Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy

Journal Article · 08 July 2022 · Journal of Structural Biology

DOI: https://doi.org/10.1016/j.jsb.2022.107881 · OSTI ID:1977405

Perez, Davis ^[1]; Dahlberg, Peter D. ^[2]; Wang, Jiarui ^[3]; Sartor, Annina M. ^[3]; Borden, Julia S. ^[4]; Shapiro, Lucy ^[3]; Moerner, William E. ^[3]

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1. Stanford Univ., CA (United States); OSTI
2. SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
3. Stanford Univ., CA (United States)
4. Univ. of California, Berkeley, CA (United States)

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., which influences 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 77 K 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. Finally, by expressing an inducible roGFP2-PopZ fusion we visualize individual microdomains in the context of their redox environment.

• Research Organization: Stanford Univ., CA (United States)
• Sponsoring Organization: National Institute of General Medical Sciences (NIGMS); USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division (CSGB)
• Grant/Contract Number: FG02-07ER15892
• OSTI ID: 1977405
• Journal Information: Journal of Structural Biology, Journal Name: Journal of Structural Biology Journal Issue: 3 Vol. 214; ISSN 1047-8477
• Publisher: ElsevierCopyright Statement
• Country of Publication: United States
• Language: English