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CHEN YU, Luis E Munoz, Sebastian Boeltz, Silvia C Finnemann; Contribution of annexin A5 to diurnal phagocytosis by the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2016;57(12):237. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by the retinal pigment epithelium (RPE) is critical for vision. POS recognition/binding by apical αvβ5 integrin receptors of the RPE triggers signaling that synchronizes POS engulfment in the eye. Annexin A5 is a Ca2+ dependent phospholipid binding protein with a binding domain for αvβ5 integrin. However, the physiological function of such interaction remains unknown. Here, we examined if and how annexin A5 contributes to RPE phagocytosis.
Annexin A5 expression in rodent retina/RPE was examined using Western blotting and immunofluorescence microscopy. Retinal cell marker labeling was used to compare retinal morphology of annexin A5 knock-out (KO) and wild-type mice. POS phagosomes in the RPE at different times after light onset were quantified to assess the phagocytic activity of annexin A5 KO RPE in situ. POS phagocytosis assays of unpassaged primary RPE or of mouse embryonic fibroblasts (MEFs) were performed to assess the effect of silencing or overexpressing annexin A5 on binding and internalization of isolated POS particles.
We found that annexin A5 is expressed in the neural retina, RPE and choroid in mouse and rat eyes. 9-month old Annexin A5 KO mice showed normal retinal morphology. However, in situ phagosome quantification revealed that annexin A5 KO RPE lacks the peak of phagosome load at light onset that is characteristic for wild-type RPE. Silencing annexin A5 decreased and overexpressing annexin A5 increased POS binding by wild-type primary RPE cells in culture. Silencing annexin A5 did not directly affect internalization or F-actin phagocytic cup formation by primary RPE. Re-expressing annexin A5 was sufficient to rescue the impaired binding capacity of annexin A5 KO MEFs and to increase POS binding by wild-type MEFs above normal. In contrast, overexpressing annexin A5 in integrin β5 KO MEFs had no effect on POS binding.
Our results demonstrate that annexin A5 plays an important role in diurnal outer segment renewal. Moreover, our cell culture assays indicate that annexin A5 specifically of the RPE contributes to this process. Like αvβ5 integrin receptors, manipulating annexin A5 alters POS binding but not POS internalization. Our results further suggest that the function in phagocytosis of annexin A5 is dependent on αvβ5 integrin.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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