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 contextmore »
- Authors:
- Publication Date:
- Research Org.:
- SLAC National Accelerator Laboratory (SLAC), 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. 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. 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 = {Mon Jun 08 00:00:00 EDT 2020},
month = {Mon Jun 08 00:00:00 EDT 2020}
}
https://doi.org/10.1073/pnas.2001849117
Web of Science
Works referenced in this record:
Multicolor localization microscopy and point-spread-function engineering by deep learning
journal, January 2019
- Hershko, Eran; Weiss, Lucien E.; Michaeli, Tomer
- Optics Express, Vol. 27, Issue 5
Novel organic dyes for multicolor localization-based super-resolution microscopy
journal, May 2015
- Lehmann, Martin; Lichtner, Gregor; Klenz, Haider
- Journal of Biophotonics, Vol. 9, Issue 1-2
Single-molecule high-resolution imaging with photobleaching
journal, April 2004
- Gordon, M. P.; Ha, T.; Selvin, P. R.
- Proceedings of the National Academy of Sciences, Vol. 101, Issue 17, p. 6462-6465
Automated segmentation of electron tomograms for a quantitative description of actin filament networks
journal, January 2012
- Rigort, Alexander; Günther, David; Hegerl, Reiner
- Journal of Structural Biology, Vol. 177, Issue 1
Caulobacter PopZ forms a polar subdomain dictating sequential changes in pole composition and function
journal, April 2010
- Bowman, Grant R.; Comolli, Luis R.; Gaietta, Guido M.
- Molecular Microbiology, Vol. 76, Issue 1
Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy
journal, December 2006
- Hess, Samuel T.; Girirajan, Thanu P. K.; Mason, Michael D.
- Biophysical Journal, Vol. 91, Issue 11
Imaging Intracellular Fluorescent Proteins at Nanometer Resolution
journal, September 2006
- Betzig, E.; Patterson, G. H.; Sougrat, R.
- Science, Vol. 313, Issue 5793
Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states
journal, November 2009
- Subach, Fedor V.; Malashkevich, Vladimir N.; Zencheck, Wendy D.
- Proceedings of the National Academy of Sciences, Vol. 106, Issue 50
Identification of PAmKate as a Red Photoactivatable Fluorescent Protein for Cryogenic Super-Resolution Imaging
journal, September 2018
- Dahlberg, Peter D.; Sartor, Annina M.; Wang, Jiarui
- Journal of the American Chemical Society, Vol. 140, Issue 39
Nanometer-localized multiple single-molecule fluorescence microscopy
journal, July 2004
- Qu, X.; Wu, D.; Mets, L.
- Proceedings of the National Academy of Sciences, Vol. 101, Issue 31, p. 11298-11303
Location and architecture of the Caulobacter crescentus chemoreceptor array
journal, July 2008
- Briegel, Ariane; Ding, H. Jane; Li, Zhuo
- Molecular Microbiology, Vol. 69, Issue 1
Selective sequestration of signalling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus
journal, January 2020
- Lasker, Keren; von Diezmann, Lexy; Zhou, Xiaofeng
- Nature Microbiology, Vol. 5, Issue 3
Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins
journal, February 2019
- Tuijtel, Maarten W.; Koster, Abraham J.; Jakobs, Stefan
- Scientific Reports, Vol. 9, Issue 1
Universal architecture of bacterial chemoreceptor arrays
journal, September 2009
- Briegel, A.; Ortega, D. R.; Tocheva, E. I.
- Proceedings of the National Academy of Sciences, Vol. 106, Issue 40
A Self-Associating Protein Critical for Chromosome Attachment, Division, and Polar Organization in Caulobacter
journal, September 2008
- Ebersbach, Gitte; Briegel, Ariane; Jensen, Grant J.
- Cell, Vol. 134, Issue 6
Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging
journal, May 2012
- Ha, Taekjip; Tinnefeld, Philip
- Annual Review of Physical Chemistry, Vol. 63, Issue 1
Localizing Exciton Recombination Sites in Conformationally Distinct Single Conjugated Polymers by Super-resolution Fluorescence Imaging
journal, February 2015
- Park, Heungman; Hoang, Dat Tien; Paeng, Keewook
- ACS Nano, Vol. 9, Issue 3
A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus
journal, November 2007
- Thanbichler, M.; Iniesta, A. A.; Shapiro, L.
- Nucleic Acids Research, Vol. 35, Issue 20
Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells
journal, January 2020
- Hoffman, David P.; Shtengel, Gleb; Xu, C. Shan
- Science, Vol. 367, Issue 6475
An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology
journal, May 2016
- Thompson, Rebecca F.; Walker, Matt; Siebert, C. Alistair
- Methods, Vol. 100
Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)
journal, August 2006
- Rust, Michael J.; Bates, Mark; Zhuang, Xiaowei
- Nature Methods, Vol. 3, Issue 10
Sequential evolution of bacterial morphology by co-option of a developmental regulator
journal, January 2014
- Jiang, Chao; Brown, Pamela J. B.; Ducret, Adrien
- Nature, Vol. 506, Issue 7489
ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging
journal, May 2014
- Ovesný, Martin; Křížek, Pavel; Borkovec, Josef
- Bioinformatics, Vol. 30, Issue 16
Metallothionein as a clonable tag for protein localization by electron microscopy of cells: METALLOTHIONEIN FOR PROTEIN LOCALIZATION IN CELLS
journal, May 2015
- Morphew, M. K.; O'Toole, E. T.; Page, C. L.
- Journal of Microscopy, Vol. 260, Issue 1
Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT
journal, December 2017
- Schueder, Florian; Lara-Gutiérrez, Juanita; Beliveau, Brian J.
- Nature Communications, Vol. 8, Issue 1
An intracellular compass spatially coordinates cell cycle modules in Caulobacter crescentus
journal, October 2016
- Lasker, Keren; Mann, Thomas H.; Shapiro, Lucy
- Current Opinion in Microbiology, Vol. 33
Quantitative Multicolor Subdiffraction Imaging of Bacterial Protein Ultrastructures in Three Dimensions
journal, February 2013
- Gahlmann, Andreas; Ptacin, Jerod L.; Grover, Ginni
- Nano Letters, Vol. 13, Issue 3
Bacterial chemoreceptors: high-performance signaling in networked arrays
journal, January 2008
- Hazelbauer, Gerald L.; Falke, Joseph J.; Parkinson, John S.
- Trends in Biochemical Sciences, Vol. 33, Issue 1
Superresolution Imaging of Multiple Fluorescent Proteins with Highly Overlapping Emission Spectra in Living Cells
journal, September 2011
- Gunewardene, Mudalige S.; Subach, Fedor V.; Gould, Travis J.
- Biophysical Journal, Vol. 101, Issue 6
Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography
journal, May 2014
- Chang, Yi-Wei; Chen, Songye; Tocheva, Elitza I.
- Nature Methods, Vol. 11, Issue 7
A Ferritin-Based Label for Cellular Electron Cryotomography
journal, February 2011
- Wang, Qing; Mercogliano, Christopher P.; Löwe, Jan
- Structure, Vol. 19, Issue 2
Oligomerization and higher-order assembly contribute to sub-cellular localization of a bacterial scaffold: Mutational analysis of PopZ
journal, October 2013
- Bowman, Grant R.; Perez, Adam M.; Ptacin, Jerod L.
- Molecular Microbiology, Vol. 90, Issue 4
FerriTag is a new genetically-encoded inducible tag for correlative light-electron microscopy
journal, July 2018
- Clarke, Nicholas I.; Royle, Stephen J.
- Nature Communications, Vol. 9, Issue 1
New Insights into Bacterial Chemoreceptor Array Structure and Assembly from Electron Cryotomography
journal, March 2014
- Briegel, Ariane; Wong, Margaret L.; Hodges, Heather L.
- Biochemistry, Vol. 53, Issue 10
Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition
journal, November 2006
- Ortiz, Julio O.; Förster, Friedrich; Kürner, Julia
- Journal of Structural Biology, Vol. 156, Issue 2
Identification of macromolecular complexes in cryoelectron tomograms of phantom cells
journal, October 2002
- Frangakis, A. S.; Bohm, J.; Forster, F.
- Proceedings of the National Academy of Sciences, Vol. 99, Issue 22
Computer Visualization of Three-Dimensional Image Data Using IMOD
journal, January 1996
- Kremer, James R.; Mastronarde, David N.; McIntosh, J. Richard
- Journal of Structural Biology, Vol. 116, Issue 1
Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009
- Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
- Nature Methods, Vol. 6, Issue 5, p. 343-345
Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging
journal, February 2017
- Hagen, Wim J. H.; Wan, William; Briggs, John A. G.
- Journal of Structural Biology, Vol. 197, Issue 2
Convolutional neural networks for automated annotation of cellular cryo-electron tomograms
journal, August 2017
- Chen, Muyuan; Dai, Wei; Sun, Stella Y.
- Nature Methods, Vol. 14, Issue 10
Marker-free method for accurate alignment between correlated light, cryo-light, and electron cryo-microscopy data using sample support features
journal, January 2018
- Anderson, Karen L.; Page, Christopher; Swift, Mark F.
- Journal of Structural Biology, Vol. 201, Issue 1