In addition to templating the nucleation of autophagosomal membranes and promoting the degradation of ubiquitylated protein aggregates, Sqstm1 also plays a role in the oxidative-stress response. Because the characteristic properties of Sqstm1 are so diverse, it is difficult to determine its roles in living cells. To facilitate such investigations, Satoshi Waguri, Masaaki Komatsu and colleagues create a Sqstm1–GFP knock-in (KI) mouse and describe its attributes in this issue's Tools and Techniques article (p. 4453). Using mouse embryonic fibroblasts (MEFs) isolated from Sqstm1-GFPKI/+ mice, they show that, upon nutrient deprivation, Sqstm1–GFP colocalises with LC3-positive puncta, which have a life-time that is similar to that of an autophagosome. Furthermore, the authors observe that Sqstm1–GFP is present in restricted parts of the LC3 cup-shaped structures and inside the region engulfed by the autophagosome. As expected, treatment of Sqstm1–GFPKI/+ MEFs with arsenite increases Sqstm1–GFP levels and phosphorylation at S351, which activates Nrf2, a master regulator of the antioxidant response. Interestingly, in autophagy-deficient Sqstm1-GFPKI/+;Atg7−/− MEFs, phosphorylation of Sqstm1–GFP at S351 is decreased following arsenite washout, indicating the presence of Sqstm1-specific phosphatases. In agreement with a role for Sqstm1 in selective autophagy, the authors reveal that Sqstm1-positive aggregates observed upon arsenite treatment appear later, and are cleared more quickly and efficiently, in Sqstm1-GFPKI/+ MEFs than in Sqstm1-GFPKI/+;Atg7−/− MEFs. Finally, they show that Sqstm1-GFPKI/+ mice can be used to assess Sqstm1 dynamics in vivo. These mice, therefore, provide a valuable resource for studying endogenous Sqstm1 in living cells and whole animals.
- © 2015. Published by The Company of Biologists Ltd