Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division
André Arashiro Pulschen, Delyan R. Mutavchiev, S J Culley, Kim Nadine Sebastian, Jacques Roubinet, Marc Roubinet, Gabriel Tarrason Risa, Marleen van Wolferen, Chantal Roubinet, Uwe Schmidt, G.K. Dey, Sonja‐Verena Albers, Ricardo Henriques, Buzz Baum
Abstract
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 [1Bisson-Filho A.W. Zheng J. Garner E. Archaeal imaging: leading the hunt for new discoveries.Mol. Biol. Cell. 2018; 29: 1675-1681Crossref PubMed Scopus (13) Google Scholar, 2Eun Y.J. Ho P.Y. Kim M. LaRussa S. Robert L. Renner L.D. Schmid A. Garner E. Amir A. Archaeal cells share common size control with bacteria despite noisier growth and division.Nat. Microbiol. 2018; 3: 148-154Crossref PubMed Scopus (41) Google Scholar, 3Walsh J.C. Angstmann C.N. Bisson-Filho A.W. Garner E.C. Duggin I.G. Curmi P.M.G. Division plane placement in pleomorphic archaea is dynamically coupled to cell shape.Mol. Microbiol. 2019; 112: 785-799Crossref PubMed Scopus (15) Google Scholar, 4Delpech F. Collien Y. Mahou P. Beaurepaire E. Myllykallio H. Lestini R. Snapshots of archaeal DNA replication and repair in living cells using super-resolution imaging.Nucleic Acids Res. 2018; 46: 10757-10770PubMed Google Scholar, 5Li Z. Kinosita Y. Rodriguez-Franco M. Nußbaum P. Braun F. Delpech F. Quax T.E.F. Albers S.V. Positioning of the Motility Machinery in Halophilic Archaea.MBio. 2019; 10: e00377-19Crossref PubMed Scopus (21) Google Scholar], 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 [6Wagner M. van Wolferen M. Wagner A. Lassak K. Meyer B.H. Reimann J. Albers S.V. Versatile Genetic Tool Box for the Crenarchaeote Sulfolobus acidocaldarius.Front. Microbiol. 2012; 3: 214Crossref PubMed Scopus (120) Google Scholar, 7Wagner M. Berkner S. Ajon M. Driessen A.J. Lipps G. Albers S.V. Expanding and understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus.Biochem. Soc. Trans. 2009; 37: 97-101Crossref PubMed Scopus (64) Google Scholar, 8Zhang C. Phillips A.P.R. Wipfler R.L. Olsen G.J. Whitaker R.J. The essential genome of the crenarchaeal model Sulfolobus islandicus.Nat. Commun. 2018; 9: 4908Crossref PubMed Scopus (40) Google Scholar, 9Zebec Z. Manica A. Zhang J. White M.F. Schleper C. CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus.Nucleic Acids Res. 2014; 42: 5280-5288Crossref PubMed Scopus (73) Google Scholar]. Additionally, studies using Sulfolobus were among the first to reveal striking similarities between the cell biology of eukaryotes and archaea [10Lindås A.C. Bernander R. The cell cycle of archaea.Nat. Rev. Microbiol. 2013; 11: 627-638Crossref PubMed Scopus (58) Google Scholar, 11Duggin I.G. McCallum S.A. Bell S.D. Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius.Proc. Natl. Acad. Sci. USA. 2008; 105: 16737-16742Crossref PubMed Scopus (50) Google Scholar, 12Robinson N.P. Dionne I. Lundgren M. Marsh V.L. Bernander R. Bell S.D. Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus.Cell. 2004; 116: 25-38Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 13Lindås A.C. Karlsson E.A. Lindgren M.T. Ettema T.J. Bernander R. A unique cell division machinery in the Archaea.Proc. Natl. Acad. Sci. USA. 2008; 105: 18942-18946Crossref PubMed Scopus (221) Google Scholar, 14Samson R.Y. Obita T. Freund S.M. Williams R.L. Bell S.D. A role for the ESCRT system in cell division in archaea.Science. 2008; 322: 1710-1713Crossref PubMed Scopus (254) Google Scholar, 15Takemata N. Samson R.Y. Bell S.D. Physical and Functional Compartmentalization of Archaeal Chromosomes.Cell. 2019; 179: 165-179.e18Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar]. 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.