Monitoring Stub1-Mediated Pexophagy

Bo-Hua Chen; Wei Yuan Yang

doi:10.3791/65010

JoVE Journal
Biology

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JoVE Journal Biology

Monitoring Stub1-Mediated Pexophagy

スタブ1を介したペキソファジーのモニタリング

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08:26 min

May 12, 2023

DOI: 10.3791/65010-v

Bo-Hua Chen¹, Wei Yuan Yang^1,2

¹Institute of Biological Chemistry,Academia Sinica, ²Institute of Biochemical Sciences, College of Life Sciences,National Taiwan University

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Overview

This study investigates the mechanism of Stub1-mediated pexophagy in response to ROS-stressed peroxisomes in live human SHSY5Y and mouse NIH 3T3 cells. The methodology highlights a novel approach to monitor pexophagy and the role of various proteins during this process, demonstrating that ROS-stressed peroxisomes are targeted for degradation in a ubiquitin-dependent manner.

Key Study Components

Research Area

Pexophagy
Cellular response to oxidative stress
Protein degradation pathways

Background

Peroxisomes are organelles involved in metabolic processes, including beta oxidation.
They can be damaged by reactive oxygen species (ROS), necessitating cellular responses.
Stub1 is an E3 ubiquitin ligase known to participate in pexophagy.

Methods Used

Live imaging techniques with confocal microscopy
Human SHSY5Y and mouse NIH 3T3 cells as model systems
Transfection and dye-assisted ROS generation protocols

Main Results

Localized ROS generation leads to the identification of stressed peroxisomes.
Stub1 and other proteins like Hsp70, p62, and LC3B translocate to ROS-stressed peroxisomes.
Ubiquitin-dependent degradation pathways are confirmed in the removal of peroxisomes.

Conclusions

This study demonstrates a novel methodology for monitoring pexophagy in live cells.
Findings are relevant for understanding cellular responses to oxidative stress and organelle quality control.

Frequently Asked Questions

What is pexophagy?

Pexophagy is the selective degradation of peroxisomes by autophagy, a critical process for cellular maintenance and metabolic regulation.

Why is ROS damage significant?

ROS damage can impair cellular function and lead to diseases; thus, studying how cells manage ROS-stressed organelles is crucial for understanding cell health.

What role does Stub1 play in pexophagy?

Stub1 acts as an E3 ubiquitin ligase that tags damaged peroxisomes for degradation through the autophagic pathway.

How are live imaging techniques used in this research?

Live imaging allows researchers to observe real-time cellular responses and protein dynamics during pexophagy processes.

What are the implications of this study?

The insights gained may lead to better understanding and treatment strategies for diseases associated with oxidative stress.

What experimental techniques were used?

Techniques include cell transfection, dye-assisted ROS generation, and confocal microscopy for live cell imaging.

Is this methodology applicable to other organelles?

Yes, the methodology can potentially be adapted to study other organelles affected by stress or damage.

このプロトコルは、生細胞におけるStub1を介したペキソファジーをトリガーおよびモニタリングするための指示を提供します。

ペルオキシソームはベータ酸化を行い、ROS損傷を受けやすい。この方法は、細胞がROSストレスペルオキシソームをどのように扱うかを調べることを可能にする。このプロトコルは、細胞集団内のすべてのペルオキシソームをグローバルに標的化するために使用されるだけでなく、単一細胞内の個々のペルオキシソームの操作を可能にすることもできます。

色素支援ROS生成は、ペルオキシソーム以外の細胞小器官を標的にして、細胞が細胞小器官損傷にどのように対処するかを調べるためにも使用できます。まず、直径20ミリメートルのガラスマイクロウェルを備えたガラス底の35ミリメートル培養皿に、840マイクロリットルの培養液に10から5番目のヒトSHSY5Y細胞を2倍、または4番目のマウスNIH H3T3細胞に10の6倍を播種します。5%下で細胞を摂氏37度で24時間増殖させます。

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