Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging.
Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging.
复制标题
DOI：
10.1091/mbc.e17-03-0161
复制
发表时间：
2017-10-15
影响因子：
3.3
通讯作者：
Elia N
中科院分区：
文献类型：
--
作者：
Schvartz T;Aloush N;Goliand I;Segal I;Nachmias D;Arbely E;Elia N
这项工作描述了一个优雅的方法，直接，位点特异性标记的蛋白质与荧光染料活细胞成像。通过整合一个非规范的氨基酸，能够结合到微管蛋白的荧光染料，我们直接和特异性标记微管蛋白与荧光染料和成像微管活的细胞。遗传密码扩展和生物正交标记首次提供了一种在活细胞中用荧光染料对蛋白质进行直接、位点特异性标记的方法。虽然荧光染料的小尺寸和极好的物理参数为高分辨率显微镜提供了独特的优势，但这种方法尚未被视为活细胞成像的工具。在这里，我们通过将其应用于α-微管蛋白标记来评估这种方法的可行性。经过一系列的校准，我们在活的哺乳动物细胞中以有效和稳健的方式用硅罗丹明（SiR）位点特异性标记α-微管蛋白。SiR标记的微管蛋白成功地以高密度掺入内源性微管中，使得能够视频记录间期和有丝分裂细胞中的微管动力学。将这种标记方法应用于结构化照明显微镜导致分辨率增加，突出了使用更小，更亮标签的优势。因此，使用我们优化的测定，遗传密码扩展提供了一个有吸引力的工具，用于在定量高分辨率成像中用最小的明亮标签标记蛋白质。
This work describes an elegant approach for direct, site-specific labeling of proteins with fluorescent-dyes for live cell imaging. By integrating a noncanonical amino acid that is capable of binding a fluorescent dye into tubulin, we directly and specifically labeled tubulin with a fluorescent-dye and imaged microtubules in live mammlian cells. Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging.