Visualization of reticulophagy in living cells using an endoplasmic reticulum-targeted p62 mutant

Abstract

Reticulophagy is a type of selective autophagy in which protein aggregate-containing and/or damaged endoplasmic reticulum (ER) fragments are engulfed for lysosomal degradation, which is important for ER homeostasis. Several chemical drugs and mutant proteins that promote protein aggregate formation within the ER lumen can efficiently induce reticulophagy in mammalian cells. However, the exact mechanism and cellular localization of reticulophagy remain unclear. In this report, we took advantage of the self-oligomerization property of p62/SQSTM1, an adaptor for selective autophagy, and developed a novel reticulophagy system based on an ER-targeted p62 mutant to investigate the process of reticulophagy in living cells. LC3 conversion analysis via western blot suggested that p62 mutant aggregate-induced ER stress triggered a cellular autophagic response. Confocal imaging showed that in cells with moderate aggregation conditions, the aggregates of ER-targeted p62 mutants were efficiently sequestered by autophagosomes, which was characterized by colocalization with the autophagosome precursor marker ATG16L1, the omegasome marker DFCP1, and the late autophagosomal marker LC3/GATE-16. Moreover, time-lapse imaging data demonstrated that the LC3- or DFCP1-positive protein aggregates are tightly associated with the reticular structures of the ER, thereby suggesting that reticulophagy occurs at the ER and that omegasomes may be involved in this process.