Report
Live Imaging of a Hyperthermophilic Archaeon
Reveals Distinct Roles for Two ESCRT-III Homologs
in Ensuring a Robust and Symmetric Division
Graphical Abstract
Highlights
d Development of a heating chamber for live imaging of
hyperthermophiles
d Live imaging of cell division in the archaeon Sulfolobus
acidocaldarius
d DNA reorganization is tightly coupled to cell division in
Sulfolobus acidocaldarius
d ESCRT-III homologs play distinct roles in ensuring robust and
symmetric division
Authors
Andre Arashiro Pulschen,
Delyan R. Mutavchiev, Siaˆ n Culley, ...,
Sonja-Verena Albers,
Ricardo Henriques, Buzz Baum
Correspondence
b.baum@ucl.ac.uk
In Brief
Pulschen et al. describe the
‘‘Sulfoscope,’’ a microscopy platform that
allows live imaging of hyperthermophiles.
By imaging cell division in the archaeon
Sulfolobus, they observe tight coupling
between DNA reorganization and
cytokinesis and complementary roles for
two ESCRT-III homologs in ensuring
robust and symmetric cytokinesis.
Pulschen et al., 2020, Current Biology 30, 1–8
July 20, 2020 ª 2020 The Authors. Published by Elsevier Inc.
https://doi.org/10.1016/j.cub.2020.05.021
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Report
Live Imaging of a Hyperthermophilic Archaeon
Reveals Distinct Roles for Two ESCRT-III Homologs
in Ensuring a Robust and Symmetric Division
Andre Arashiro Pulschen,1 Delyan R. Mutavchiev,1 Siaˆ n Culley,1 Kim Nadine Sebastian,2 Jacques Roubinet,6
Marc Roubinet,7 Gabriel Tarrason Risa,1 Marleen van Wolferen,2 Chantal Roubinet,1 Uwe Schmidt,3,4 Gautam Dey,1
Sonja-Verena Albers,2 Ricardo Henriques,1 and Buzz Baum1,5,8,*
1MRC-Laboratory for Molecular Cell Biology, UCL, Gower Street, London WC1E 6BT, UK
2Molecular Biology of Archaea, Institute of Biology II - Microbiology, University of Freiburg, 79104 Freiburg, Germany
3Center for System Biology Dresden (CSBD), 01307 Dresden, Germany
4Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), 01307 Dresden, Germany
5Institute for the Physics of Living Systems, UCL, London WC1E 6BT, UK
6Gratentour, France
7Figeac, France
8Lead Contact
*Correspondence: b.baum@ucl.ac.uk
https://doi.org/10.1016/j.cub.2020.05.021
SUMMARY
Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukary-
otes. Although similar techniques have been applied to the study of halophilic archaea [1–5], 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 [6–9]. Additionally, studies using Sulfolobus were among the first to reveal
striking similarities between the cell biology of eukaryotes and archaea [10–15]. 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, segrega-
tion, and cell division. Furthermore, by imaging deletion mutants, we observed functional differences be-
tween the two ESCRT-III proteins implicated in cytokinesis, CdvB1 and CdvB2. The deletion of cdvB1
compromised cell division, causing occasional division failures, whereas the DcdvB2 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.
RESULTS
High-Temperature Live Imaging of Fluorescently
Labeled Sulfolobus Cells
In order to achieve the stable high temperatures (70�C–80�C)
required for live imaging of thermophilic archaea, like Sulfolobus
acidocaldarius, we designed a chamber consisting of two indi-
vidual heating elements: a cap and stage (Figures 1A–1E). Our
first-generation design employed a heated stage and a metal
chamber (Figure 1A, bottom part). However, this led to large
(>10�C) temperature gradients across the chamber because of
the poor conductivity of the glass coverslip and thermal losses
to the surrounding room-temperature air. Furthermore, the
open top led to the rapid loss of water from the chamber through
evaporation. This could not be resolved using a simple seal,
because this resulted in evaporated water condensing onto the
underside of the lid. To overcome both problems, inspired by
PCR machines, we designed and built a heated cap for the
chamber (Figures 1A and 1E), which could be sealed using an
O-ring (Figure 1B). To further improve the temperature control,
we custom-built a heated stage (Figures 1C and 1E) that
perfectly fit the chamber and cap (Figure 1D). By applying a tem-
perature gradient, so that the cap was raised to a temperature
5�C higher than the base, the medium quickly reached a stable
equilibrium temperature that could be maintained for long pe-
riods of time with minimal loss of water (Figures 1F and 1G).
In order to image Sulfolobus acidocaldarius cells live using
this setup, cells were pre-labeled using dyes (Nile Red for
membrane and SYBR Safe for DNA) that retain their optical
properties at high temperature and low pH. Cell immobilization
proved the greater challenge. Although Sulfolobus cells could
be imaged without immobilization in heated chambers, only a
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small number of cells remained static long enough to allow for
accurate quantitative measurements to be made. Additionally,
to be sure that observed changes in DNA reorganization during
division were not due to cell movement, cells had to be held in
place. Unlike bacteria cells, however, Sulfolobus cells appear
to be soft and sensitive to mechanical stress (Figure S1D)—in
line with observations made in other archaea [1, 2]. So, to pro-
vide a soft support sufficient to prevent cells from moving, we
placed cells under a semi-solid, preheated Gelrite pad (see
STAR Methods for details). 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. Whereas the membrane
dye proved non-toxic, the DNA dye, as reported for many other
cells, reduced the rate of cell growth (Figure 2A). Therefore,
where possible (e.g., for the study of division symmetry and
failures), measurements were performed using Nile Red alone.
Comparisons of cell division rates under these different condi-
tions can be found in Figure S1. The fastest division times
were recorded for cells imaged in the absence of a DNA dye
without immobilization—conditions closest to those found in
liquid culture (Figure 2B; Figure S1).
Live Imaging Reveals Coordination of DNA Segregation,
Compaction, and Cytokinesis in Dividing Sulfolobus
acidocaldarius Cells
Using the Sulfoscope, we were able to assess the dynamics
of events accompanying cell division in the thermophilic archaeon
Sulfolobusacidocaldarius.Intheseexperiments,S.acidocaldarius
cells were found to be near spherical and to divide to generate two
ovaldaughter cells (Figures 2B–2D).Imaging also revealed coordi-
nated changes in DNA organization and cell division (Videos S1
and S2). The first evidence that cells were about to divide was a
Figure 1. Heating Elements and Characterization of the Sulfoscope
(A) Heating cap and Attofluor chamber, disconnected.
(B) Assembled cap with Attofluor chamber.
(C) Heating stage view from the top.
(D) Fully assembled system.
(E) Schematic of the heating system.
(F) Temperature measurements performed inside the system. Temperature was recorded using a probe inserted into the imaging chamber through the sealable
inlet.
(G) Evaporation measurements after 6 h at the desired temperature. Error bars show mean and SD.
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change in DNA organization prior to the start of membrane furrow-
ing (Figure 2D). During this period, the DNA underwent a transition
fromadiffusestatetoasemi-compactstateinwhichthenucleoids
appeared partially separated (Figure 2E). The onset of furrowing
was accompanied by further compaction of the DNA, leading to
the formation of two distinct, spatially separated chromosomes
(Figures 2C–2E). Importantly, we observed the exact same order
of events and the same changes in DNA changes over time in
the presence or absence of a Gelrite pad (Figure 2E; Figure S1C).
Reassuringly, a similar sequence of events was also seen in
Figure 2. Live-Cell Division of Sulfolobus acidocaldarius DSM 639
(A) Growth curve of S. acidocaldarius treated with Nile Red, SYBR Safe, and control. Error bars show mean and SD.
(B) Time-lapse of a non-immobilized Sulfolobus cell stained with Nile Red alone. * = start of cytokinesis. ** = end of cytokinesis, orange arrowhead = cell sep-
aration.
(C) Time-lapse microscopy showing immobilized cells segregating their DNA and dividing.
(D) Time-lapse imaging of an immobilized cell as it divides, showing changes in the membrane and DNA organization.
(E) Changes in DNA organization that accompany division in immobilized cells (n = 50) and non-immobilized cells (n = 20). Cells were separated into three different
classes based upon their DNA organization: Cells with a single diffuse structure (blue), two diffuse structures (purple), or compact and well-defined structures
(pink).
Scale bars: 1 mm. Error bars show mean and SD. See also Figures S1 and S2 and Videos S1 and S2.
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movies of dividing Saccharolobus solfataricus cells (formerly
Sulfolobus solfataricus [16]) (Figures S2A and S2B), imaged at
75�C using the same setup, suggesting that the process is
conserved.
The Role of the Contractile ESCRT-III Proteins, CdvB1
and CdvB2, in S. acidocaldarius Cell Division
Risa and collaborators [17] recently proposed a model of
ESCRT-III-mediated division in S. acidocaldarius based on
data acquired from fixed cells. Under this model, a non-contrac-
tile CdvB division ring is formed at the equatorial plane of dividing
Sulfolobus cells that templates the assembly of a contractile
ESCRT-III heteropolymer based on two other ESCRT-III pro-
teins, CdvB1 and CdvB2. The sudden proteasome-mediated
degradation of CdvB then triggers division by allowing the
constriction of the CdvB1 and CdvB2 ring [17]. An untested
assumption of this model is that CdvB1 and CdvB2 are equiva-
lent in their ability to drive scission [17]. Although this fits with
their co-localization, and is in keeping with previous studies
that identified CdvB1 and CdvB2 as close paralogs that share
65% amino-acid identity and 80% similarity, we wondered if
we could dissect their individual function using the Sulfoscope.
To test this, we generated in-frame deletion mutants of cdvB1
and cdvB2 in the S. background strain MW001 and imaged these
cells live (Figures 3A and 3B; Video S3). Interestingly, although
growth was severely impaired in the cdvB2 deletion strain, the
deletion of cdvB1 only had a modest impact on growth at 75�C
(Figure 3A). Moreover, DcdvB1 cells had an average size that
Figure 3. Effects of the Deletion of cdvB1 and cdvB2 in Cell Division and Cell Growth in Sulfolobus acidocaldarius
(A) Growth of MW001 (background strain), DcdvB1, and DcdvB2 on BNS medium plates, showing that the growth of DcdvB2 is compromised.
(B) Western blots of MW001, DcdvB1, and DcdvB2 using CdvB1 and CdvB2 antibodies. The DNA binding protein Alba was used as a loading control.
(C) Time-lapse microscopy showing MW001, DcdvB1, and DcdvB2 cells. Purple dashed lines encircle cells undergoing successful divisions, whereas cells
undergoing failed divisions are encircled by red dashed lines. The blue arrowheads point to small cells that drift across the imaging field.
(D) Time-lapse imaging of DcdvB1 cells that following membrane furrowing fail division, resulting in the fusion of daughter cells.
(E) Quantification of the frequency of division failures in immobilized wild-type, MW001, DcdvB1, and DcdvB2 cells. Five independent movies were used to
evaluate the frequency of division failure from 130 to 180 divisions per strain, in total.
Scale bars: 1 mm. Error bars show mean and SD. All live imaging shown in this figure was performed using immobilized cells. See also Figure S2 and Videos S3
and S4.
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was similar to cells from the background strain, MW001 (Fig-
ure 3C; Video S3).
When imaged live, a subpopulation of DcdvB1 cells failed
midway through the division process (Figures 3D–3E; Video
S4). In such cases, a halt in cytokinesis caused the nascent
daughter of DcdvB1 cells to fuse back together to generate
single cells that carry both copies of the spatially separated
and compact chromosomes (Figure 3D). Under our imaging
conditions such failures accounted for 16% of DcdvB1 divi-
sions but were very rare (1%–2%) in the other strains analyzed
(Figure 3E). However, the growth of DcdvB1 colonies was
much more severely impaired at 65�C (Figure S2C). At this
lower temperature, we observed a significant population of
>2N cells and an increase in the number of cells unable to
complete cytokinesis (Figures S2D–S2H). Thus, although
CdvB1 plays only a minor role in Sulfolobus acidocaldarius di-
vision under optimum growth conditions, it appears necessary
to ensure that division remains fail-safe and is robust under
suboptimal conditions.
As suggested by the colony phenotype, the impact of CdvB2
loss on division was much more profound than the loss of CdvB1
at 75�C. At a first glance, this was seen by an increase in the vari-
ation in cell size in the population, as well as by the presence of
small, irregularly shaped cells not properly immobilized by the
pad (Figure 3C; Video S3). Upon further examination, such phe-
notypes were found to result from asymmetric divisions (Videos
S3 and S4). Thus, the site of division in DcdvB2 cells proved
extremely variable (Figures 4A–4D). In most cases, each of the
two daughter cells generated by an asymmetric division retained
one of the two separated chromosomes (Figure 4B). However, in
some cases, extreme mispositioning of the furrow led to the for-
mation of ghost cells (Figure 4C) and a corresponding population
of large cells with extra chromosomes (Figures S3A and S3B).
The asymmetry was also visible as a high variance in the size
of newly born G1 cells (and to a lesser extent G2 cells; Fig-
ure S3C), as indicated by the analysis of fixed cells labeled
with a DNA dye by flow cytometry (Figures 4E and 4F). Impor-
tantly, both the division asymmetries and the cell size variation
in DcdvB2 cells could be rescued by the ectopic expression of
CdvB2 from an inducible promoter (Figures 4D–4F; Figures
S3D and S3E; Video S5), demonstrating that these defects are
due to a lack of CdvB2.
To determine the cause of the error in division symmetry in
DcdvB2 cells, we used immunostaining of different components
of the ESCRT-III ring (CdvB, CdvB1, and CdvB2), to determine
where in the previously described division sequence [17] defects
in ring positioning first arise (Figure 4G). CdvB, the first compo-
nent of the ESCRT-III ring to assemble during division, was found
correctly positioned in the center of both MW001 and DcdvB2
cells (Figure 4H). Furthermore, with few exceptions, the same
was true for cells that co-stained for CdvB and CdvB1 (Fig-
ure 4G). However, following proteasome-mediated degradation
of CdvB [17], while CdvB1 was correctly positioned at the cell
center in the MW001 control, CdvB1 rings were found at variable
positions in DcdvB2 cells starting to divide (Figures 4G and 4H).
These data suggested that CdvB2 is recruited (along with
CdvB1) to the correct position at the cell center by the CdvB
ring and functions, following the loss of CdvB, to prevent the di-
vision ring from slipping as it constricts.
DISCUSSION
Here, we describe the development of the ‘‘Sulfoscope,’’ an im-
aging platform that makes it possible to image Sulfolobus cell di-
visions live. 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
(both S. acidocaldarius and S. solfataricus; Figures S2A and
S2B). This begins as replicated chromosomes lose their diffuse
organization as they separate and is followed by a relatively sud-
den DNA compaction, which coincides with the onset of furrow-
ing. Thus, DNA compaction may help to ensure that the segre-
gated chromosomes remain out of the way of the furrow as it
closes. These findings are in line with observations in fixed cells
of Sulfolobus [18] and are similar to DNA segregation behavior
observed in fixed cells of Halobacterium salinarium [19].
By combining molecular genetics and live-cell imaging, we were
also able to use the Sulfoscope to define homolog-specific roles
for the two ESCRT-III proteins (cdvB1 and cdvB2) that form part
of the contractile division ring [17]. Unlike cdvB, which is an essen-
tial gene [20], cdvB1 and cdvB2 deletion strains could be obtained
and their individual roles investigated using live-cell imaging. This
analysis revealed that, although CdvB1 and CdvB2 are co-local-
izedinthecontracting divisionring(Figure4H)and areclosehomo-
logs, they perform distinct functions. The loss of CdvB2 had the
most striking phenotype, which was characterized by a profound
failure in division symmetry. Remarkably, our analysis suggests
that the wide variance in the size of daughter DcdvB2 cells, which
included a proportion of ghost cells and daughter cells with twice
the normal DNA content (Figure 4C; Figure S3D), resulted from
slippage of the CdvB1 ring as it constricted (Figures 4D and 4G).
By contrast, although division failures were only occasionally
observed in the corresponding DcdvB1 mutant cells, these divi-
sions remained symmetric. These data show that, though it is
possible for the two proteins to form a co-polymer in the wild
type, as has been suggested to be the case for many of their eu-
karyotic ESCRT-III counterparts [21, 22], they likely perform
distinct but complementary functions in ring contraction. Our
data suggest that CdvB2 fixes the contracting ring in place
following the loss of the CdvB scaffold, perhaps by binding tightly
to the membrane, whereas CdvB1 may be required to aid force
generation to ensure that the process is fail-safe and may be
particularly important under stressful conditions, e.g., following
changes in temperature (which might alter the biophysics of the
membrane) or mechanical resistance. Strikingly, despite these dif-
ferences, our data also show that either one of the two contractile
ESCRT-III rings is sufficient to drive division in many cells.
Previous work provides support for the idea that CdvB1 and
CdvB2 play distinct functional roles in division. For example,
an earlier study found that the deletion of cdvB2 rendered cells
more slower growing than did a cdvB1 deletion [20]. Similarly,
in other studies, Sulfolobus islandicus strains with reduced levels
of the CdvB1 protein were found to be viable [23], as was a dele-
tion strain [8], whereas cdvB2 was found to be essential [8, 23].
Most notably, Liu and collaborators [23] observed distinct
morphological defects when overexpressing truncated versions
of CdvB1 and CdvB2 in S. islandicus. In this case, the truncated
versions of the CdvB2 version generated cells that the authors
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suggested were arrested in the final stages of abscission [23],
with bridges that resembled eukaryotic midbodies, whereas
the overexpression of truncated CdvB1 generated populations
of cells that connected cells to one another in chains. Although
some of the differences in the findings in these different studies
may reflect strain differences, it is clear from our analysis that
live-cell imaging greatly aids the mechanistic study of dynamic
processes like division.
Importantly, our findings have striking parallels in eukaryotes.
In
mammalian
cells,
for
example,
the
two
ESCRT-III
proteins CHMP2A and CHMP2B, which were thought to be
functional homologs, were recently found to contribute differ-
ently to membrane remodeling and to have different affinities
for the membrane [24]. Moreover, in in vitro studies, different
eukaryotic ESCRT-III proteins have been shown to work together
to increase membrane binding of the heteropolymer [25]. Our
Figure 4. The ESCRT-III Protein CdvB2 Ensures Division Symmetry in Sulfolobus acidocaldarius
(A) Asymmetric divisions, from moderate (I and II) to strong (III) asymmetries. Red arrowhead shows a very asymmetric cell division.
(B) Asymmetric division in which both daughters inherit one copy of the genome.
(C) Asymmetric division of a cell leading to the formation of a ghost cell.
(D) Daughter cell area ratios for different strains. A value of 1 represents a perfect symmetric division, whereas smaller values indicate increasing asymmetry. At
least three independent movies and at least 50 cells were evaluated for each strain (****p < 0.0001, n.s., not significant. statistical test: non-parametric Mann-
Whitney).
(E) Variation in the size of newly divided ethanol fixed cells as estimated by flow-cytometry. The plot shows robust coefficient of variance (rCV = st.dev/median) for
the ConA-alexa 647 signal, which marks the cell periphery (S-layer).
(F) Plots based upon an analysis using flow cytometry indicating variation in cell size for MW001, DcdvB2+pSvA (Induced), and DcdvB2+pSvA-CdvB2 (Induced).
Note that the expression of CdvB2 from a plasmid partially rescues the division phenotype. Newly divided cells have a 1N DNA content.
(G) Quantification of the position of ESCRT-III division rings in fixed MW001 and DcdvB2 cells that were immunolabelled for CdvB and CdvB1. A value of 1
represents the center of the cell. Ring positions (n = 50 to 60) were estimated for different stages in the division process [17] (****p < 0.0001, n.s., not significant.
statistical test: non-parametric Mann-Whitney).
(H) Immunolocalization of CdvB, CdvB1, and CdvB2 in MW001 and DcdvB2 cells. White arrowheads show asymmetric divisions.
Scale bars: 1 mm. Error bars show mean and SD. All live imaging shown in this figure was performed using immobilized cells. See also Figure S3 and Videos S4 and S5.
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data suggest that the same might apply in Sulfolobus, where two
different ESCRT-III homologs, CdvB1 and CdvB2, act coopera-
tively to ensure proper membrane binding and constriction—
preventing ring slippage or cytokinesis failure. We think this
work also makes the case for Sulfolobus being a simple, well-
defined system in which to study ESCRT-III-dependent division.
Finally, though we have used the Sulfoscope to reveal funda-
mental novel aspects of cell division, we anticipate that this type
of high-temperature live-imaging platform can be used to shed
light on other exciting areas of Sulfolobus cell biology, including
DNA remodeling [15, 26], swimming [27], conjugation [28, 29],
viral infection [23, 30], competition [31, 32], and cell-cell fusion
[33]. In addition, this system can now also be applied to the study
of other thermophilic microbes, from eukaryotes to bacteria
[34, 35]. Although we hope that our work can open new avenues
of research and lead further developments in the field of live im-
aging of thermophiles [36, 37], the further development of ther-
mophile cell biology will also depend on the establishment of a
thermostable fluorescent proteins [38, 39] and the use of micro-
fluidics, to allow for the tracking of cells over long periods across
multiple generations, as currently performed in Haloarchaea [40].
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead Contact
B Materials Availability
B Data and Code Availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
B Strain constructions and growth conditions
B Cap/stage construction and development
B Chamber measurements
B Live imaging methodology
B Imaging acquisitions
B Immunofluorescence labeling
B Immunofluorescence microscopy
B Flow cytometry
B Western blotting
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
cub.2020.05.021.
ACKNOWLEDGMENTS
The authors would like to thank the entire Baum lab for their input throughout
the project. We would like to thank Juan Manuel Garcia-Arcos for help early on
in this project; Tobias H€artel, Pedro Matos Pereira, and Alexandre Bisson for
discussions about fluorescent probes; Alexander Wagner for providing the
plasmid pSVAaraFX-stop; Alexandre Bisson and Jan Lo¨ we for comments on
the manuscript; and Mateusz Trylinski for helping with movies. A.A.P. was sup-
ported by the HFSP LT001027/2019 fellowship. D.R.M. was supported by the
BBSRC (BB/K009001/1). G.T.R. was supported by the MRC PhD studentship
award (MC_CF12266). G.D. was funded by a European Union Marie
Sklodowska-Curie Individual Fellowship (704281-CCDSA). U.S. was sup-
ported by the BMBF grant SYSBIO II (031L0044). In addition, A.A.P., S.C.,
and G.D. all received generous support from the Wellcome Trust (203276/Z/
16/Z). Research was supported by Wellcome Trust (203276/Z/16/Z) and
Volkswagen Foundation (Az 96727) with additional support for B.B. and R.H.
provided by UCL and the MRC (MC_CF12266).
AUTHOR CONTRIBUTIONS
A.A.P., D.R.M., G.D., R.H., and B.B. conceived the study. Microscope design
and construction and commercial heating stage testing was carried out by
A.A.P., S.C., G.D., D.R.M., and R.H. Design of the heating cap and heating
stage was carried out by A.A.P., J.R., C.R., and M.R. J.R. and M.R. con-
structed the heating cap and heating stage. A.A.P. and D.R.M. established
the imaging methodology and dyes used. Live imaging and imaging analysis
were performed by A.A.P. and D.R.M. with help from S.C. and U.S. Strains
were generated by A.A.P, M.v.W. and K.N.S. Immunofluorescence and west-
ern blots were performed by A.A.P. Flow cytometry and its analysis were per-
formed by G.T.R. Chamber measurements were performed by A.A.P. The bulk
of data analysis was carried out by A.A.P., and the figures and text were pre-
pared by A.A.P. and B.B., with input from all other authors.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: February 14, 2020
Revised: April 15, 2020
Accepted: May 6, 2020
Published: June 4, 2020
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Ensuring a Robust and Symmetric Division, Current Biology (2020), https://doi.org/10.1016/j.cub.2020.05.021
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Ensuring a Robust and Symmetric Division, Current Biology (2020), https://doi.org/10.1016/j.cub.2020.05.021
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
Rabbit Anti-CdvB
[17]
N/A
Chicken Anti-CdvB1
[17]
N/A
Guinea pig Anti-CdvB2
[17]
N/A
Mouse Anti-Alba
[17]
N/A
Goat anti-rabbit IRDye 680CW
LI-COR
Cat# 926-68021; RRID:AB_10706309
Donkey anti-chicken IRDye 800CW
LI-COR
Cat# 926-32218; RRID:AB_1850023
Donkey anti-guinea pig 800CW
LI-COR
Cat# 926-32411; RRID:AB_1850024
Goat anti- mouse IRDye 800CW
LI-COR
Cat# 926-32210; RRID:AB_621842
Goat anti-rabbit Alexa Fluor 546
Invitrogen
Cat# A11035; RRID:AB_143051
Goat anti-guinea pig Alexa Fluor 488
Invitrogen
Cat# A11073; RRID:AB_2534117
Goat anti-chicken Alexa Fluor 488
Invitrogen
Cat# A11039; RRID:AB_142924
Chemicals, Peptides, and Recombinant Proteins
SYBR safe
Thermo Fisher Scientific
Cat# S33102
Nile red
Sigma-Aldrich
Cat #72485
Concanavalin A Alexa Fluor 647 Conjugated
Thermo Fisher Scientific
Cat# C21421
DNase I
Thermo Fisher Scientific
Cat# EN0521
EDTA-free protease inhibitor cocktail
Roche
Cat# 11836170001
Hoechst
Invitrogen
Cat# H3570
Experimental Models: Organisms/Strains
S. solfataricus P2 (Wild type)
DSMZ
DSM1616
S. acidocaldarius DSM639 (Wild type)
DSMZ
DSM639
MW001 Uracil auxotrophic background strain DpyrE (Dbp 91–412),
background: S. acidocaldarius DSM639
[6]
N/A
DcdvB1 (Saci_0451) (background MW001)
This work
N/A
DcdvB2 (Saci_1416) (background MW001)
This work
N/A
DcdvB2+pSVA (DcdvB2 transformed with pSVA empty plasmid)
This work
N/A
DcdvB2+pSVA-CdvB2 (DcdvB2 transformed with pSVA containing
the wild-type gene of CdvB2, under an inducible promoter controlled
by Arabinose)
This work
N/A
Recombinant DNA
Plasmid pSVA431 (Backbone of deletion plasmid containing a pyrEF
and LacS cassette from S. solfataricus)
[6]
N/A
Plasmid pSVA1895
Used for deletion of cdvB1 (Saci_0451)
This work
N/A
Plasmid pSVA1896
Used for deletion of cdvB2 (Saci_1416)
This work
N/A
Plasmid pSVAaraFX-stop
Backbone of expression plasmid harboring an arabinose inducible
promoter containing a pyrEF cassette from S. solfataricus
[41]
N/A
Plasmid pSVAaraCdvB2 Used for expression of CdvB2 (Saci_1416)
This work
N/A
Software and Algorithms
Adobe Ilustrator
Adobe Inc.
https://www.adobe.com/products/
illustrator
Prism v.8
GraphPad
https://www.graphpad.com/scientific-
software/prism/
(Continued on next page)
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RESOURCE AVAILABILITY
Lead Contact
Further information and requests for resources, Sulfolobus strains and reagents should be directed to and will be fulfilled by the Lead
Contact, Buzz Baum (b.baum@ucl.ac.uk).
Materials Availability
All unique/stable reagents generated in this study are available from the Lead Contact without restriction.
Data and Code Availability
This study did not generate any unique dataset or code.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Sulfolobus acidocaldarius wild-type (DSM639), Saccharolobus solfataricus wild-type, and mutants (DcdvB2+pSVAaraFX-stop
(empty plasmid control) and DcdvB2+pSVA-CdvB2) were grown at 75�C in Brock Salts medium supplemented with 0.1% NZ-amine
and 0.2% Sucrose (BNS), final pH corrected to 3.5 with H2SO4. Uracil was added to the final concentration of 20 mg/mL when working
with uracil auxotrophic strains MW001, DcdvB1 and DcdvB2 strains. The density of liquid cell cultures was maintained at OD600nm
values between 0.05 and 0.3 as measured by a spectrophotometer.
METHOD DETAILS
Strain constructions and growth conditions
Deletion of genes was performed using the ‘‘pSVA431 method’’ as previously described by Wagner and collaborators [10]. For
CdvB2 overexpression, a synthetic gene containing restriction sites (NcoI and XhoI) flanking the cdvB2 gene (Saci_1416) was
used (Thermo Scientific�) and cloned into the pSVAaraFX-stop plasmid. For the DcdvB2+pSVAaraFX-stop and DcdvB2+pSVA-
CdvB2 rescue experiments, expression was induced by adding 0.15% w/v (final concentration) of L-arabinose to freshly diluted cul-
tures. Cells were grown overnight with L-arabinose and collected the next morning for live-imaging, immunofluorescence and flow
cytometry, at an OD600nm of 0.1 to 0.25. The experiment was repeated three times on independent days.
Cap/stage construction and development
The heating system was designed to be combined with the Attofluor� chamber. The metal elements in the cap and the heating el-
ements 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. Screws used are stainless steel 304. The insulating disk was built with carbon fiber. 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. The temperature used in the cap is a thermocouple type T; the one used for the chamber is a miniPT100. Detailed
information about the design of the chamber, controlling system and materials is available upon request.
Chamber measurements
For measurements of the temperature inside the chamber, a digital thermometer with a probe (Signstek 6802 II with a 2k type probe)
was used. The probe was inserted inside the chamber trough the sealable inlet (Figure 1) and positioned exactly at the middle of the
chamber, touching the glass. 500 mL of Ultrapure water was added to the chamber, since in our imaging conditions, liquid media is
always present. Readings were taken initially every 10 min (for the first 30 min) then followed up for 30 min. A more comprehensive
follow up was also performed in which the temperature was followed continuously for half an h for every tested temperature. We
never observed a temperature variation greater than 0.5�C during the duration of these measurements. For the evaporation assays,
500 mL of BNS media was added to the chamber, and the weight of the chamber was measured using an analytical scale. The pre-
heated system was then closed. After six hours, the heating system was turned off. After cooling back to room temperature, the Atto-
fluor chamber was removed and the weight was recorded again. The difference in the final weight calculated and considered to
reflect the loss of liquid due to evaporation.
Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
ImageJ (FiJi)
NIH
https://imagej.nih.gov/ij/
ImageJ plugin StackReg
[42]
http://bigwww.epfl.ch/thevenaz/stackreg
FlowJo v10.1
FlowJo
https://www.flowjo.com/solutions/flowjo
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Live imaging methodology
All the manipulation of cells and materials prior to live imaging at high temperature were conducted in a way to minimize temperature
loss during the transport from the incubators to the microscope. In order to achieve this, metal beads were placed inside a container
that was heated overnight at 75�C. Aliquots of the cultures (5mL to 10mL) were transferred to previously clean and heated flasks
placed inside the metal beads container, and then transported to the microscope. When done properly, cells suffer from minimal
heat stress, and cell division can be imaged immediately after the start of the image acquisitions. The commercial Attofluor chamber
and coverslips were washed thoroughly with BNS media, followed by a wash with ultrapure water before use. We noticed that dirty
coverslips can damage the membrane of Sulfolobus cells. The assembled chamber containing the clean coverslip needs to be pre-
heated before imaging starts. This can be achieved during the washing/drying steps.
For live imaging of S. acidocaldarius and S. solfataricus, Nile red (Sigma) was used at 2.5 mg/mL final concentration and SYBR safe
(Thermo Fisher Scientific), 10,000x concentrated was used at a final dilution of 1x to 0.5x. Cells were imaged at an OD600nm of 0.1 to
0.35. For imaging non-immobilized cells, 400ul of culture was added to the chamber. Cells were allowed to settle for 5 min before
commencing image acquisition. For imaging immobilized cells, we used heated semi-solid BNS pads (0.6% Gelrite, 50% non-pH
BNS, with 20mM final concentration of CaCl2). Pads were prepared by cutting circular (8mm) disks from a semi-solid BNS media
Petri dish, prepared as described above (25mL per plate). To facilitate handling, a 13mm circular coverslip was used to support
the pad after it was removed from the plate. The pads, now inside a closed Petri dish, were placed at an incubator at 75�C degrees
for 20 min prior to imaging and transported inside a container full of heated metal beads (as described above) to the microscope. After
loading 400 mL of the cell culture inside the chamber, the heated pads were placed on top of the cells (using forceps, holding the
13mm coverslip with the pad on top). The chamber was quickly closed and positioned for imaging. We observed that heating up
the pads prior to imaging not only prevented heat shock, but also slightly dried the pads. This causes the cells to accumulate at
the border of the pads once placed inside the chamber. Movies were therefore acquired within these regions where many cells
can be found that are only subject to moderate levels of confinement. Imaging immobilized cells, with DNA dye, membrane dye under
constant light exposure was limited to two hours, since the combined effect as found to be toxic over periods longer than this. Im-
aging with Nile Red only can be performed for longer periods.
Imaging acquisitions
Images were acquired using a Nikon Eclipse Ti-2 inverted microscope, equipped with a with a Prime 95B sCMOS camera (Photo-
metrics). A 60x air objective (Plan Apo 60x/0.95 objective, Nikon) combined with the 1.5x additional magnification from the Ti-2 mi-
croscope body was used, resulting in an effective magnification of 90x (Pixel size = 0.1235 mm). For membrane imaging, Nile red was
illuminated with 550nm LED illumination at 20% of maximum intensity, while for DNA imaging SYBR safe was illuminated with 470nm
LED at 25% of the maximum intensity (pE-4000 LED illuminator, CoolLED). Exposure time was limited to about 40 ms for each chan-
nel. Whenever DNA was imaged together with Nile red, images were acquired every 2 min. For measurement of division asymmetry
and failure division events, only Nile red was used, and images were acquired every 30 s to every 1 min. For correction of XY axis
drifting in the images, was used ImageJ plugin StackReg [42]. Focal drift was avoided by using the inbuilt Perfect Focus System.
Immunofluorescence labeling
Cells were fixed sequentially in ice-cold ethanol until reaching the final concentration of 70%, starting at 30%. For the analysis, cells
were centrifuged for 3min at 8,000xg and resuspended in PBSTA (PBS + 0.2% Tween20 + 1% BSA) for 5 min for rehydration. After
rehydration, cells were incubated with 100 mL of primary antibodies diluted in PBSTA for 2 h, at room temperature and in a small ther-
momixer at 500rpm. Cells were washed twice in PBSTA and them incubated in 100ml of secondary antibodies diluted in PBSTA, with
200 mg/mL of Concavalin A conjugated with 647 Alexa Fluor for 1 h, 500rpm, at room temperature. Cells were washed twice again,
resuspended in PBSTA and 1 mg/mL of Hoechst to visualize DNA.
Immunofluorescence microscopy
Microscopy slides were prepared by coating LabTek chambered slides with 2% polyethyleneimine (PEI) for 30 min at 30�C. Slides
were then washed with distilled water and 200 mL of stained cell suspension was added and spun for 30 min at 500 - 1000 RCF in a
swing-bucket rotor. Images were captured for 50ms to 100ms using a Nikon Ti2, 1.39NA 100x Objective, and Photometrics Prime
95B SCMOS camera.
Flow cytometry
Flow cytometry analysis was carried out on BD LSR II and BD Fortessa with immunostained cells going through the following lasers
and filters. Lasers: 355 nm, 488 nm, 561 nm, 633 nm Filters: 450/50UV, 525/50 Blue, 582/15 YG, 474 710/50 Red. Side Scatter and
Forward scatter was also recorded. Analysis were performed using FlowJo software v10.
Western blotting
S. acidocaldarius pellets were lysed in 100-250 mL lysis buffer (TK150 buffer, supplemented with DNase I, and EDTA-free protease
inhibitor cocktail, 0.1% Triton X-100). Cells were disrupted in a sonicator (Diagenode) and subsequently centrifuged at 4�C (10,000 3
g for 15 min). Supernatant was collected and protein concentration was measured using Bradford reagent (Bio-Rad). About 8-15 mg
of total protein extract was resolved on NuPAGE 4%–12% Bis-Tris gels (Invitrogen) using MES SDS running buffer (Invitrogen).
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Proteins were transferred to a 0.45 mm nitrocellulose membrane. Membranes were blocked with PBST (PBS, 0.2% Tween20) with 5%
non-fat milk. Primary and secondary antibodies were diluted in this same buffer. Membranes were incubated with primary antibodies
overnight at 4�C. Primary antibodies anti-CdvB1, anti-CdvB2 and anti-Alba were used as described in Risa, 2019. Next, the mem-
branes were washed with PBST and incubated with Li-COR IRDye 680CW or 800CW secondary antibodies diluted 1:10000 for 1 h at
room temperature. Finally, membranes were washed with PBST and developed using Li-COR Odyssey Infrared Imaging System.
Images were analyzed on ImageJ.
QUANTIFICATION AND STATISTICAL ANALYSIS
All statistical tests were conducted using GraphPad Prism. Statistical test used: non-parametric Mann–Whitney. Changes in DNA
organization that accompany division in immobilized cells were estimated by using 50 cells for the immobilized treatment and 20 cells
for non-immobilized cells. For quantification of division failure (75�C), five independent movies were used to evaluate the frequency of
division failure from 130 to 180 divisions per strain, in total. For comparison of cell division speeds and failure rates at 65�C, cells from
two independent movies were used. Division time was estimated as the duration from the start of constriction until the two cells were
physically separated and between 42 to 66 events were quantified for each strain, in total. For the asymmetry measurements, cells
area were measured using Fiji (ImageJ). At least three independent movies and the area of at least 50 cells divisions were evaluated
for each strain. For comparison of the impact of different treatments on cell division, cells were segmented using StarDist [43] and
division events were detected using Trackmate [44] on these segmentation results. By fitting a sigmoidal curve to the length of the
short axis of the cell over time, the period of time corresponding to division-associated morphological changes could be defined as
the time between the two inflection points of the fitted sigmoid function (Figure S1E, shaded gray region on plots), using between 15
to 20 cells per treatment. Ring positions (n = 50 to 60 rings) were estimated using ImageJ, for different stages in the division process.
Quantification of number of division rings per 1000 cells in MW001 and DcdvB1 at 65�C was performed manually. At least 3000 cells
for each strain was analyzed in total (at least 1000 from each independent triplicate) and the number of rings quantified. For correction
of XY axis drifting in the images, was used ImageJ plugin StackReg [42]. Focal drift was avoided by using the inbuilt Perfect Focus
System.
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Current Biology 30, 1–8.e1–e4, July 20, 2020
Please cite this article in press as: Pulschen et al., Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in
Ensuring a Robust and Symmetric Division, Current Biology (2020), https://doi.org/10.1016/j.cub.2020.05.021
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