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

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.

Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2-autoreceptor activity

“…Localizations were fit using ThunderStorm as described above. Single molecule trajectories were found using the program swift (Turkowyd et al, 2020). Parameter used beyond the default were: ”expdisplacement” : 300,”expnoiserate”: 10,”maxblinkingduration” : 2,”pblink” : 0.5,”preappear” : 0.5,”pswitch” : 0.001,”precision”: 30,”pruningbase” : 2,”pruningrate” : 0.2,”randomseed” : 42,”wdiffusion” : 2,”wdirected” : 1,”wfield” : 0,”wimmobile” : 1.…”

Open issues for protein function assignment inHaloferax volcaniiand other halophilic archaea

“…Its genome has been sequenced and carefully annotated (Hartman et al, 2010, Pfeiffer et al, 2008a, Pfeiffer and Oesterhelt, 2015. A plethora of biological aspects have been successfully tackled in this species, with examples including DNA replication (Perez-Arnaiz et al, 2020); cell division and cell shape (Turkowyd et al, 2020, Walsh et al, 2019, de Silva et al, 2021, Duggin et al, 2015, Liao et al, 2021; metabolism (Brasen and Schonheit, 2001, Johnsen et al, 2009, Pickl et al, 2012, Sutter et al, 2016, Reinhardt et al, 2019, Kuprat et al, 2021, Kuprat et al, 2020, Sutter et al, 2020, Tästensen et al, 2020; protein secretion (Abdul-Halim et al, 2020, Abdul Halim et al, 2018, Abdul Halim et al, 2013, Storf et al, 2010; motility and biofilms (Schiller et al, 2020, Pohlschroder and Esquivel, 2015, Li et al, 2020, Collins et al, 2020, Quax et al, 2018, Nussbaum et al, 2020; mating (Shalev et al, 2017); signalling (Braun et al, 2019); virus defence (Maier et al, 2019); proteolysis (Reuter and Maupin-Furlow, 2004, Reuter et al, 2010, Prunetti et al, 2014, Cerletti et al, 2014, Costa et al, 2018; posttranslati...…”