gap Junctions / Q000378:metabolism* / Q000648:ultrastructure; D006258:Head and Neck Neoplasms / Q000378:metabolism* / Q000473:pathology; D006801:Humans
D018031:Connexin 43