Sqstm1-GFP knock-in mice reveal dynamic actions of Sqstm1 during autophagy and under stress conditions in living cells

Abstract

Sqstm1 serves as a signaling hub and receptor for selective autophagy. Consequently, dysregulation of Sqstm1 causes imbalances in signaling pathways and disrupts proteostasis, thereby contributing to the development of human diseases. Environmental stresses influence the level of Sqstm1 by altering its expression and/or autophagic degradation, and also changes the localization of Sqstm1, making it difficult to elucidate the actions and roles of this protein. In this study, we developed knock-in mice expressing Sqstm1 fused to GFP (Sqstm1-GFPKI/+). Using these Sqstm1-GFPKI/+ mice, we revealed for the first time the dynamics of endogenous Sqstm1 in living cells. Sqstm1–GFP was translocated to a restricted area of LC3-positive structures, which primarily correspond to the inside of autophagosomes, and then degraded. Moreover, exposure to arsenite induced expression of Sqstm1–GFP, followed by accumulation of the fusion protein in large aggregates that were degraded by autophagy. Furthermore, suppression of autophagy in Sqstm1-GFPKI/+ mouse livers caused accumulation of Sqstm1–GFP and formation of GFP-positive aggregate structures, leading to severe hepatic failure. These results indicate that Sqstm1-GFPKI/+ mice are a useful tool for analyzing Sqstm1 in living cells and intact animals.

NBR 1‐mediated p62‐liquid droplets enhance the Keap1‐Nrf2 system

“…Alternatively, the persistent phosphorylation of p62 at Serine 349 by the overexpression of NBR1 may inhibit the transition of FIP200 to LC3‐II. Actually, p62 is de‐phosphorylated at Serine 349 in response to the removal of As (III) , and the phospho‐defective mutant of p62 is resistant to autophagy , suggesting the involvement of de‐phosphorylation in LC3 binding. Another possibility is that NBR1 titrates ATG components that are essential for the degradation of p62 droplets.…”

“…Isolation and immortalization of the MEFs employed in this study was described previously . Cells were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 5 U/ml penicillin, and 50 μg/ml streptomycin.…”

p62/SQSTM1 – steering the cell through health and disease

“…Next, ubiquitylated proteins are recruited to these filaments, fragmenting them into shorter and less compact forms that are engulfed by the nascent autophagosomal membrane (Bjørkøy et al, 2005;Ciuffa et al, 2015;Clausen et al, 2010;Ichimura et al, 2008). Finally, these large structures are degraded through autophagy, with their accumulation a typical hallmark of impaired autophagy (Eino et al, 2015;Komatsu et al, 2007).…”

Negative Regulation of the Keap1-Nrf2 Pathway by a p62/Sqstm1 Splicing Variant

“…To elucidate the biological relevance of the variant, we used p62-GFP knock-in mice ( p62-GFP KI/KI ), in which the p62 splicing variant cannot be produced ( 31 ) ( Fig. 6A ).…”

“…Atg7 f/f , Atg7 f/f ; albumin- Cre , and p62-GFP KI/KI mice in the C57BL/6 genetic background were used in this study ( 30 , 31 ). Mice were housed in specific-pathogen-free facilities, and the Ethics Review Committee for Animal Experimentation of Niigata University approved the experimental protocol.…”