A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.
A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.
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DOI：
10.1371/journal.pbio.1001041
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发表时间：
2011-04
期刊：
影响因子：
9.8
通讯作者：
Tsien RY
中科院分区：
文献类型：
--
作者：
Shu X;Lev-Ram V;Deerinck TJ;Qi Y;Ramko EB;Davidson MW;Jin Y;Ellisman MH;Tsien RY
电子显微镜（EM）在蛋白质定位方面实现了最高的空间分辨率，但特异性的蛋白质EM标记缺乏普遍适用的基因编码标签，无法在细胞和组织中进行原位可视化。在这里，我们介绍“miniSOG”（迷你单线态氧气发生器），这是一种从拟南芥促光素2中提取的荧光黄蛋白。MiniSOG含有106个氨基酸，小于绿色荧光蛋白的一半大小。在miniSOG的照射下，产生足够的单线态氧，局部催化二氨基联苯胺聚合成可被EM分解的亲锇反应产物。在哺乳动物细胞、完整的线虫和啮齿动物中，miniSOG与许多特性良好的蛋白质的融合在哺乳动物细胞、完整的线虫和啮齿动物中都能正确定位，在强醛固定后，无需外源配体、探针或破坏性的渗透洗涤剂，就能从大量组织中获得相关的荧光和EM。MiniSOG允许通过电子断层扫描或连续切片块面扫描电子显微镜进行高质量的超微结构保存和三维蛋白质定位。EM显示，在培养的皮质神经元中，minogg标记的SynCAM1是突触前的，而在培养和完整小鼠中，minogg标记的SynCAM2是突触后的。因此SynCAM1和SynCAM2可能是异性恋伴侣。MiniSOG对EM的作用就像绿色荧光蛋白对荧光显微镜的作用一样。电子显微镜（EM）曾经彻底改变了细胞生物学，它以几十纳米的分辨率揭示亚细胞解剖结构，远低于光学显微镜的衍射极限。在过去的二十年里，由于自发荧光蛋白的发展，光学显微镜重新焕发了活力，这使得几乎任何感兴趣的蛋白质都可以通过遗传融合被特异性标记。EM缺乏普遍适用的可比较的遗传标签。在这里，我们介绍了“miniSOG”，一种小的（106个残基）荧光黄蛋白，在蓝光照射下有效地产生单线态氧。在固定组织中，光生成的单线态氧局部聚合二氨基联苯胺，形成可被锇染色的沉淀物，因此可以很容易地通过EM以高分辨率成像。因此，miniSOG是一种通用的标记，用于细胞、组织和生物体（包括完整的线虫和小鼠）中遗传标记的蛋白质的相关光和电子显微镜。为了证明miniSOG的能力，通过在神经元培养和完整小鼠脑中对miniSOG融合物的电镜分析，解决了关于突触细胞粘附分子定位的争议。
Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce “miniSOG” (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy. Electron microscopy (EM) once revolutionized cell biology by revealing subcellular anatomy at resolutions of tens of nanometers, well below the diffraction limit of light microscopy. Over the past two decades, light microscopy has been revitalized by the development of spontaneously fluorescent proteins, which allow nearly any protein of interest to be specifically tagged by genetic fusion. EM has lacked comparable genetic tags that are generally applicable. Here, we introduce “miniSOG”, a small (106-residue) fluorescent flavoprotein that efficiently generates singlet oxygen when illuminated by blue light. In fixed tissue, photogenerated singlet oxygen locally polymerizes diaminobenzidine into a precipitate that is stainable with osmium and therefore can be readily imaged at high resolution by EM. Thus miniSOG is a versatile label for correlated light and electron microscopy of genetically tagged proteins in cells, tissues, and organisms including intact nematodes and mice. As a demonstration of miniSOG's capabilities, controversies about the localization of synaptic cell adhesion molecules are resolved by EM of miniSOG fusions in neuronal culture and intact mouse brain.
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影响因子：
56.9
作者：
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Waterman-Storer, Clare M.
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Sills, M
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