Visualizing Yeast Organelles with Fluorescent Protein Markers
Visualizing Yeast Organelles with Fluorescent Protein Markers
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使用荧光蛋白标记可视化酵母细胞器
DOI：
10.3791/63846
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Zhiping Xie
中科院分区：
文献类型：
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作者：
Chang-Yue Liu;Jing Zhu;Zhiping Xie
芽殖酵母是研究细胞器功能和动力学的经典模型系统。在我们之前的工作中，我们已经构建了主要细胞器和内膜结构的荧光蛋白标记物，包括细胞核，内质网（ER），高尔基体，内体，液泡，线粒体，过氧化物酶体，脂滴和自噬体。本文介绍的方案描述了在酵母中使用这些标记的程序，包括酵母转化的DNA制备、转化体的选择和评价、荧光显微镜观察和预期结果。文本是面向研究人员谁正在进入酵母细胞器研究领域从其他背景。涵盖了基本步骤，以及有关显微镜硬件考虑因素和几个常见陷阱的技术说明。它为人们通过活细胞荧光显微镜观察酵母亚细胞实体提供了一个起点。这些工具和方法可用于识别蛋白质亚细胞定位并在延时成像中跟踪感兴趣的细胞器。
The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.