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

Autor:	Turkowyd B; Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany., Schreiber S; Department of Biology II, Ulm University, Ulm, Germany., Wörtz J; Department of Biology II, Ulm University, Ulm, Germany., Segal ES; Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel., Mevarech M; Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel., Duggin IG; The ithree Institute, University of Technology Sydney, Ultimo, NSW, Australia., Marchfelder A; Department of Biology II, Ulm University, Ulm, Germany., Endesfelder U; Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany.; Department of Physics, Mellon College of Science, Carnegie-Mellon University, Pittsburgh, PA, United States.
Jazyk:	angličtina
Zdroj:	Frontiers in microbiology [Front Microbiol] 2020 Nov 20; Vol. 11, pp. 583010. Date of Electronic Publication: 2020 Nov 20 (Print Publication: 2020).
DOI:	10.3389/fmicb.2020.583010
Abstrakt:	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. (Copyright © 2020 Turkowyd, Schreiber, Wörtz, Segal, Mevarech, Duggin, Marchfelder and Endesfelder.)
Databáze:	MEDLINE
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