Modulation of autophagy by RTN-1C: role in autophagosome biogenesis

Manuela D’ Eletto,Federica Di Sano,Bisan Mehdawy,Serafina Oliverio,Roberta Nardacci,Anna Risuglia,Matteo Bordi

doi:10.1038/s41419-019-2099-7

Abstract

The endoplasmic reticulum (ER) is a key organelle fundamental for the maintenance of cellular homeostasis and to determine the cell’s fate under stress conditions. Among the known proteins that regulate ER structure and function there is Reticulon-1C (RTN-1C), a member of the reticulon family localized primarily on the ER membrane. We previously demonstrated that RTN-1C expression affects ER function and stress condition. ER is an essential site for the regulation of apoptotic pathways and it has also been recently recognized as an important component of autophagic signaling. Based on these evidences, we have investigated the impact of RTN-1C modulation on autophagy induction. Interestingly we found that reticulon overexpression is able to activate autophagic machinery and its silencing results in a significative inhibition of both basal and induced autophagic response. Using different experimental approaches we demonstrated that RTN-1C colocalizes with ATG16L and LC3II on the autophagosomes. Considering the key role of reticulon proteins in the control of ER membrane shaping and homeostasis, our data suggest the participation of RTN-1C in the autophagic vesicle biogenesis at the level of the ER compartment. Our data indicate a new mechanism by which this structural ER protein modulates cellular stress, that is at the basis of different autophagy-related pathologies.

Highlights

Endoplasmic reticulum (ER) is an organelle formed by a continuous membrane system comprising the nuclear envelope as well as the peripheral network of tubules and sheets[1,2]
We analyzed the impact of RTN-1C overexpression on autophagy by the use of flow cytometry and a Cyto-ID Autophagy Detection Kit[17]; flow cytometry profile clearly demostrated the up-regulation of autophagic activity in the presence of cloroquine after RTN-1C induction as indicated by the increase of the fluorescence signals (Fig. 1c)
Cells with increased levels of autophagic activity had a greater number of autophagosomes and this in turn was associated with a greater variability in pixel intensity; in cells where autophagy was not observed this variation in pixel intensity was lower due to GFP-LC3 signal uniformly distributed throughout the cytosol

Summary

Introduction

Endoplasmic reticulum (ER) is an organelle formed by a continuous membrane system comprising the nuclear envelope as well as the peripheral network of tubules and sheets[1,2]. Several findings demonstrated that there are specific proteins regulating ER shaping and morphology[3], and among these there are reticulons that have been recently indicated as key molecules which are able to stabilize ER tubules curvature[3]. They have a role in the nuclear envelope. It has recently been suggested that reticulon proteins may play a role in autophagic process. Reticulonrelated proteins are emerging as novel regulators of autophagic processes 8,9

Methods

Results

Conclusion