Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy.
Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy.
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DOI：
10.1016/j.jsb.2022.107881
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发表时间：
2022-09
影响因子：
3
通讯作者：
Moerner, W. E.
中科院分区：
文献类型：
--
作者：
Perez, Davis;Dahlberg, Peter D.;Wang, Jiarui;Sartor, Annina M.;Borden, Julia S.;Shapiro, Lucy;Moerner, W. E.
低温相关光学和电子显微镜（cryo-CLEM）旨在利用两种强大的成像模式中存在的正交信息。虽然低温电子显微镜（cryo-EM）的最新进展允许在纳米尺度上观察和识别细胞内的结构，但关于细胞环境的信息，如pH值，膜电位，离子强度等，影响观察到的结构仍然缺乏。荧光显微镜可以潜在地用于揭示这一信息时，特定的标签，称为荧光生物传感器，使用，但有这样的生物传感器在冷冻CLEM的最小使用日期。在这里，我们证明了一个这样的生物传感器，荧光蛋白roGFP 2，冷冻CLEM实验的适用性。在室温下，已知当在425 nm或488 nm激发时roGFP 2发射亮度的比率报告局部氧化还原电位。当含有roGFP 2的样品以与cryo-EM相容的方式快速冷却至77 K时，激发峰的比率仍然是冷冻时氧化还原电位的忠实指标。使用纯化的蛋白质在不同的氧化/还原环境中，我们生成的校准曲线，可用于分析原位测量。作为一个证明的原则演示，我们调查玻璃化新月柄杆菌细胞内的氧化/还原状态。极性组织蛋白Z（PopZ）定位于C. Crescentus，其中已知形成独特的微区。通过表达可诱导的roGFP 2-PopZ融合体，我们可以在其氧化还原环境的背景下可视化各个微结构域。
Cryogenic correlative light and electron microscopy (cryo-CLEM) seeks to leverage orthogonal information present in two powerful imaging modalities. While recent advances in cryogenic electron microscopy (cryo-EM) allow for the visualization and identification of structures within cells at the nanometer scale, information regarding the cellular environment, such as pH, membrane potential, ionic strength etc. that influence the observed structures remains absent. Fluorescence microscopy can potentially be used to reveal this information when specific labels, known as fluorescent biosensors, are used, but there has been minimal use of such biosensors in cryo-CLEM to date. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. At room temperature, the ratio of roGFP2 emission brightness when excited at 425 nm or 488 nm is known to report on the local redox potential. When samples containing roGFP2 are rapidly cooled to 77K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. By expressing an inducible roGFP2-PopZ fusion we can visualize individual microdomains in the context of their redox environment.
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影响因子：
15
作者：
Dahlberg PD;Sartor AM;Wang J;Saurabh S;Shapiro L;Moerner WE
通讯作者：
Moerner WE
影响因子：
2.8
作者：
Metskas, Lauren Ann;Briggs, John A. G.
通讯作者：
Briggs, John A. G.
影响因子：
3
作者：
Bilan, Dmitry S.;Matlashov, Mikhail E.;Gorokhovatsky, Andrey Yu.;Schultz, Carsten;Enikolopov, Grigori;Belousov, Vsevolod V.
通讯作者：
Belousov, Vsevolod V.
影响因子：
3
作者：
Tang, Guang;Peng, Liwei;Ludtke, Steven J.
通讯作者：
Ludtke, Steven J.
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.