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Pei Xu, Zhen-Yang Zhao, Bo Yu, Yiqin Zuo, Jiyang Cai, Yan Chen; Functional Interactions between Autophagy and Phagocytosis Pathways in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):343.
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Retinal pigment epithelial (RPE) cells are professional phagocytes and are responsible for the removal of daily shed photoreceptor outer segments (POS). Incomplete degradation of phagocytosed POS results in accumulation of undigested materials and RPE lipofuscin. Emerging evidence suggests that the maturation of phagosomes is dependent on molecular components of the autophagy pathway. The goal of the present study was to determine whether inhibiting autophagy will influence the trafficking and lysosome-dependent turnover of phagocytosed POS in cultured ARPE-19 cells.
Bovine POS was purified by sucrose gradient centrifugation. Cultured ARPE-19 cells were fed with POS for 24 hours and chased for an additional 24 hours for monitoring the turnover of phagocytosed POS. The amount and subcellular localization of rhodopsin were measured by Western blot analyses and confocal microscopy. To inhibit autophagy, cells were either treated with chemical inhibitors, bafilomycin A or chloroquine (CQ), or transfected with siRNA targeting either LC3 A/B or Atg5.
Phagocytosed POS were co-localized with the autophagosome marker LC3 and were quickly degraded by ARPE-19 cells within 24 hours. Bafilomycin A and chloroquine inhibited the degradation of POS. The remaining POS formed aggregates in lysosomes, which were stained by the marker protein LAMP1. Similar effects were achieved by downregulating LC3 A/B or Atg5.
Inhibiting autophagy in ARPE-19 cells led to reduced degradation of POS. The results indicate that autophagy is essential for both the cellular self-renewal process and the clearance of phagocytosed POS. Crosstalk between phagocytosis and autophagy pathways is an important regulatory mechanism of RPE function.
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