title: A fluorescence-microscopic and cytofluorometric system for monitoring the turnover of the autophagic substrate p62/SQSTM1.
abstract: Autophagic flux can be measured by determining the declining abundance of autophagic substrates such as sequestosome 1 (SQSTM1, better known as p62), which is sequestered in autophagosomes upon its direct interaction with LC3. However, the total amount of p62 results from two opposed processes, namely its synthesis (which can be modulated by some cellular stressors including autophagy inducers) and its degradation. To avoid this problem, we generated a stable cell line expressing a chimeric protein composed by p62 and the HaloTag (®) protein, which serves as a receptor for fluorescent HaloTag (®) ligands. Upon labeling with HaloTag (®) ligands (which form covalent, near-to-undissociable bonds with the Halotag (®) receptor) and washing, the resulting fluorescent labeling is not influenced by de novo protein synthesis, therefore allowing for the specific monitoring of the fusion protein decline without any interference by protein synthesis. We demonstrate that a HaloTag (®) -p62 fusion protein stably expressed in suitable cell lines can be used to monitor autophagy by flow cytometry and automated fluorescence microscopy. We surmise that this system could be adapted to high-throughput applications.
authors: INSERM, Institut Gustave Roussy, Villejuif, France.
authors: Amena
authors: A
authors: BenYounès
authors: Nicolas
authors: N
authors: Tajeddine
authors: Maximilien
authors: M
authors: Tailler
authors: Shoaib Ahmad
authors: SA
authors: Malik
authors: Shensi
authors: S
authors: Shen
authors: Didier
authors: D
authors: Métivier
authors: Oliver
authors: O
authors: Kepp
authors: Ilio
authors: I
authors: Vitale
authors: Maria Chiara
authors: MC
authors: Maiuri
authors: Guido
authors: G
authors: Kroemer
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D001343:Autophagy; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D015388:Organelles / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D048868:Adaptor Proteins, Signal Transducing