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Feng He, Melina A Agosto, Ralph M Nichols, Lavanya Kailasam, Theodore G Wensel; Vps34 and PI(3)P are critical for autophagy, phagocytosis, and endosome processing in RPE cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4016.
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© ARVO (1962-2015); The Authors (2016-present)
Vps34 and its product, PI(3)P, play important roles in autophagy and phagocytosis. We investigated the functions of Vps34 and PI(3)P in retinal pigment epithelium (RPE) cells, which employ both of these pathways.
An RPE cell-specific knockout (KO) of Vps34 was generated by crossing Vps34 “floxed” and Best1-Cre transgenic mice. Structural and functional changes in the RPE and retina were determined by immunoblotting, immunofluorescence and electron microscopy. Localization of phosphoinositides was determined using exogenously expressed fluorescent phosphoinositide-binding proteins.
The KO mice had a subset of RPE cells with significantly altered morphology, as well as disordered photoreceptor outer segments. In the RPE, RPE65, ZO-1, and F-actin staining was lost, indicating dead and dying cells. Autophagy markers LC3 and p62, ubiquitinated proteins, and late endosome marker Rab7 accumulated, indicating failure of autophagosomes and late endosomes to fuse with lysosomes. Lipidated LC3-II was found in autophagosome membranes, despite depletion of PI(3)P, but was found in phagosome membranes in WT RPE only. In hRPE1 cells treated with chloroquine, which inhibits autophagosome and phagosome degradation, we found not only PI(3)P but also PIP2 co-localized with LC3-II puncta. Furthermore, treatment of hRPE1 cells with the Vps34 inhibitor Vps34INI caused the PI(3)P binding domain from Hrs, but not WIPI2, the putative PI(3)P binding protein that initiates the process of LC3-II formation, to dissociate from autophagosome membranes. These data reveal an alternative pathway by which WIPI2 can bind to autophagosome membranes and promote LC3-II formation in the absence of PI(3)P. In contrast, Vps34 KO RPE contained abnormal phagosomes lacking LC3-II, indicating the alternative LC3-II formation pathway cannot occur in phagosome membranes.
Loss of PI(3)P disrupts lysosomal fusion and degradation of autophagosomes, phagosomes, and late endosomes, leading to death of RPE cells and underlying photoreceptors. Although both autophagy and phagocytosis involve PI(3)P, LC3-II, and lysosomal degradation, they are distinct pathways. PI(3)P is essential for LC3 lipidation in phagosomes, but not in autophagosomes, revealing an alternative pathway for WIPI2 and LC3 recruitment.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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