Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division

Abstract

Summary Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. Although similar techniques have been applied to the study of halophilic archaea [ 1 , 2 , 3 , 4 , 5 ], our ability to explore the cell biology of thermophilic archaea has been limited by the technical challenges of imaging at high temperatures. Sulfolobus are the most intensively studied members of TACK archaea and have well-established molecular genetics [ 6 , 7 , 8 , 9 ]. Additionally, studies using Sulfolobus were among the first to reveal striking similarities between the cell biology of eukaryotes and archaea [ 10 , 11 , 12 , 13 , 14 , 15 ]. However, to date, it has not been possible to image Sulfolobus cells as they grow and divide. Here, we report the construction of the Sulfoscope , a heated chamber on an inverted fluorescent microscope that enables live-cell imaging of thermophiles. By using thermostable fluorescent probes together with this system, we were able to image Sulfolobus acidocaldarius cells live to reveal tight coupling between changes in DNA condensation, segregation, and cell division. Furthermore, by imaging deletion mutants, we observed functional differences between the two ESCRT-III proteins implicated in cytokinesis, CdvB1 and CdvB2. The deletion of cdvB1 compromised cell division, causing occasional division failures, whereas the ΔcdvB2 exhibited a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus , as is the case in eukaryotes, and that two contractile ESCRT-III polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division.

Tracing back variations in archaeal cell division to protein domain architectures

“…Thus, there is one major difference in the PPIs of Thaumarchaeota to the ones of Crenarchaeota: while in Thaumarchaeota we predict CdvA to bind to CdvC, in Crenarchaeota we suggest that CdvA binds to CdvB. This finding matches well to the differences between the two phyla found in fluorescence microscopy studies 1,5–7,11,12,14 . In the Thaumarchaeon N. maritimus , fluorescence bands of CdvA at the division site are followed by bands of CdvC, whilst in Crenarchaeota CdvA bands are followed by CdvB bands.…”

“…While in both groups fluorescence microscopy studies showed that initially CdvA proteins enrich at the future division site, probably binding to the membrane, the subsequent steps seem to differ. In Crenarchaeota, CdvB homologs successively join CdvA, together with CdvC 1,5,6,11,13,15 . While the exact mechanism is unknown, it is mostly suggested that CdvB homologs form a ring-like higher order structure, tethered to the membrane by CdvA 1,11,13 .…”

“…In Euryarchaeota, most organisms do not possess a Cdv system at all, but some few species show CdvB and/or CdvC proteins encoded in their genome. In Asgard archaea, only little is known because it was just recently possible to cultivate a member of this super-phylum 5,10 , but metagenome data and protein prediction indicates that genes encoding for CdvB and CdvC proteins are common within this group. In TACK archaea, all groups except Thermoproteales possess a full system of CdvA, CdvB and CdvC.…”

Studying life at the extremes

“…They recruited a lab member's father and brother, both aerospace engineers, to fabricate a chamber out of aircraft aluminium. The researchers have published diagrams for this 'Sulfoscope' 5 , but any double-heated chamber should do, says Baum. The system allows the team to investigate proteins that maintain symmetrical cell division.…”

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

“…very high or low temperature or high saline, acidic, alkaline or anaerobic environments), b) their colorful pigments causing autofluorescence and c) the limited range of available model organisms and molecular tools (Kramm et al, 2019). The set of fluorescence tools for imaging archaea available today includes several optimized fluorescent protein versions, a codon-optimized and a split version of GFP (Reuter and Maupin-Furlow, 2004;Duggin et al, 2015;Winter et al, 2018;Li et al, 2019) and a mCherry (Duggin et al, 2015;Liao et al, 2020) for imaging of Haloarchaea and a heat-tolerant GFP for Sulfolobus (Pulschen et al, 2020). Extrinsic stains include DNA staining with Acridine Orange, propidium iodide, Hoechst 33258 or 33342, DAPI and SYTOX Green (Samson et al, 2008, Liao, 2020#2010Delmas et al, 2013) or BrdU and EdU labeling of nascent DNA (Gristwood et al, 2012;Delpech et al, 2018), FM4-64Xa staining of membranes (Samson et al, 2008) or non-permeable dyes that stain the cell wall (Wirth et al, 2011), a new thermostable protein tag (H5) for Sulfolobus (Visone et al, 2017) and some immunofluorescence staining of fixed cells (Poplawski et al, 2000;Lindas et al, 2008;Samson et al, 2008;Ettema et al, 2011;Zenke et al, 2015).…”