Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

Journal article

Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures.

Authors: Kuzma-Kuzniarska, M; Yapp, C; Pearson-Jones, T; Jones, A; Hulley, P

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Society of Photo-optical Instrumentation Engineers (SPIE)
• Journal: Journal of Biomedical Optics
• Volume: 19
• Issue: 1
• Pages: 15001
• Publication date: 2014-01-03
• DOI: 10.1117/1.JBO.19.1.015001
• EISSN: 1560-2281
• ISSN: 1083-3668
• Language: English
• Keywords: fluorescence recovery after photobleaching; human tenocytes; SBTMR; three dimensional; gap junctions