Live cell imaging of the hyperthermophilic archaeonSulfolobus acidocaldariusidentifies complementary roles for two ESCRTIII homologues in ensuring a robust and symmetric cell division

Abstract

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we report the construction of the Sulfoscope, a heated chamber that enables live-cell imaging on an inverted fluorescent microscope. Using this system combined with thermostable fluorescent probes, we were able to image Sulfolobus cells as they divide, revealing a tight coupling between changes in DNA compaction, segregation and cytokinesis. By imaging deletion mutants, we observe important differences in the function of the two ESCRTIII proteins recently implicated in cytokinesis. The loss of CdvB1 compromises cell division, causing occasional division failures and fusion of the two daughter cells, whereas the deletion of cdvB2 leads to 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 ESCRTIII polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division. Taken together, the Sulfoscope has shown to provide a controlled high temperature environment, in which cell biology of Sulfolobus can be studied in unprecedent details. AAP, DRM, GD, RH and BB conceived the study. Microscope design and construction and commercial heating stage testing was carried out by AAP, SC, GD, DRM and RH. Design of the heating cap and heating stage was carried out by AAP, JR, CR and MR. JR and MR constructed the heating cap and heating stage. AAP and DRM established the imaging methodology and dyes used. Live-imaging and imaging analysis were performed by AAP and DRM with help from SC. Genetics and strains were performed by AAP, MVW and KNS. Immunofluorescence and Western-Blots were performed by AAP. Flow Cytometry were performed by GTR. Chamber measurements were performed

The archaeal division protein CdvB1 assembles into polymers that are depolymerized by CdvC

“…The action of CdvC may then remodel the CdvB1 filaments whereupon the membrane shrinks, while CdvB2 keeps the ring in place. This idea is in line with previous findings (11) where mutants lacking CdvB1 were less able to perform the fission of the membrane, indicating its role in membrane deformation, whereas mutants without CdvB2 would lead to asymmetric divisions, suggesting its guiding role.…”

“…It was shown that Sulfolobus cells lacking CdvB were unable to grow, while cells lacking the CdvB paralogs were still viable, albeit with a lower growth rate or aberrant daughter cells (10), and therefore the CdvB1-3 paralogs were deemed non-essential for cell division. Recently however, developments in hightemperature microscopy techniques (11,12) together with high-resolution imaging of fixed cells (13) shed valuable light onto how the CdvB paralogs participate in the cell division. It was shown that CdvB1 and B2 are recruited to the CdvB ring right before the cytokinesis, whereupon CdvB disappears from the membrane, while CdvB1 and CdvB2 carry out the deformation of the membrane needed for the division of the cell (11,13).…”

“…Recently however, developments in hightemperature microscopy techniques (11,12) together with high-resolution imaging of fixed cells (13) shed valuable light onto how the CdvB paralogs participate in the cell division. It was shown that CdvB1 and B2 are recruited to the CdvB ring right before the cytokinesis, whereupon CdvB disappears from the membrane, while CdvB1 and CdvB2 carry out the deformation of the membrane needed for the division of the cell (11,13). Additionally, it was shown that Sulfolobus mutants without any CdvB2 undergo asymmetric cell divisions yielding differently sized daughter cells, while cells without CdvB1 occasionally failed to divide, yielding multiploid cells with 2 genomes (11).…”

“…It was shown that CdvB1 and B2 are recruited to the CdvB ring right before the cytokinesis, whereupon CdvB disappears from the membrane, while CdvB1 and CdvB2 carry out the deformation of the membrane needed for the division of the cell (11,13). Additionally, it was shown that Sulfolobus mutants without any CdvB2 undergo asymmetric cell divisions yielding differently sized daughter cells, while cells without CdvB1 occasionally failed to divide, yielding multiploid cells with 2 genomes (11). All this changed the view of the basic mechanism of the Cdv system.…”

SiCTeC: An inexpensive, easily assembled Peltier device for rapid temperature shifting during single-cell imaging

“…The flexibility of our device in terms of programming near-arbitrary temperature dynamics is also unmatched. While other modified devices have recently been constructed to allow for imaging at extreme temperatures [ 34 , 35 ], these instruments are more expensive than SiCTeC, highly customized, and limited to fluorescence imaging. Our device is entirely compatible with both brightfield and fluorescence imaging, which can be utilized to study protein localization and expression changes during temperature shifts as long as the temperature dependence of fluorescence is accounted for [ 36 ].…”