Spermidine and Rapamycin Reveal Distinct Autophagy Flux Response and Cargo Receptor Clearance Profile.

Sholto De Wet,Andre Du Toit,Ben Loos

doi:10.3390/cells10010095

Abstract

Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic interplay between receptor proteins such as p62/Sequestosome-1 and neighbour of BRCA1 gene 1 (NBR1) has gained attention. However, the extent of receptor protein recruitment and subsequent clearance alongside autophagosomes under different autophagy activities remains unclear. Here, we dissect the concentration-dependent and temporal impact of rapamycin and spermidine exposure on receptor recruitment, clearance and autophagosome turnover over time, employing micropatterning. Our results reveal a distinct autophagy activity response profile, where the extent of autophagosome and receptor co-localisation does not involve the total pool of either entities and does not operate in similar fashion. These results suggest that autophagosome turnover and specific cargo clearance are distinct entities with inherent properties, distinctively contributing towards total functional autophagy activity. These findings are of significance for future studies where disease specific protein aggregates require clearance to preserve cellular proteostasis and viability and highlight the need of discerning and better tuning autophagy machinery activity and cargo clearance.

Highlights

Macroautophagy is a major intracellular degradation pathway critical in protein removal and the maintenance of cellular homeostasis [1]
Whilst L Rapa treatment caused a significant increase in light chain 3 (LC3)-II at 2 h and 8 h, this effect was lost at 24 h, suggesting rapid re-establishment of basal autophagy flux after 24 h
Following 2 h of h post-treatment using μM Rapamycin (H Rapa), there was a significant difference between LC3-II abundance at 2 h +bafilomycin A1 (Baf) and 2 h −Baf, demonstrating effective autophagy induction

Summary

Introduction

Macroautophagy is a major intracellular degradation pathway critical in protein removal and the maintenance of cellular homeostasis [1]. Autophagy is typically characterised as a sequential process which is initiated by the synthesis of the pre-autophagosome structure; the phagophore, which matures into an autophagosome [29,30] These will subsequently sequester cytoplasmic components, including proteinaceous cargo, and be delivered to hydrolase-containing lysosomes where degradation takes place [1]. A plethora of complex and dynamically interactive “receptor” proteins exist and engages with the autophagosome machinery, facilitating cargo-specific degradation [34,35,36] These proteins include domains that allow binding to ubiquitinated cytoplasmic components with LC3-II, thereby enabling the targeting of ubiquitinated proteinaceous cargo to the autophagosome [37]. Major cross talk between p62 and NBR1 has been revealed, with NBR1 levels increasing upon p62 knockout [35], suggesting a complex interplay between receptor recruitment, cargo clearance and autophagy activity

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