Semi-in situ atomic force microscopy imaging of intracellular neurofilaments under physiological conditions through the 'sandwich' method
Semi-in situ atomic force microscopy imaging of intracellular neurofilaments under physiological conditions through the 'sandwich' method
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DOI：
10.1093/jmicro/dfw006
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发表时间：
2016-08-01
期刊：
影响因子：
1.8
通讯作者：
Terada, Sumio
中科院分区：
文献类型：
--
作者：
Sato, Fumiya;Asakawa, Hitoshi;Terada, Sumio
神经丝是神经元特异性的中间丝蛋白，其特征是在体内形成生化稳定的专性杂聚物。虽然纯化或重新组装的神经丝已通过电子显微镜和原子力显微镜进行形态学分析，但仍然需要以最小的人工化风险对细胞质真正的中间丝进行直接成像。在本研究中，我们应用改良的“玻璃三明治上的细胞”方法将细胞内神经丝外化，降低了样品制备造成伪影的风险。 SW13vim(-)细胞用神经丝介质多肽(NF-M)和α-internexin(α-inx)双重转导。用单宁酸预稳定后，用阳离子化盖玻片覆盖培养的细胞，形成三明治，然后分成两部分。在确认神经丝可以沉积在通过去顶暴露的腹侧质膜上后，我们在水溶液中进行了原子力显微镜半原位成像。观察到的细丝被认为保留了神经丝的天然结构，沿其长度表现出大约 50-60 nm 的周期性。它们的结构特性似乎反映了它们的成分（即 NF-M 和 alpha-inx）形成的形态。通过分离三明治，对暴露的真实组装的神经丝进行半原位原子力显微镜的成功表明，它可以成为通过原子力显微镜研究生理条件下细胞质中间丝的有效和替代方法。
Neurofilaments are intermediate filament proteins specific for neurons and characterized by formation of biochemically stable, obligate heteropolymers in vivo. While purified or reassembled neurofilaments have been subjected to morphological analyses by electron microscopy and atomic force microscopy, there has been a need for direct imaging of cytoplasmic genuine intermediate filaments with minimal risk of artefactualization. In this study, we applied the modified' cells on glass sandwich' method to exteriorize intracellular neurofilaments, reducing the risk of causing artefacts through sample preparation. SW13vim(-) cells were double transduced with neurofilament medium polypeptide (NF-M) and alpha-internexin (alpha-inx). Cultured cells were covered with a cationized coverslip after prestabilization with tannic acid to form a sandwich and then split into two. After confirming that neurofilaments could be deposited on ventral plasma membranes exposed via unroofing, we performed atomic force microscopy imaging semi-in situ in aqueous solution. The observed thin filaments, considered to retain native structures of the neurofilaments, exhibited an approximate periodicity of 50-60 nm along their length. Their structural property appeared to reflect the morphology formed by their constituents, i.e. NF-M and alpha-inx. The success of semi-in situ atomic force microscopy of exposed bona fide assembled neurofilaments through separating the sandwich suggests that it can be an effective and alternative method for investigating cytoplasmic intermediate filaments under physiological conditions by atomic force microscopy.