Abstract
Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.
Highlights
Macroautophagy is a cellular process that leads to the inclusion of cytoplasmic contents by double-membraned vesicles called autophagic vacuoles (AV; called autophagosomes) and fusion to lysosomes for degradation, and is the prevalent process in a more general pathway; here we will refer to this process as autophagy throughout
SQSTM1/p62 is a selective autophagy receptor, which sequesters ubiquitinated proteins into AVs by interacting with LC3.[2]. In addition, p62 is a substrate for autophagic degradation, its degradation can be used as a marker of autophagic clearance.[2,3,4]
Compounds that upregulate autophagy induce close proximity of the LC3II antibodies resulting in an accumulation of signal as AVs are formed; that signal is reduced over time as AVs are turned over
Summary
Macroautophagy is a cellular process that leads to the inclusion of cytoplasmic contents by double-membraned vesicles called autophagic vacuoles (AV; called autophagosomes) and fusion to lysosomes for degradation, and is the prevalent process in a more general pathway; here we will refer to this process as autophagy throughout. Genetic studies in yeast have identified autophagy related genes that are essential to induction, formation or maturation of AVs, or degradation of substrates. LC3 is the best characterized mammalian core autophagy protein and plays an essential role in initiation and formation of AVs. LC3I is found in the cytoplasm and is conjugated to the lipid phosphatidylethanolamine to form LC3II, which binds to the AV membrane and can be used as a marker of AVs.[1] SQSTM1/p62 is a selective autophagy receptor, which sequesters ubiquitinated proteins into AVs by interacting with LC3.[2] In addition, p62 is a substrate for autophagic degradation, its degradation can be used as a marker of autophagic clearance.[2,3,4].
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