A rapid autophagosome-isolation workflow distinguished starvation and exercise cargo profiles in mouse skeletal muscle.
Evidence
Method-development and quantitative proteomics work in GFP-LC3 transgenic mouse tissues found distinct cargo selection patterns, with higher and during starvation.
Caveat
The findings come from a reporter mouse workflow and identify TEX264 and RETREG1/FAM134B as potential mediators rather than proven mechanisms.
Simplified
is a cellular process to clear unwanted and dysfunctional cellular cargoes, which are sequestered in autophagosomes before their delivery to lysosomes for degradation. Autophagy cargo selection, mediated by cargo receptors, varies across cell types and conditions. Understanding the cargo features is essential for elucidating autophagy's function in specific physiological or pathological contexts. Here, we present a simple and rapid method for isolating LC3B-positive autophagosomes from the tissues of GFP-LC3 transgenic mice, a widely used autophagy reporter model, without relying on the complex ultracentrifugation steps required by traditional methods. When combined with quantitative proteomics, this approach enables efficient in vivo characterization of autophagy cargoes. We applied this method to establish autophagy cargo profiles in skeletal muscle during starvation and exercise, two physiological conditions that activate autophagy, and identified distinct cargo selection patterns, with significantly higher levels of and observed during starvation. We further revealed the ER-phagy receptors TEX264 and RETREG1/FAM134B as potential mediators of the elevated ER-phagy under starvation. In summary, we report an efficient workflow for in vivo autophagy cargo characterization and provide detailed analysis and comparison of cargo profiles under starvation and exercise conditions.
Key numbers
1.9×
Increase in GFP-LC3 II Levels during Starvation
GFP-LC3 II levels increased compared to basal condition.
1.8×
Increase in GFP-LC3 II Levels during Exercise
GFP-LC3 II levels increased compared to basal condition.
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