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

Abstract

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.

Intercellular communication controls agonist-induced calcium oscillations independently of gap junctions in smooth muscle cells

“…This recovery is similar to the 7.89% recovery we measured in an isolated cell. Similar minimal recovery has been noted in the literature 28 , indicating that transport across gap junctions was blocked. We then measured histamine-induced Ca 2+ oscillations in confluent clusters of SMCs.…”

“…The gap-FRAP assay: Gap Fluorescent Recovery After Photobleaching (FRAP) is an experimental technique that has been established as an effective method of observing gap-junctional communication of small fluorescent molecules between adjacent cells 28,55 . SMCs were cultured on NuSil gels of E=0.3 kPa and E=13 kPa until confluence was achieved, and then serum-starved for at least 24 hours prior to the experiment.…”

Lovastatin‐Mediated Changes in Human Tendon Cells

“…Zeiss LSM 710 scanhead (Zeiss GmbH, Jena, Germany) coupled to an inverted Zeiss Axio Observer.Z1 microscope (Zeiss GmbH) was used for photobleaching and fluorescence imaging. FRAP was performed as described previously (Kuzma‐Kuzniarska et al, ). In brief, a region of interest (ROI) was manually drawn around whole fluorescent cells selected for bleaching and subsequently bleached at 100% laser power.…”

“…The only source of fluorescent calcein in this experiment is the neighbouring cells as the dye is not present in the medium during acquisition. Cells that either do not have gap junctions or their gap junctions have been blocked by inhibitors are unable to recover after photobleaching (Kuzma‐Kuzniarska et al, ).…”

“…However, their precise role has not been sufficiently studied. Recently, we showed that fluorescence recovery after photobleaching (FRAP) can be used to measure gap junction function in human tendon cells (Kuzma‐Kuzniarska et al, ).…”