A reporter cell system to monitor autophagy based on p62/SQSTM1

Abstract

M acroautophagy (hereafter referred to as autophagy) is a catabolic pathway to isolate and transport cytosolic components to the lysosome for degradation. Recently, autophagy receptors, like p62/SQSTM1 and NBR1, which physically link autophagic cargo to ATG8/MAP1-LC3/GABARAP family members located on the forming autophagic membranes, have been identified. To identify conditions or compounds that affect autophagy, cell systems that efficiently report on autophagic flux are required. Here we describe reporter cell systems based on induced expression of GFP-p62, GFP-NBR1 or GFP-LC3B. The degradation of the fusion proteins was followed after promoter shut-off by flow cytometry of live cells. All three fusion proteins were degraded at a basal rate by autophagy. Surprisingly, the basal degradation rate varied for the three reporter fusion proteins. GFP-LC3B was the most stable protein. GFP-NBR1 was most efficiently degraded under basal conditions while degradation of GFPp62 displayed the strongest response to amino acid starvation. GFP-p62 was found to perform the best of the tested reporters. Single cell analysis of autophagic flux by flow cytometry allows estimates of heterogeneous cell populations. The feasibility of this approach was demonstrated using transient overexpression of a dominant negative ULK1 kinase and siRNA-mediated knockdown of LC3B to inhibit autophagic degradation of GFP-p62. The inducible GFP-p62 cell system allows quantification by several approaches and will be useful in screening for compounds or conditions that A reporter cell system to monitor autophagy based on p62/SQSTM1

Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity

“…In the process, SQSTM1 is degraded through autophagy; accordingly, a decrease in SQSTM1 is considered indicative of enhanced autophagic flux. 38,39 We observed that treatment with AMA led to an increase in SQSTM1 levels in HL-1 cells and that coincubation with rapamycin decreased its accumulation (Fig. 4B).…”

p62 and NDP52 Proteins Target Intracytosolic Shigella and Listeria to Different Autophagy Pathways

“…S1A). To confirm this, we applied a novel reporter cell system based on induced expression of p62-GFP, where the degradation of GFP can be followed after promoter shutoff by flow cytometry (16). In accordance with the autophagic flux assay (Fig.…”

“…p62-GFP cells were cultured in DMEM plus GlutaMAX supplemented with 15 g/ml hygromycin, 200 g/ml blasticidin, and 10% FCS (16). Cells were grown at 37°C and 10% CO 2 .…”

Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors

“…Recently, a reporter system to monitor autophagy in mammalian cells based on p62 has been proposed. 69 According to the authors, the GFP-p62 performed the best among the three tested reporter fusions (GFP-LC3, GFP-NBR1 and GFP-p62) and was the most useful in screening for compounds or conditions that affected the rate of autophagy.…”