Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Emily Jie-Ning Yang; Istvan R. Boldogh; Haojie Ji; Liza Pon; Theresa C. Swayne

doi:10.3791/65428

JoVE Journal
Biology

This content is Free Access.

JoVE Journal Biology

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

mtHyPer7（一种用于线粒体过氧化物的比例生物传感器）在活酵母细胞中的成像

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09:47 min

June 2, 2023

DOI: 10.3791/65428-v

Emily Jie-Ning Yang¹, Istvan R. Boldogh^1,2, Haojie Ji², Liza Pon^1,2, Theresa C. Swayne^1,2

¹Department of Pathology and Cell Biology,Columbia University Irving Medical Center, ²Confocal and Specialized Microscopy Shared Resource in the Herbert Irving Comprehensive Cancer Center,Columbia University Irving Medical Center

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Overview

This study investigates mitochondrial function in the budding yeast Saccharomyces cerevisiae, focusing on the role of hydrogen peroxide (H₂O₂) as both a damaging agent and a signaling molecule. Using a genetically encoded ratiometric biosensor, mtHyPer7, the researchers describe a method to quantitatively measure mitochondrial H₂O₂ in live cells.

Key Study Components

Research Area

Mitochondrial function
Oxidative stress
Aging in yeast

Background

Mitochondria are essential for cellular energy production and signaling.
H₂O₂ can cause oxidative damage but also acts in signaling pathways.
Mitochondrial fitness is linked to cell lifespan and health.

Methods Used

Live cell imaging with mtHyPer7 biosensor
Model organism: Saccharomyces cerevisiae
Fluorescence microscopy for quantitative analysis

Main Results

Successful imaging of mitochondrial H₂O₂ levels in live yeast cells.
Established optimal conditions for imaging and imaging analysis.
Demonstrated that mtHyPer7 does not affect cellular function or fitness.

Conclusions

This study showcases a reliable method for measuring mitochondrial H₂O₂ and its implications for cellular health.
It contributes to understanding mitochondrial quality control and its effects on aging.

Frequently Asked Questions

What is mtHyPer7?

mtHyPer7 is a genetically encoded ratiometric biosensor used to detect hydrogen peroxide levels in mitochondria.

Why is hydrogen peroxide important in cellular biology?

Hydrogen peroxide is both a potential source of oxidative damage and a key signaling molecule within cells.

What is the significance of using live cell imaging?

Live cell imaging allows researchers to observe dynamic processes in real-time, providing valuable insights into cellular function.

How does mtHyPer7 compare to other ROS detection methods?

mtHyPer7 offers advantages like high affinity for H₂O₂, stability, and does not interfere with cellular functions.

What organism is used as the model system in this study?

Saccharomyces cerevisiae, or budding yeast, is used as a model organism to study mitochondrial function.

What role do mitochondria play in aging?

Mitochondria are crucial for energy production and metabolic processes, affecting cellular health and lifespan.

How can this research contribute to aging studies?

By understanding mitochondrial function and oxidative stress, researchers can gain insights into the mechanisms of aging and potential interventions.

过氧化氢（H 2O₂）既是氧化损伤的来源，也是信号分子。该协议描述了如何使用线粒体靶向HyPer7（mtHyPer7），一种遗传编码的比率生物传感器，在活酵母中测量线粒体H 2 O₂。它详细介绍了如何使用免费软件优化成像条件并执行定量细胞和亚细胞分析。

线粒体是细胞内的细胞器，可促进能量动员、中枢代谢、重要大分子的产生、钙储存和信号转导。因此，它们在细胞功能、健康和寿命控制中起着至关重要的作用。我们的研究重点是使用出芽酵母酿酒酵母作为模式生物来研究线粒体对衰老的影响。

活细胞成像是我们观察线粒体功能变化的最佳工具。在这里，我们描述了一种利用称为线粒体HyPer7的遗传编码比率度量光谱位移生物传感器的方法。mtHyPer7 可检测过氧化氢（一种活性氧），可破坏细胞成分，但也可作为活细胞线粒体中的信号分子发挥作用。

mtHyPer7 对过氧化氢具有高亲和力，对 pH 值的敏感性低。它被整合到酵母基因组中，从而在酵母细胞群中产生稳定和一致的表达。最后，它定量靶向线粒体，对细胞或线粒体功能或健康没有可检测的影响。

像mtHyPer7这样的生物传感器对于研究亚细胞分辨率的线粒体功能、寻找线粒体质量控制的机制以及研究该过程如何影响细胞适应性、进料和寿命至关重要。与用于活性氧检测的有机染料相比，mtHyPer7 染色剂提供了一致的工作效果。与基于 FRET 的传感器相比，mtHyPer7 更紧凑，避免了交叉激励和渗漏等问题，也避免了 FRET 的精确定位和方向要求。