Department of Cellular and Molecular Neuropathology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Laboratory of Morphology and Image Analysis, Biomedical Research Core Facilities, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Sci Rep. 2022 Jul 1;12(1):11130. doi: 10.1038/s41598-022-15438-6.
Biotin ligases have been developed as proximity biotinylation enzymes for analyses of the interactome. However, there has been no report on the application of proximity labeling for in-resin correlative light-electron microscopy of Epon-embedded cells. In this study, we established a proximity-labeled in-resin CLEM of Epon-embedded cells using miniTurbo, a biotin ligase. Biotinylation by miniTurbo was observed in cells within 10 min following the addition of biotin to the medium. Using fluorophore-conjugated streptavidin, intracellular biotinylated proteins were labeled after fixation of cells with a mixture of paraformaldehyde and glutaraldehyde. Fluorescence of these proteins was resistant to osmium tetroxide staining and was detected in 100-nm ultrathin sections of Epon-embedded cells. Ultrastructures of organelles were preserved well in the same sections. Fluorescence in sections was about 14-fold brighter than that in the sections of Epon-embedded cells expressing mCherry2 and was detectable for 14 days. When mitochondria-localized miniTurbo was expressed in the cells, mitochondria-like fluorescent signals were detected in the sections, and ultrastructures of mitochondria were observed as fluorescence-positive structures in the same sections by scanning electron microscopy. Proximity labeling using miniTurbo led to more stable and brighter fluorescent signals in the ultrathin sections of Epon-embedded cells, resulting in better performance of in-resin CLEM.
生物素连接酶已被开发为用于相互作用组分析的邻近生物素化酶。然而，尚未有关于邻近标记在包埋在环氧树脂中的细胞的树脂内共聚焦光电子显微镜中的应用的报道。在这项研究中，我们使用 biotin ligase miniTurbo 建立了包埋在环氧树脂中的邻近标记的树脂内共聚焦光电子显微镜。在向培养基中添加生物素后 10 分钟内，可以观察到 miniTurbo 的生物素化。使用荧光素缀合的链霉亲和素，在用包含多聚甲醛和戊二醛的混合物固定细胞后，可标记细胞内生物素化的蛋白质。这些蛋白质的荧光对锇四氧化物染色具有抗性，并可在包埋在环氧树脂中的细胞的 100nm 超薄切片中检测到。在相同的切片中，细胞器的超微结构得到了很好的保存。切片中的荧光比表达 mCherry2 的包埋在环氧树脂中的细胞的切片中的荧光亮约 14 倍，并且可检测到 14 天。当在线粒体中定位的 miniTurbo 在细胞中表达时，在切片中检测到类似线粒体的荧光信号，并且通过扫描电子显微镜，在线粒体的相同切片中观察到线粒体的超微结构作为荧光阳性结构。使用 miniTurbo 进行的邻近标记导致包埋在环氧树脂中的细胞的超薄切片中产生更稳定和更亮的荧光信号，从而提高了树脂内共聚焦光电子显微镜的性能。
