Schvartz Tomer, Aloush Noa, Goliand Inna, Segal Inbar, Nachmias Dikla, Arbely Eyal, Elia Natalie
Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
The National Institute for Biotechnology in the Negev, Beer Sheva 84105, Israel.
Mol Biol Cell. 2017 Oct 15;28(21):2747-2756. doi: 10.1091/mbc.E17-03-0161. Epub 2017 Aug 23.
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.
遗传密码扩展和生物正交标记首次为在活细胞中用荧光染料对蛋白质进行直接、位点特异性标记提供了一种方法。尽管荧光染料的小尺寸和出色的光物理参数为高分辨率显微镜提供了独特优势，但这种方法尚未被用作活细胞成像的工具。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。经过一系列校准后，我们以高效且稳健的方式在活的哺乳动物细胞中用硅罗丹明（SiR）对α-微管蛋白进行了位点特异性标记。SiR标记的微管蛋白成功地以高密度掺入内源性微管中，从而能够对间期和有丝分裂细胞中的微管动力学进行视频记录。将这种标记方法应用于结构照明显微镜可提高分辨率，突出了使用更小、更亮标签的优势。因此，使用我们优化的检测方法，遗传密码扩展为在定量高分辨率成像中用最小、最亮的标签标记蛋白质提供了一种有吸引力的工具。
