Man Huizi, Bian Hui, Zhang Xinfu, Wang Chao, Huang Zhenlong, Yan Yu, Ye Zhiwei, Xiao Yi
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, 116024, China.
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, 116024, China.
Biosens Bioelectron. 2021 Oct 1;189:113378. doi: 10.1016/j.bios.2021.113378. Epub 2021 May 29.
The endoplasmic reticulum (ER) transforms its morphology to fit versatile cellular functions especially under stress conditions. Since various ER stresses are critical pathophysiological factors, the precise observations of ER can provide insights into disease diagnoses and biological researches. Live-cell super-resolution imaging is highly expected for uncovering microstructures of ER. However, to achieve this, there remains a big challenge in how to efficiently label ER with advanced fluorophores. Herein, we report a new SNAP-tag fluorescent probe, namely, CLP-TMR, for specific and high-density labeling of the newly constructed dual ER-signal (targeting and retention) peptides fused-SNAP proteins. This hybrid labeling system integrating chemical probes with genetically encoded techniques enables molecular position reconstructions of ER morphologies through direct stochastic optical reconstruction microscopy (dSTORM) imaging. The super-resolution imaging reveals several never-known ultrastructural changes responding to different ER stresses, i.e. the formation of peripheral ER sheets to restore the immunogenicity, and the long flattened ER tubules under inflammation.
内质网（ER）会改变其形态以适应多种细胞功能，尤其是在应激条件下。由于各种内质网应激是关键的病理生理因素，因此对内质网的精确观察可为疾病诊断和生物学研究提供见解。人们高度期望通过活细胞超分辨率成像来揭示内质网的微观结构。然而，要实现这一点，在如何用先进的荧光团高效标记内质网方面仍存在巨大挑战。在此，我们报告了一种新的SNAP标签荧光探针，即CLP-TMR，用于对新构建的融合了双内质网信号（靶向和保留）肽的SNAP蛋白进行特异性和高密度标记。这种将化学探针与基因编码技术相结合的混合标记系统，能够通过直接随机光学重建显微镜（dSTORM）成像对内质网形态进行分子位置重建。超分辨率成像揭示了几种对不同内质网应激做出反应的前所未知的超微结构变化，即形成外周内质网片层以恢复免疫原性，以及在炎症状态下出现长而扁平的内质网管。
