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Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeon Haloferax volcanii

Bartosz Turkowyd, Sandra Schreiber, Julia Wörtz, Ella Shtifman Segal, Moshe Mevarech, View ORCID ProfileIain G. Duggin, Anita Marchfelder, View ORCID ProfileUlrike Endesfelder
doi: https://doi.org/10.1101/2020.07.27.222935
Bartosz Turkowyd
1Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology (SYNMIKRO), 35043 Marburg, Germany
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Sandra Schreiber
2Department of Biology II, Ulm University, 89069 Ulm, Germany
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Julia Wörtz
2Department of Biology II, Ulm University, 89069 Ulm, Germany
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Ella Shtifman Segal
3Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978-01, Israel
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Moshe Mevarech
3Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978-01, Israel
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Iain G. Duggin
4The ithree institute, University of Technology Sydney, Ultimo, NSW, 2007, Australia
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Anita Marchfelder
2Department of Biology II, Ulm University, 89069 Ulm, Germany
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  • For correspondence: anita.marchfelder@uni-ulm.de uendesfelder@cmu.edu
Ulrike Endesfelder
1Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology (SYNMIKRO), 35043 Marburg, Germany
5Department of Physics, Mellon College of Science, Carnegie-Mellon University, Pittsburgh, PA 15213, USA
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  • ORCID record for Ulrike Endesfelder
  • For correspondence: anita.marchfelder@uni-ulm.de uendesfelder@cmu.edu
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Abstract

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology.

Here we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA. This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted July 27, 2020.
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Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeon Haloferax volcanii
Bartosz Turkowyd, Sandra Schreiber, Julia Wörtz, Ella Shtifman Segal, Moshe Mevarech, Iain G. Duggin, Anita Marchfelder, Ulrike Endesfelder
bioRxiv 2020.07.27.222935; doi: https://doi.org/10.1101/2020.07.27.222935
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Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeon Haloferax volcanii
Bartosz Turkowyd, Sandra Schreiber, Julia Wörtz, Ella Shtifman Segal, Moshe Mevarech, Iain G. Duggin, Anita Marchfelder, Ulrike Endesfelder
bioRxiv 2020.07.27.222935; doi: https://doi.org/10.1101/2020.07.27.222935

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