A reporter cell system to monitor autophagy based on p62/SQSTM1.
Macroautophagy (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 GFPp62, 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 GFP-p62 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 affect the rate of autophagy. Inducers of autophagy can be identified using rich medium whereas inhibitors are identified under starvation conditions.
Larsen
Kenneth Bowitz
KB
BioImaging Core Facility, Medical Faculty, Institute of Medical Biology, University of Tromsø, Tromsø, Norway.
Lamark
Trond
T
Øvervatn
Aud
A
Harneshaug
Ingvill
I
Johansen
Terje
T
Bjørkøy
Geir
G
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D001343:Autophagy; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000071456:Sequestosome-1 Protein
D048868:Adaptor Proteins, Signal Transducing; D011993:Recombinant Fusion Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D049452:Green Fluorescent Proteins