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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 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.