A Validated Set of Ascorbate Peroxidase-Based Organelle Markers for Electron Microscopy of Saccharomyces cerevisiae.
A Validated Set of Ascorbate Peroxidase-Based Organelle Markers for Electron Microscopy of Saccharomyces cerevisiae.
复制标题
一套经过验证的基于抗坏血酸过氧化物酶的细胞器标记，用于酿酒酵母电子显微镜检查
作者：
遗传编码的标签，如工程抗坏血酸过氧化物酶APEX 2，为电子显微镜（EM）中亚细胞结构的特异性标记提供了独特的优势。然而，APEX 2在酵母菌EM研究中的应用受到限制。在这里，我们描述了APEX 2为基础的酿酒酵母细胞器标记的发展。我们发现，对于APEX 2催化形成的二氨基联苯胺沉淀，在样品制备过程中细胞壁的去除并不重要，但APEX 2嵌合体中荧光蛋白的存在具有负面影响。我们发现，主要的细胞器，包括内质网，早期高尔基体，晚期高尔基体/早期内体，晚期内体，线粒体，过氧化物酶体，和脂滴可以标记适当的APEX 2嵌合体。我们的APEX 2嵌合体的亚细胞定位通过EM可视化进行验证，并在必要时补充免疫荧光共定位分析，验证其作为细胞器标记物的可行性。酵母是研究基本细胞过程的优秀单细胞模型系统。然而，酵母细胞比大多数动物和植物细胞小得多，这使得观察和识别酵母亚细胞结构具有挑战性。在这里，我们开发了一套酵母细胞器标记用于电子显微镜和记录我们的技术方法，使用这种方法。
Genetically encoded tags, such as engineered ascorbate peroxidase APEX2, offer unique advantages for the specific labeling of subcellular structures in electron microscopy (EM). However, the use of APEX2 in EM investigation of yeast has been limited. Here we describe the development of APEX2-based organelle markers for Saccharomyces cerevisiae. We found that with regard to APEX2 -catalyzed formation of diaminobenzidine precipitation, cell wall removal was not essential during sample preparation, yet the presence of fluorescent proteins in APEX2 chimeras had a negative impact. We showed that major organelles including endoplasmic reticulum, early Golgi, late Golgi/early endosomes, late endosomes, mitochondria, peroxisomes, and lipid droplets could be labeled by appropriate APEX2 chimeras. The subcellular localization of our APEX2 chimeras was verified by EM visualization and supplemented with immunofluorescence colocalization analysis when necessary, validating their feasibility as organelle markers. IMPORTANCE Yeast is an excellent single cellular model system for studying basic cellular processes. However, yeast cells are much smaller than most animal and plant cells, making the observation and recognition of yeast subcellular structures challenging. Here we developed a set of yeast organelle markers for use in electron microscopy and documented our technical approach for using this method.
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影响因子：
3.5
作者：
Li J;Wang Y;Chiu SL;Cline HT
通讯作者：
Cline HT
影响因子：
3.6
作者：
Kuipers, Jeroen;van Ham, Tjakko J.;Kalicharan, Ruby D.;Veenstra-Algra, Anneke;Sjollema, Klaas A.;Dijk, Freark;Schnell, Ulrike;Giepmans, Ben N. G.
通讯作者：
Giepmans, Ben N. G.
影响因子：
9.8
作者：
Shu X;Lev-Ram V;Deerinck TJ;Qi Y;Ramko EB;Davidson MW;Jin Y;Ellisman MH;Tsien RY
通讯作者：
Tsien RY
DOI：
10.1007/978-1-4939-1363-3_1
发表时间：
2014-01-01
期刊：
YEAST GENETICS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Gietz, R. Daniel
通讯作者：
Gietz, R. Daniel
影响因子：
5.8
作者：
Arganda-Carreras, Ignacio;Kaynig, Verena;Seung, H. Sebastian
通讯作者：
Seung, H. Sebastian