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Julia Kristiina Johansson, Teemu Ihalainen, Heli Skottman, Soile Nymark; Connexin 43 translocation in retinal pigment epithelium during phagocytosis of photoreceptor outer segments. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1045.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptor cells of the retina undergo continuous renewal by constantly generating new outer segment from their base and by shedding the aged outer segment fragments from their distal tips. These fragments are eventually engulfed and digested by the retinal pigment epithelium (RPE) through diurnally coordinated phagocytosis, a process which is still not completely understood. Recent reports have suggested that gap junctions participate in this process, however more detailed studies are missing. We investigated the effect of phagocytosis on connexin 43 (Cx43), the major gap junction protein, localization both in human embryonic stem cell (hESC) derived RPE and mouse RPE.
The hESC-RPE were spontaneously differentiated and cultured as previously described. Mouse RPE was prepared for immunolabeling by enucleating the eyes and bisecting along the equator. The eyecups were sectioned in Ames’ solution buffered with HEPES. The retina was gently removed from the eyecup leaving the RPE firmly attached to the eyecup preparation.The phagocytosis was assayed by incubating the purified porcine outer segments on hESC-RPE cultures with and without 18α-glycyrrhetinic acid, a blocker for gap junctions. Phagocytosis was assessed ex vivo by preparing the mouse eyes with the retina intact at light onset and 15min, 2h and 10h after it. To detect the bound POS particles, large fields were imaged with Zeiss LSM780 confocal system on inverted Zeiss Cell Observer microscope.
Our results showed that after 10h from light onset, when phagocytosis process is not ongoing, Cx43 localizes to cell-cell junctions as bead-like structures. However, during the first 2h of phagocytosis they were partially translocated from the junctions and also localized around the cell-bound POS particles. Interestingly, when phagocytosis was assayed with 18α-glycyrrhetinic acid, the blocker of gap junctions, we observed a potentiation in the translocation effect.
Our results are consistent with the previous hypothesis that phagocytosis promotes gap junctional disassembly. The data indicates that this occurs through partial translocation from the specific loci in cell-cell junctions to more diffuse distribution in the cell membrane. Furthermore, this translocation was sensitive to the blocking of the gap junctions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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