Abstract
SummaryLive-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.
Highlights
We identified conditions under which it was possible to combine this soft immobilization with dyes and two-color fluorescent imaging to follow S. acidocaldarius cells for up to 2 h, after which cell divisions under these conditions became rare
Our first use of the Sulfoscope revealed a tight coupling between DNA reorganization, nucleoid separation, and membrane deformation during division (Figure 2D)
These events appear to occur in a defined order in the wild-type cell
Summary
METHOD DETAILSStrain constructions and growth conditions Deletion of genes was performed using the ‘‘pSVA431 method’’ as previously described by Wagner and collaborators [10]. For the DcdvB2+pSVAaraFX-stop and DcdvB2+pSVACdvB2 rescue experiments, expression was induced by adding 0.15% w/v (final concentration) of L-arabinose to freshly diluted cultures. Cap/stage construction and development The heating system was designed to be combined with the AttofluorÒ chamber. The metal elements in the cap and the heating elements were built with aircraft aluminum (AL 7075 for the cap and stage and AL 2024 for the metal collars) using a lathe to obtain the final shape. The top part of the heating cap was built using fiberglass and a silicon rubber, which separates the metal and the fiberglass and holds the electric resistance (thin film resistance). The temperature of the cap and the chamber were controlled by two independent controlling systems attached to the heating elements. Detailed information about the design of the chamber, controlling system and materials is available upon request
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