Immuno- and Correlative Light Microscopy-Electron Tomography Methods for 3D Protein Localization in Yeast
Immuno- and Correlative Light Microscopy-Electron Tomography Methods for 3D Protein Localization in Yeast
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DOI：
10.1111/tra.12192
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发表时间：
2014-10-01
期刊：
影响因子：
4.5
通讯作者：
Reggiori, Fulvio
中科院分区：
文献类型：
--
作者：
Mari, Muriel;Geerts, Willie J. C.;Reggiori, Fulvio
真核细胞的区隔化是通过包括膜转运和细胞器生物发生在内的膜重排来产生和维持的。三维重建与纳米级分辨率结合蛋白质定位是必要的准确分子解剖这些过程。酿酒酵母是鉴定细胞超微结构组织所必需的基因和途径的关键模型系统。然而，酵母的电子显微镜研究一直受到细胞壁的阻碍，这阻碍了树脂和低温保护剂的渗透，并且由于蛋白质密集的细胞质模糊了膜的细节。在这里，我们提出了一种免疫电子断层扫描（IET）方法，该方法可以确定酵母重建细胞器上的蛋白质分布模式。此外，我们将这种IET方法扩展到相关的光学显微镜-电子断层扫描程序中，其中通过荧光信号定位的特定蛋白质的阳性结构在3D中得到解决。这些新的酵母研究工具将有助于促进我们对真核细胞内膜系统组织的理解。
Compartmentalization of eukaryotic cells is created and maintained through membrane rearrangements that include membrane transport and organelle biogenesis. Three-dimensional reconstructions with nanoscale resolution in combination with protein localization are essential for an accurate molecular dissection of these processes. The yeast Saccharomyces cerevisiae is a key model system for identifying genes and characterizing pathways essential for the organization of cellular ultrastructures. Electron microscopy studies of yeast, however, have been hampered by the presence of a cell wall that obstructs penetration of resins and cryoprotectants, and by the protein dense cytoplasm, which obscures the membrane details. Here we present an immuno-electron tomography (IET) method, which allows the determination of protein distribution patterns on reconstructed organelles from yeast. In addition, we extend this IET approach into a correlative light microscopy-electron tomography procedure where structures positive for a specific protein localized through a fluorescent signal are resolved in 3D. These new investigative tools for yeast will help to advance our understanding of the endomembrane system organization in eukaryotic cells.