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Title: Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter

Abstract

Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ~30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context formore » localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.« less

Authors:
; ORCiD logo; ; ORCiD logo; ; ORCiD logo; ; ORCiD logo
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States); Stanford Univ., CA (United States). Dept. of Chemistry; Stanford Univ. School of Medicine, CA (United States). Dept. of Developmental Biology; Stanford Univ., CA (United States). Dept. of Bioengineering
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1632358
Alternate Identifier(s):
OSTI ID: 1647237
Grant/Contract Number:  
FWP100463; AC02-76SF00515; R35GM118067; R35GM118071; P01NS092525
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; CLEM; superresolution; cryogenic electron tomography; correlative microscopy; CIASM

Citation Formats

Dahlberg, Peter D., Saurabh, Saumya, Sartor, Annina M., Wang, Jiarui, Mitchell, Patrick G., Chiu, Wah, Shapiro, Lucy, and Moerner, W. E. Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter. United States: N. p., 2020. Web. doi:10.1073/pnas.2001849117.
Dahlberg, Peter D., Saurabh, Saumya, Sartor, Annina M., Wang, Jiarui, Mitchell, Patrick G., Chiu, Wah, Shapiro, Lucy, & Moerner, W. E. Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter. United States. doi:https://doi.org/10.1073/pnas.2001849117
Dahlberg, Peter D., Saurabh, Saumya, Sartor, Annina M., Wang, Jiarui, Mitchell, Patrick G., Chiu, Wah, Shapiro, Lucy, and Moerner, W. E. Mon . "Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter". United States. doi:https://doi.org/10.1073/pnas.2001849117.
@article{osti_1632358,
title = {Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter},
author = {Dahlberg, Peter D. and Saurabh, Saumya and Sartor, Annina M. and Wang, Jiarui and Mitchell, Patrick G. and Chiu, Wah and Shapiro, Lucy and Moerner, W. E.},
abstractNote = {Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ~30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.},
doi = {10.1073/pnas.2001849117},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = ,
volume = ,
place = {United States},
year = {2020},
month = {6}
}

Journal Article:
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DOI: https://doi.org/10.1073/pnas.2001849117

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