Single-Molecule Localization Microscopy to study Archaea

In our new study, we and the Marchfelder group from Ulm have laid the groundwork for expanding the use of single-molecule localization microscopy (SMLM) to archaea — organisms known for their ability to live in harsh, inhospitable conditions. Our research was now published in the journal Frontiers of Microbiology.

The beauty of the SMLM technique we are using is we can localize single molecules in living cells. This represents a sharp break from previous biochemistry work on the structures and mechanisms of cells that required purifying the cellular components of interest in test tubes to study them. While it's much more complicated and data intensive to observe live cellular structures and mechanisms the benefits are clear: It gives us the strong advantage that we really can observe the molecules in their native environment and see what they naturally do.

So far, SMLM has been widely used on bacteria and yeast for years but has not been used before in archaea. Originally thought to be a type of bacteria, there is still much debate in the scientific community about how exactly to classify archaea, which can be found around the world but especially well known for surviving in conditions no other life could tolerate.

To develop a protocol for studying them, we thus joined our expertise and forces.  The Marchfelder lab in Germany has long studied Haloferax volcanii, which is found living in warm and extremely salty environments such as the Dead Sea, while our group is specialized in SMLM studies in microorganisms. We first had to balance keeping conditions ideal for both the archaea and microscope and develop fluorescent proteins that could be inserted into the archaea to image their structures. After testing various options, we found that codon-optimized versions of photoactivatable mCherry1 and photoconvertible Dendra2 worked the best in this species.

Furthermore, we got help from Moshe Mevarechs group in Tel Aviv and modified the archaeon's genes to stop it from producing the carotenoid pigments. Finally, building on the fluorescence work Iain Duggin in Sydney, we ended up successfully imaging two intracellular proteins in the archaeon as proof of SMLM's possibility in studying the organism and could show that their levels and organization strongly depends on the cellular growth conditions. Our study opens up lots of possibilities for more closely studying archaea.