Purchase this article with an account.
Edward Chaum, Christina S Winborn, Sujoy Bhattacharya, Jinggang Yin; Prominin 1 Plays a Central Role in Regulating Stress-induced Autophagy in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6041.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Prominin1 (Prom1) is a transmembrane glycoprotein that localizes to plasma membrane protrusions and is a known cancer stem cell marker. It also plays a role in photoreceptor disk morphogenesis; mutations in the Prom1 gene cause Type 4 Stargardt disease. Our previous studies have shown that Prom1 is also a novel regulator of autophagy in the RPE. The purpose of this study was to examine the cellular mechanisms of Prom1-mediated autophagy. We show that loss of Prom1 expression impairs stress-induced autophagy in the human RPE.
We used the CRISPR/CAS9 nuclease system to knockout (KO) Prom1 in ARPE19 cells. FlowSight imaging cytometry and confocal immunofluorescence assays were used to quantify LC3 puncta accumulation in the presence and absence of chemical modulators of autophagy in control APRE19 cells and in Prom1 KO cells. We also monitored autophagosome formation in response to autophagic regulators using a tandem RFP-GFP-LC3 construct containing a GFP marker that is quenched when the autophagosome fuses with the lysosome.
We show that knocking out Prom1 expression significantly reduces starvation-induced LC3-puncta accumulation in the human RPE. Flow cytometry and confocal microscopy demonstrated that Prom1 KO; 1) decreases LC3 puncta accumulation in response to starvation or rapamycin, 2) reduces autophagy flux and increases chloroquine-induced LC3-puncta accumulation, and 3) delays autophagosome maturation.
Prom1 expression plays in important role in the activation of stress-induced autophagy in the human RPE.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
This PDF is available to Subscribers Only