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

doi:10.3389/fmicb.2020.583010

Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon Haloferax volcanii

Front Microbiol. 2020 Nov 20:11:583010. doi: 10.3389/fmicb.2020.583010. eCollection 2020.

Authors

Bartosz Turkowyd¹, Sandra Schreiber², Julia Wörtz², Ella Shtifman Segal³, Moshe Mevarech³, Iain G Duggin⁴, Anita Marchfelder², Ulrike Endesfelder^{1

5}

Affiliations

¹ Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany.
² Department of Biology II, Ulm University, Ulm, Germany.
³ Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
⁴ The ithree Institute, University of Technology Sydney, Ultimo, NSW, Australia.
⁵ Department of Physics, Mellon College of Science, Carnegie-Mellon University, Pittsburgh, PA, United States.

Abstract

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and are 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 (FOV) 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.

Keywords: FtsZ1 division ring; Haloferax volcanii; RNA polymerase dynamics; advanced fluorescence microscopy; archaeal cell imaging; imaging technology in microbiology; single-molecule imaging and tracking.