3-D Ultrastructure of O.

tauri: Electron Cryotomography

of an Entire Eukaryotic Cell
Gregory P. Henderson, Lu Gan, Grant J. Jensen*

Division of Biology, California Institute of Technology, Pasadena, California, United States of America

The hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles.
Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light
microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging
technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state.
Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is
the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at
various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula,
Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to
open long before mitosis, and while one microtubule (or two in some predivisional cells) was consistently present, no mitotic
spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple
chromosomes.
Citation: Henderson GP, Gan L, Jensen GJ (2007) 3-D Ultrastructure of O. tauri: Electron Cryotomography of an Entire Eukaryotic Cell. PLoS ONE 2(8):
e749. doi:10.1371/journal.pone.0000749

INTRODUCTION taken an alternative approach by identifying and imaging the

The history of cell biology has been punctuated by major advances smallest known eukaryotic cell, Ostreococcus tauri.
in imaging technologies. Following the invention of the electron O. tauri is a unicellular green alga (a ‘‘picoplankton’’) with only
microscope in the early 1930s, what we would now call the a single mitochondrion, chloroplast, and Golgi body [12]. It has been
‘‘conventional’’ specimen preparation methods of chemical reported to be less than a micron in diameter, and because of its
fixation, dehydration, plastic-embedding, sectioning, and staining small size and presumed simplicity, it has been put forward as
were developed to allow the visualization of biological material. a model eukaryotic organism [13]. Nevertheless when O. tauri’s 12.6
While producing some artifacts, these methods have been megabase nuclear genome was sequenced [13], it was found to have
profoundly successful, filling our textbooks with information about more genes (,8,200) than Saccharomyces cerevisiae (,6,600). These
the structure and positions of cell walls, internal membranes, genes are distributed on 20 linear chromosomes. The Prasinophy-
cytoskeletal filaments, and even large cytoplasmic particles like ceae lineage to which O. tauri belongs represents an early branch
ribosomes. High-pressure freezing/freeze substitution fixation from the green lineage that includes the land plants [14,15]. Unlike
techniques have since improved specimen preservation [1]. other model plants O. tauri has only one copy of each class of cyclin-
Recent technological developments have created the opportunity dependent kinases and cyclins [16], making it a powerful system to
to realize two major improvements: imaging cells in three study the cell cycle without concerns of genetic redundancy. O. tauri’s
dimensions (3-D) and in nearly-native states. Cells can be imaged cell cycle can be partially synchronized by growing the cells in
in 3-D through tomography [2], a technique wherein specimens are a twelve hour light-and-dark cycle and further synchronization can
imaged iteratively while being incrementally tilted around one or two be achieved pharmacologically [17]. Because of its small size, it may
axes. Full 3-D reconstructions of the sample can then be calculated. be the only eukaryotic cell known that can be effectively imaged
Computationally stacking such 3-D reconstructions of serial (thick) intact throughout its entire life cycle by ECT.
sections has made it possible to reconstruct even large regions of
fixed cells [3]. With conventional sample preparation methods,
however, image contrast arises from heavy metal salts surrounding Academic Editor: Paul Digard, University of Cambridge, United Kingdom
cross-linked molecules held in place by a resin support. It has now
Received May 21, 2007; Accepted July 16, 2007; Published August 15, 2007
also become possible to image non-chemically-fixed cells in a more
nearly native state through plunge-freezing. Plunge-freezing pre- Copyright: ß 2007 Henderson et al. This is an open-access article distributed
serves cells in a life-like, ‘‘frozen-hydrated’’ state free of stains and under the terms of the Creative Commons Attribution License, which permits
with minimal artifacts [4]. Electron cryotomography (ECT) unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
combines these two advances to produce 3-D reconstructions of
nearly-native biological material to ‘‘molecular’’ resolution [5]. Funding: This work was supported in part by NIH grants R01 AI067548 and PO1
Because of multiple scattering, however, images of samples GM66521 and DOE grant DE-FG02-04ER63785 to GJJ, a Searle Scholar Award to
GJJ, the Beckman Institute at Caltech, and gifts to Caltech from the Gordon and
thicker than about 500 nm are difficult to interpret. Thus previous Betty Moore Foundation and Agouron Institute. Lu Gan was supported by
ECT work has focused on purified macromolecules, viruses, small a fellowship from the Damon Runyon Cancer Research Foundation (DRG-1940-
prokaryotic cells [6], purified organelles [7], or cell peripheries 07). These sponsors/funders had no role in the design and conduct of the study,
in the collection, analysis, and interpretation of the data, and in the preparation,
[8,9], but the potential insight that might come from examining review, or approval of the manuscript.
whole eukaryotic cells by ECT has not yet been realized because
they are too thick. To this end methods are being developed to Competing Interests: The authors have declared that no competing interests
exist.
either cryosection frozen-hydrated tissues [10] or focused-ion-
beam mill thin slabs [11] suitable for tomography. Here we have * To whom correspondence should be addressed. E-mail: [email protected]

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 3-D Ultrastructure of O. tauri

Here we present ECT reconstructions of intact O. tauri cells frozen they had visibly dividing organelles, but some were chosen almost
at different stages throughout the cell cycle. In addition to providing randomly to broaden coverage of the cell cycle. The reconstructed
new insights into the ultrastructure of chloroplasts, mitochondria, cells were classified into two groups, ‘‘non-dividing’’ and
endoplasmic reticula (ER), Golgi bodies, peroxisomes, microtubules, ‘‘predivisional,’’ based on whether or not their organelles were
and the distribution of large macromolecular complexes, O. tauri visibly dividing (Figure 2 and in particular see Video S1, which
presented a number of surprises including a nuclear envelope (NE) presents a complete 3-D tour of one representative cell and all its
that was open throughout most of the cell cycle, an unusually shaped organelles). Of the 23 reconstructed cells taken from the dark-to-
ER, and a rough ER that was confined to the outer nuclear light transition, all but one were non-dividing, consistent with their
membrane. Perhaps most intriguingly, only one or two microtubules being in G1 as predicted by Farinas et al, 2006. Of the 29
were ever observed in individual cells, suggesting that single reconstructed cells taken from the light-to-dark transition, 4 were
microtubules might be sufficient to segregate multiple chromosomes. non-dividing and 25 were predivisional.
The Results and Discussion are presented one after another together
for each topic below to improve the flow of the text, but a Conclusion
Cell shape and content
has been added to place the whole work in context.
While the limited tilt range decreased the resolution perpendicular
to the grid, it was nevertheless clear that the frozen-hydrated cells
RESULTS AND DISCUSSION were not spherical. Instead they were rather flattened, ranging
Cell Growth and Synchronization from 1.1 to 2.1 mm in diameter and 0.5 to 0.8 mm in thickness.
Cells were obtained from a strain of O. tauri isolated from the Thau The more flattened surfaces are referred to herein as the ‘‘top’’
lagoon (France) [18]. O. tauri’s growth was synchronized using and ‘‘bottom’’ of the cells, depending on their orientation on the
a twelve-hour-light, twelve-hour-dark cycle that resulted in an grid, but there were no consistent structural features that indicated
average of one cell division per day. A previous study found that any functional distinction. The diameters and flatness observed
under these growth conditions, cells at the dark-to-light transition here were consistent with earlier reports [12], but to check
were in the mid-G1 phase of the cell cycle. Cells at the light-to- whether surface tension flattened the cells beyond their normal
dark transition were found in various stages of the cell cycle state, cell shapes were also investigated by phase and differential
including approximately 50% in early G1, 10% in late G1, 25% in interference contrast light microscopy of free-floating live cells.
S, and 15% in G2/M [17]. Free-floating cells also had flat profiles, but exhibited more
complex morphologies with bends and depressions as if they were
Sample Preservation Strategies quite flexible (Figure 3). Thus while the flattened morphology is
Cells from liquid cultures were prepared for EM using three natural, suspending the cells in thin fluid across circular holes on
different preparation methods: conventional fixation and embed- an EM grid likely promoted a more round and simple shape.
ding, high-pressure freezing/freeze-substitution fixation, and All cells imaged by ECT contained a single mitochondrion,
plunge-freezing (Figure 1). Conventional EM fixation revealed Golgi body, and chloroplast. Each cell also contained a nucleus
most membranes, but the membrane structures were badly with one to three discernable nuclear pore complexes (NPCs),
distorted and any further details were difficult to resolve and/or a smooth ER, and a variety of vesicles that could not be identified.
poorly reproducible. High-pressure frozen specimens had mark- The non-dividing cells had a single peroxisome and microtubule,
edly improved membrane preservation, especially in the chloro- but some predivisional cells had two copies of each. The rough ER
plast, but macromolecular complexes were not resolved. Further, was limited only to the outer NE. One unusual cell had an oval
because of stain and the generally high-density of the plastic- shape with two terminal membrane-bound cell extensions
embedded samples, sections thick enough to contain entire cells (Figure 2E–F). This cell was thinner than the others, had the long
could not be imaged. Thus reconstructing an entire cell would axis of the chloroplast turned ninety degrees relative to the long
have required multiple serial sections. Plunge-frozen cells were axis of the nucleus, and had giant holes in the NE.
imageable intact and exhibited good ultrastructural preservation
including readily resolvable ribosome-like particles, microtubules, Cellular Organization
and smooth cellular membranes. Moreover, plunge-freezing was The organization of the non-dividing cells was remarkably
both more consistently successful and significantly faster than high- consistent. The largest organelles – the chloroplast and nucleus –
pressure freezing, allowing a large number of whole cells to be resided in opposing semi-circular halves of the cell. The other
imaged and a more comprehensive survey of O. tauri ultrastructure organelles (mitochondrion, peroxisome, ER, Golgi body, vesicles)
to be obtained. The plunge-frozen cells were, however, at the limit rested between them (Figure 4). The nucleus and chloroplast pressed
of useful thickness for ECT (500–800 nm), so data could not be so closely to the plasma membrane that macromolecular complexes
collected at tilt angles greater than ,55–60u and the resolution of like ribosomes were sterically excluded. While the mitochondrion did
the reconstructions was correspondingly anisotropic. not appear to have a fixed location and the chloroplast rested
opposite the nucleus, the nuclear envelope (NE) appeared to
Data Recorded organize the rest of the organelles. The outer nuclear membrane
Cultures at both the dark-to-light and light-to-dark transitions was contiguous with sheets of ER that were found to run parallel to
were plunge-frozen. Samples were first surveyed in the EM to the plasma membrane and around co-localized granules. The Golgi
investigate the cell sizes and morphologies present. Cells were body was always found close to the nucleus. A single microtubule
almost always round, and were seen to vary in diameter could be found in contact with the NE, near the top or bottom of the
continuously from 1 to 2 m on every grid, but cells were generally cell. The peroxisome, a large single-membrane vesicle known to be
larger at the light-to-dark transition. In many of the cells frozen at present in all eukaryotic cells, occupied the central region of the cell
the light-to-dark transition, it was possible to see in the surveys that between the nucleus and the chloroplast (discussed later).
the organelles were dividing. Full tilt-series of 52 cells were then Six of the best reconstructed cells were used to estimate the
recorded. Most of these were chosen because they were especially volumes and surface areas of key organelles. These six (shown in
thin, which results in higher resolution reconstructions, or because Figure 4) were chosen because they were among the thinnest cells

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 3-D Ultrastructure of O. tauri

Figure 1. Preservation of O. tauri. (A) Projection image through a conventionally fixed, embedded, thin-sectioned cell. (B) Projection image through
a high-pressure frozen, low-temperature embedded, thin-sectioned cell. (C) 28.8-nm thick slice through the 3-D electron cryotomographic
reconstruction of an intact, frozen-hydrated cell. The letters identify nuclei (n), chloroplasts (c), mitochondria (m), Golgi bodies (g), the Endoplasmic
Reticulum (er), and granules (gr). Scale bar = 200 nm.
doi:10.1371/journal.pone.0000749.g001

and because they were imaged along two orthogonal axes, perpendicular to the boundary between the nucleus and
a practice that minimizes the effects of the missing wedge of data chloroplast, setting up an apparent roughly two-fold-symmetric
in ECT [19]. All six came from the dark-to-light transition. These division plane (Figure 2C–D, 6C, 6D, and 7C). In most of the
presumably underwent cell division at the beginning of the 12- predivisional cells (62%), there was a space between the
hour dark period, entered G1, and then grew minimally due to chloroplast and the plasma membrane filled with either ribo-
lack of light. The average cell volume and surface area was some-like complexes or the mitochondria (Figure 5A), as if the
0.9160.07 mm3 and 8.3 mm2, respectively. The chloroplasts, forces positioning the chloroplast immediately next to the
nuclei, and mitochondria were the largest three organelles, membrane in G1 were weakened. (2) The nucleus was no longer
occupying 4763, 1762, and 2.760.4% of the cell volume, oval. In some cases the nucleus was wedge-shaped, protruding into
respectively. The cytoplasm around the organelles occupied the middle of the dividing chloroplast (Figure 2C–D). In twelve
another 2962% of the cell and granules and other vesicles other cases (46%) the NE formed extensions that streamed above/
occupied the remainder of the volume. In these thinnest of cells, below the chloroplast, sometimes accompanied by the Golgi body
the distribution of organellar volumes was remarkably consistent. or microtubule (Figure 5A–B). (3) Whereas in G1 the mitochon-
The predivisional class of cells was distinguished by four drion appeared to be randomly positioned, in dividing cells at least
ultrastructural differences. (1) The most obvious difference was the part of it consistently crossed the apparent division plane. In some
partial division of the chloroplast along a line approximately cases it seemed to be straddling the division plane (Figure 2C–D),

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 3-D Ultrastructure of O. tauri

Figure 2. Cross-sections and 3-D segmentations of O. tauri cells. Each row shows a single slice (A 21.6-nm; B 36-nm; C 9.6-nm) through the middle
(left) and a manually segmented model (two perpendicular views, right) of one particular cell: (A, B) a non-dividing cell harvested at the dark-to-light
transition; (C, D) a dividing cell harvested at the light-to-dark transition; (E, F) an unusual cell with a different internal organization and two membrane-
bound cell extensions. Only the central portion of the cell in (F) is shown due to the limited ability to completely segment thicker cells. Here and below
the letters and colors identify nuclei (n, red), nuclear envelope (ne), chloroplasts (c, green), mitochondria (m, dark purple), Golgi bodies (g, yellow),
peroxisomes (p, orange), granules (gr, dark blue), inner membranes including ER (er, light blue), microtubules (light purple), and ribosome-like particles
(r). Because the dividing cell shown in panels C and D was especially thick, only its middle region was segmentable. Scale bar 250 nm.
doi:10.1371/journal.pone.0000749.g002

in others it was elongated as if at least one edge were being drawn been present at the top and bottom of the chloroplast could not be
to the division plane (Figure 5C). (4) The oval storage granules resolved. In the midsection, grana composed of three disks (six
lined up in a symmetric ‘‘V’’ shape across the division plane membranes) hugged the inner chloroplast membrane and some-
pointing towards the chloroplast with the tip of the ‘‘V’’ located at times extended into the middle of the chloroplast as broad sheets
the cell’s center (Figure 6D). without connecting stroma-like thylakoid structures (Figure 6A–B).
Because the lumens of the two outer disks (about 7 nm across) were
consistently wider than that of the central disk, the grana had
Chloroplasts a sandwich-like appearance: the ‘‘top’’ and ‘‘bottom’’ membranes of
The chloroplast was the largest organelle and since it was also one of the stack stood apart from the inner four, which were packed so
the most dense, its internal structure was the most difficult to resolve. tightly that they were often hard to distinguish. The grana
Due to the missing wedge artifact, thylakoid grana that may have thylakoids terminated at the poles of the long axis of the chloroplast

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 3-D Ultrastructure of O. tauri

Figure 3. Light microscope images of free-floating O. tauri. Sequential

DIC images of five representative cells (1–5) tumbling freely in sea water
culture medium after six hours growth in light are shown from left to
right, demonstrating their disk-like shape.
doi:10.1371/journal.pone.0000749.g003

(Figure 6A–B), but the connectivity of the individual membranes

and the details of the presumed organizing scaffold were unclear.
No connections or vesicles between the grana thylakoids and the
inner chloroplast membrane were seen.
The chloroplasts contained two distinct classes of granules. Each
chloroplast had multiple dark and very nearly spherical granules
(Figure 6, white arrowheads) and a single larger granule that Figure 5. Cells with dividing organelles. (A) Segmentation showing
a portion of a NE streaming over the top of a chloroplast. Here the
chloroplast is separated from the plasma membrane by the mitochon-
drion and several granules. (B) 36-nm thick slice near the top of the
reconstruction of the same cell. The NE (white arrows) stretches towards
the Golgi body (black arrowheads) and microtubule (black arrows). (C)
24-nm slice through a different cell, showing an elongated mitochon-
drion near the center of the cell and duplicating chloroplast granules.
Scale bar = 150 nm.
doi:10.1371/journal.pone.0000749.g005

manifested electron beam damage artifacts (bubbling) much earlier

than the rest of the cell (Figure 6, white arrows). Radiation-
sensitive granules have also been reported in the gram-negative
bacterium Caulobacter crescentus and were found to be carbon-rich
[20]. The ‘‘bubbled’’ granule was seen stretched across the division
plane in dividing chloroplasts, as expected for the starch granule,
which was previously shown to exist as one copy that grew and
divided [21]. The onset of bubbling in the granule occurred at
,100 electrons/Å2, since in the cases of those cells where dual-
axis tilt-series were recorded, reconstructions obtained from just
the first half of the data (the first tilt ,80 electrons/Å2) did not
show bubbling. (The higher total doses were used, however,
because they led to reconstructions with more interpretable detail,
indicating that the perturbations to the rest of the cellular
ultrastructure caused by bubbling in the presumed starch granule
were minor in comparison to the advantages of higher dose.) The
chloroplasts divided along the apparent cellular division plane
(Figure 6C–D, 7C). As the thylakoids terminated at poles
(Figure 6B) away from the division plane, the division would
result in one old pole and one new pole for each half of the
chloroplast. Each half of dividing chloroplasts contained a portion
of the presumed starch granule and at least one dark granule. No
completely divided chloroplasts were observed, suggesting that cell
division quickly followed the termination of chloroplast division.
In agreement with the likely evolutionary relationship between
Figure 4. 3-D segmentations of six cells imaged at the dark-to-light
transition (mid G1). The cell in panel F is the same as in Figure 1A–B chloroplasts and cyanobacteria, the ultrastructure of the O. tauri
and 12. For scale, the diameter of the cell in panel D is approximately chloroplasts was reminiscent of cyanobacteria. Both thylakoid
1750 nm in diameter. systems are comprised almost exclusively of grana [22,23],
doi:10.1371/journal.pone.0000749.g004 positioned here near the inner chloroplast membrane and near the

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 3-D Ultrastructure of O. tauri

Figure 6. Chloroplast. (A) 7.2-nm thick slice through a non-dividing cell. The starch granule (white arrow) has suffered damage from the electron
beam. Besides it are two dark granules (white arrowhead). (B) Above: Enlarged view of the boxed area in panel A. The three thylakoid membranes
(black arrowhead) can be seen forming a stack. Below: Schematic of above. Cell membrane (black), chloroplast membranes (green), outer thylakoid
membrane (red), inner thylakoid membranes (blue). (C) 24-nm slice through an early predivisional cell, where the chloroplast is kidney-shaped rather
than oval and the starch granule is elongated. (D) 36-nm thick slice through a late predivisional cell, where the chloroplast is deeply constricted and
one dark and one starch granule is found in each side. Here the cytoplasmic granules (*) are arranged in a V-shape pointing to the division plane.
Scale bar 250 nm.
doi:10.1371/journal.pone.0000749.g006

analogous plasma membrane in cyanobacteria. The chloroplasts also almost always open, and other mitotic events differ substantially
appeared to replicate like cyanobacteria, since their thylakoids were from traditional models.
present before division and must have been pinched off at the Throughout the cell cycle the NE had gaps hundreds of
nascent poles. In contrast, higher plant chloroplasts mature from nanometers in diameter (Figure 7B, arrows). In fact only one cell
progenitor proplastids that lack differentiated internal membranes. was observed with an apparently closed NE (Figure 7A). This small
Traditional models depict the lumens of thylakoid disks as cell was taken from a culture at the light-to-dark transition, and its
discreet from the lumen between the chloroplast’s inner and outer non-dividing organelles implied that it must have just divided and
membranes [24]. A recent tomogram of a higher-plant chloroplast was in early G1. This cell could have had gaps in the top and
supports this traditional ‘‘detached’’ thylakoids model [25]. Van de bottom of the NE, but we could not verify this due to the effects of
Meene et al. showed a cyanobacteria’s thylakoid contacting the the missing wedge. Other cells imaged that were also presumably
inner plasma membrane, however, but the nature of the in early G1 were found to have an open NE. The openings in the
connection could not be determined [22]. Here O. tauri thylakoids NE consistently faced the cytoplasmic space between the nucleus
appeared detached, but unfortunately the complete chloroplast and the chloroplast; the regions facing the plasma membrane were
could not be clearly observed, transient events might not have generally closed. In certain rare cases, the nucleus was almost
been captured, and the details of the presumed organizing completely unbounded by NE (Figure 7C and 2E). In these cells
scaffolding could not be resolved. No transport mechanisms for large fragments of NE were seen along the borders of
bringing required lipids to a growing thylakoid, such as vesicles a recognizably dense nucleoplasm, implying that the nucleoplasm
between the inner membrane and thylakoid stacks, were detected. is gel-like and able to retain its unique composition.
The holes in the NE are unlikely to be an artifact of sample
Nuclei preservation; four lines of evidence support the authenticity of
Higher eukaryotes undergo open mitosis, resulting in the partial or these holes. First, no other cell membranes showed similar gaps.
complete breakdown of the NE. The onset of mitosis is marked by Second, nuclei have been previously isolated from cells and
NE breakdown, NPC disassembly, and mitotic spindle formation. imaged by ECT, without disruption of the membrane [27]. Third,
The NE reforms on the chromosomes during metaphase and the locations of the holes seen here were consistent, which suggests
completely closes after telophase [26]. In contrast, model lower they form in a regulated manner. Finally, some cells simply did
eukaryotes like yeast undergo closed mitosis, in which the NE have not enough NE to cover the entire nucleus (Figure 2E),
remains intact. Our reconstructions reveal that O. tauri’s NEs are arguing against an acute change.

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 3-D Ultrastructure of O. tauri

Previously published EM images of O. tauri do not suggest the

existence of gaps in the NE [17]. With conventional EM, however,
in some cases membranes can be severely distorted and each
image generally shows just a thin section through the nucleus
(Figure 1). Because ECT offers 3-D views of near-native structures,
the gaps were quite clear.

Nuclear Pore Complexes

NPCs within the NE were identified as ,80 nm diameter rings of
high electron density bordered by a region of the NE where the
outer and inner membranes merged (Figure 8). Each cell had one
to three visible NPCs, but there may have been additional NPCs
within the top or bottom faces of the nuclei that were not
recognized because of the missing wedge. As mentioned earlier,
the nuclei were immediately adjacent to the plasma membrane,
but the NPCs were never found facing that membrane. Instead,
the NPCs were found in regions of the NE exposed to the cell’s
interior, but the mechanisms localizing the NPCs remain unclear.
In the four best resolved NPCs, the average ‘‘outer’’ diameter of
the rings (where the outer and inner bilayers of the NE could no
longer be distinguished, Figure 8, solid lines) was 8164 nm. The
average ‘‘inner’’ diameter of the high density ring (Figure 8,
dashed lines) was 4868 nm; the greater variance being due
perhaps to the uncertainty in locating the inner boundary. Other
internal densities were seen which may represent cargo, ‘‘trans-
porters,’’ or luminal spoke rings [28].
While these may be the first measurements of NPCs in-vivo, or of
plant or algal NPCs in any state, the O. tauri NPCs observed here
Figure 7. Nuclear envelope. (A) 41-nm thick slice through a cell
harvested at the light-to-dark transition with a completely closed NE
were smaller (81 nm) than those from Dictyostelium (125 nm),
(black arrow). The cell’s small size and non-dividing organelles suggest Xenopus (100 nm), or Saccharomyces (100 nm) [27,29,30]. Unfortu-
the cell could have recently divided. Two NPCs (black arrowheads) are nately the resolution was insufficient to detect the symmetry, as
present. Close ups of the NPCs are shown at Figure 8. (B) 31-nm thick was done for isolated Dictyostelium nuclei [27], so it is unclear
slice through a cell harvested at the dark-to-light transition. The NE only whether the smaller size results from smaller or fewer constituent
covers about three-fourths of the nucleus in this section (black arrows proteins. Compared to the Dictyostelium NE (the only other intact
mark the tips). Again two NPCs (black arrowheads) are present, and at
the bottom of the nucleus the ER branches off the NE (white nucleus imaged by cryotomography), which has such a high
arrowheads). (C) 19.2-nm thick slice through a large, late predivisional concentration of NPCs (,45/mm2) that they exhibit local
cell harvested at the light-to-dark transition exhibiting an almost hexagonal packing [27,30], O. tauri NE had a much lower NPC
completely open nucleus with only small patches of NE (black arrows). concentration (,1/mm2).
Scale bar 100 nm.
doi:10.1371/journal.pone.0000749.g007
Mitochondria
Each cell had a single mitochondrion. The mitochondria were
Near the plasma membrane, the outer and inner NE generally oval (Figure 9), but were frequently deformed when
membranes were too close to be resolved. Elsewhere, these
membranes were spaced between 18 and 100 nm apart. NPCs
were present (discussed below). As noted previous, in 42% of the
predivisional cells, protrusions of the nucleus streamed over the
top of the chloroplast (Figure 5A–B). No internal structures (like
heterochromatin, nuclear lamina, or condensed chromosomes)
were recognized.
Many higher eukaryotes undergo NE breakdown during
mitosis, so the fact that a cell can have a NE with large gaps is
reasonable. Nevertheless O. tauri’s NE appeared to be open during
most of the cell cycle (from the beginning to the end of the light
cycle). This is significant because presumably, the nucleus must be
closed in order to establish the Ran nucleo-cytoplasmic gradient,
which shuttles cargo in and out of the nucleus via the NPCs. The Figure 8. Nuclear pore complex. 3.6-nm thick slices through the best-
NE had such large gaps that the role of the NPCs is unclear, resolved NPC from the ‘‘side’’ (perpendicular to the NE, left) and ‘‘top’’
although they remained embedded in the NE fragments. Perhaps (in plane of NE, right). The approximate center, inner, and outer
certain molecules like mRNAs require regulated transport and so diameters are marked by the arrow, dashed, and solid lines,
respectively. The region around the NPC in the right panel is low
are specifically targeted to the NPC. Despite being open, many of
density (whiter) because it is inside the NE lumen; the dark crescent
the nuclei must have been transcriptionally and translationally near the edge of the panel shows where the plane of the slice cuts
active since the cells were in the exponential growth phase and through the NE. Scale bar = 100 nm.
ribosomes consistently decorated parts of the NE. doi:10.1371/journal.pone.0000749.g008

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Figure 9. Mitochondrion. (A) 3-D segmentation of the mitochondrion from the cell shown in Figure 1E–F. Here, the membranes are colored purple
(outer membrane), red (inner membrane), and yellow (cristae). Dense granules within the mitochondrial matrix are shown in green. (B–D) 15-, 55-,
and 29-nm thick slices though three different mitochondria, showing crista junctions (arrowheads) and a dense granule (white arrow). Panel B is a slice
through the cell shown in panel A. Scale bar 50 nm (for panels B–D). (E) 4.8-nm thick slice through a junction or channel (black arrows) connecting
the outer and inner membranes. Here c-cytoplasm, m-mitochondrion. Scale bar 50 nm.
doi:10.1371/journal.pone.0000749.g009

situated adjacent to other organelles. In some dividing cells the consisted of sheets that were found adjacent to the plasma
mitochondria were elongated (Figure 5C) or constricted in their membrane and at the four regions that projected extra-cellular
center (Figure 2C–D). The inner and outer membranes were filaments, as discussed later.
smooth rather than corrugated as sometimes observed by Current descriptions of ER ultrastructure assert the notions that
traditional EM [31]. While the spacing between the inner and (1) the lumens of different ER sheets are interconnected with each
outer membrane was generally ,12 nm (Figure 9C–D), it was other and with the NE lumen, and (2) rough ER forms sheets and
sometimes significantly greater (top of Figure 9B), or so small that all other ER (smooth ER) is tubular [36,37]. O. tauri’s ER differed
the inner and outer membranes appeared to touch (Figure 9E). in both these characteristics. While some ER tubes and sheets
The mitochondria positioned near the thinner edges of cells were appeared to be contiguous with the NE, others had no observable
the best resolved and exhibited both lamellar and tubular cristae, connection. Rapid fission and fusion of membranes can make
typically with one or two junctions with the inner membrane compartments functionally interconnected, like the Golgi body’s
(arrowheads in Figure 9B–D). These crista junctions were circular many cisternae, but our ‘‘snapshots’’ of flash-frozen cells suggest
or slot-shaped and ranged in size from 15–55 nm (Video S1). The that some ER compartments are physically isolated at least part of
spacing between the two membranes of the cristae was also 15 nm the time. Less likely but still possibly, connections might have
or greater. In some mitochondria (40%), multiple (4–10) dark existed but were not observed due to the missing wedge artifact or
granules with complex shapes were observed in the matrix limits in resolution. Surprisingly, while clusters of ribosome-like
(Figure 9A–B). particles were found on the cytoplasmic face of the outer nuclear
Electron tomographic reconstructions of mitochondria have membrane, no ribosome-decorated ER (rough ER) could be
already discredited the baffle model of membrane folding, where found. This contradicts earlier speculations that ribosomes are
cristae were proposed to fold in much like the bellows of an necessary to maintain the ER’s flat shape [37,38].
accordion. Instead, both isolated and in-situ mitochondria have
been shown to have tubular and laminar cristae that connect to
the inner membrane by circular or slot-shaped crista junctions
[7,32]. Our observations in O. tauri add to this body of literature
and highlight the consistency of mitochondrial ultrastructure.
Notwithstanding the evolutionary pressures that shrunk O. tauri
and its mitochondria, the arrangement of its cristae has essentially
not changed. This strengthens the idea that the structure of the
cristae plays an important role in oxidative phosphorylation,
including the possibility that it creates local environments
separated by bottlenecks of restricted diffusion [33–35].

Endoplasmic Reticula
Sheets and tubes of membranes branching off the NE were
identified as ER (Figure 7B, white arrowheads). The inter-
membrane spacing within each ER sheet ranged from 14 nm to
80 nm. The ER tubes had diameters of ,14 nm – a value much
smaller than the 25–100 nm typically described for smooth ER
Figure 10. Endoplasmic reticulum. 3-D segmentation of a cell
[36,37]. Interestingly, some ER sheets were perforated by the dark harvested at the dark to light transition. ER (light blue) forms a sheet
oval granules (Figure 7B and 10). Other structures looked just like near the edge of the cell, perforated by four granules (dark blue).
the ER, but connections to the NE were not found. These doi:10.1371/journal.pone.0000749.g010

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 3-D Ultrastructure of O. tauri

Golgi bodies having a volume similar to the cis-cisterna. None of the cisternae
Golgi bodies were identified as stacks of flat, membranous were connected.
cisternae positioned next to the nucleus and usually extending The origins and destinations of vesicles in the Golgi are of
towards the chloroplast. No cell had more than one Golgi body, considerable interest [40]. From the nucleus outwards, occasion-
but in some cells (23%, including both non-dividing and ally vesicles were seen budding out of the NE towards the cis-
predivisional) no Golgi body was found, probably because it was cisternae (8% of cells). There were also free-standing vesicles
positioned in such a way that the missing wedge artifacts made it observed between the NE and cis-cisternae. The tubular vesicles in
impossible to recognize. In mammalian cells the Golgi body front of the cis-surface (Figure 11, light blue) might have been the
disperses prior to cell division [39]; here the Golgi was frequently fusion of two or more NE-derived vesicles that formed a vesicular
observed in predivisional O. tauri. tubular cluster. Vesicles were not seen between cisternae, but they
In O. tauri, the Golgi bodies typically consisted of five uniformly were frequently present along the sides of the stacks. The trans-
spaced cisternae (Figure 11A–B). Some Golgi bodies exhibited up cisterna often abutted the chloroplast, leaving no room for vesicles
to seven cisternae, but the extra cisternae were often not trans to the cisterna. No vesicles were ever found trans to the Golgi
consistently spaced or appeared as if they were beginning to leave body, but the trans-cisterna often had an appearance of slipping
(Figure 11C, dark blue). The NE was used to determine cis to trans off the stack (Figure 11A–B, green cisternae), and occasionally had
direction because the resolution was insufficient to resolve the an additional cisterna beyond it (Figure 11C, dark blue cisterna).
protein coats on the vesicles that are normally used to determine Vesicles were not observed between the Golgi body and the non-
the Golgi polarity. Most cisternae were roughly disk shaped nuclear ER.
(Figure 11A–B), but more elongated cisternae were also seen These observations are consistent with the maturation model of
(Figure 11C). All were slightly curved. The cisterna nearest the Golgi body transport, but the lack of trans vesicles is surprising.
nucleus was consistently the smallest, with a diameter and volume Some of the vesicles along the side of the Golgi body could have
of about 150 nm and 561028 pL, respectively. Its concave (cis) originated from the trans surface and moved off to the side,
surface faced towards the nucleus. The middle three cisternae sterically deflected by the chloroplast. It may also be possible that
were larger, measuring 200 nm in diameter and 7.561028 pL in instead of having many small trans vesicles, here at least Figure 11C
volume. The trans-cisterna was 200 nm in diameter and thinner, suggests that the entire trans-cisternae (dark blue) is one transport

Figure 11. Golgi body. The Golgi bodies from three different cells are shown (rows A–C). In column I, slices through the reconstructions are shown,
with cisternae marked by arrowheads. In column II, 3-D segmentations of the Golgi bodies are shown in situ within their cellular contexts. Column III
shows the isolated 3-D segmentations from a view perpendicular to the one in column II. The five ‘‘core’’ cisternae common to all Golgi bodies seen
here are colored purple, red, gold, yellow, and green (cis to trans). Surrounding vesicles are colored either light or dark blue. Some vesicles in column
III do not appear in column II because they are ‘‘below’’ the cellular slice shown. The blue-green vesicle in IIC was removed from IIIC to create an
unobstructed view of the Golgi body. Scale bars 100 nm.
doi:10.1371/journal.pone.0000749.g011

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 3-D Ultrastructure of O. tauri

vesicle. Many cells rely on microtubules to transport Golgi associated discern whether bridging proteins connected the two (Figure 12B,
vesicles [41], but as in Schizosaccharomyces pombe [3], no microtubules insert). There was neither an obvious microtubule organizing
were ever found contacting the Golgi body or its neighboring center nor a flared or capped end to conclusively identify the
vesicles. Perhaps other, smaller cytoskeletal filaments were present to minus or plus end, respectively. It is possible that the microtubules
guide vesicles but were not resolved in our reconstructions. are organized similarly to those of higher plants, in which the
minus end interacts closely with the NE (as discussed later).
Vesicles Surprisingly, none of the cells exhibited a mitotic spindle even
A variety of vesicles were seen. The most numerous type (1–10 or though many stages of division were seen and microtubules were
more per cell) were pleomorphic, typically ,200 nm diameter, consistently resolved in our reconstructions. Unlike metazoans and
and occurred in clusters sometimes configured as a ‘‘V’’ in yeast, plants lack a structurally distinct microtubule organizing
predivisional cells (Figure 2, 4, 5, 6, 10). Their high internal center, but their NEs do organize mitotic spindles that segregate
density prevented us from determining if there was a surrounding their chromosomes [46]. Three of the reconstructed O. tauri cells
membrane. Presumably these are some type of storage granule. had two microtubules, and in one of these cells they were partially
Smaller vesicles were also seen whose size and random locations embedded within the open nucleus (the cell in Figure 7C, though
suggest they could have been involved in transport. In addition to the microtubules are not present in the slice shown), but because
these storage and transport vesicles, all the cells (n = 52) contained O. tauri has 20 independent linear chromosomes [13], a canonical
at least one characteristically low-density, nearly spherical, second mitotic spindle would have been expected to have more than 40
type of vesicle surrounded by a single membrane (Figure 4A and F, microtubules. While it is possible that in O. tauri the spindle
10) measuring 166620 nm in diameter. Some dividing cells had appears so transiently that by chance none of the 26 predivisional
two. Remarkably, these special vesicles were almost always in cells reconstructed here contained one, its absence suggests the
contact with both the chloroplast and the nucleus (Figure 10), even intriguing possibility that some evolutionary pressure (like size
when they were found far from the main body of the nucleus. In minimization) has caused O. tauri to adapt a novel mechanism for
these cases they were found touching long, thin extensions of the chromosome segregation. Perhaps the twenty pairs of chromo-
NE streaming over the chloroplast (Figure 5A), suggesting somes are segregated one at a time by the two mitotic
connections to both the NE and chloroplast. Because (i) microtubules. Alternatively, the chromosomes may be physically
peroxisomes have been described as single-membraned vesicles, linked during mitosis and be co-segregated.
(ii) small O. tauri cells are likely to need one and only one
peroxisome but multiple storage granules, and (iii) Ostreococcus Ribosome-like complexes
genomes contain peroxisomal genes [42], these vesicles were The cytoplasm contained many discrete large macromolecular
assumed to be peroxisomes. As dividing peroxisomes were never complexes, which were presumably mostly ribosomes. The
seen, perhaps O. tauri only produces peroxisomes de novo [43–45]. cytoplasms of three non-dividing cells (D, E, and F in Figure 4)
were searched by cross-correlation for ribosome-like particles
Microtubules using a low-pass-filtered ribosome model as template. Visual
Most O. tauri cells had a single, hollow, tubular structure 2461 nm inspection of the search results using different thresholds showed
wide and 200–700 nm long assumed here to be a microtubule. the search had located large protein complexes within the
The microtubule always rested between the outer nuclear cytoplasm, but as expected, there was no clear cutoff in the
membrane and the plasma membrane against one of the ‘‘top’’ distribution of cross-correlation coefficients that could distinguish
or ‘‘bottom’’ flat faces of the cell (Figure 4, purple rods). Both ends between ribosomes and other large particles (Figure 13A).
of the microtubule were open and blunt (Figure 12, arrowheads). Moreover, because the peak-search algorithm excluded adjacent
The microtubules were slightly curved and near one end the shaft peaks closer together than a ribosome diameter, clustered complexes
made contact with the NE, but the resolution was insufficient to were not accurately parsed. Nevertheless because a threshold of the
top 500 picks clearly excluded many large protein complexes and
a threshold of 2000 clearly included many smaller complexes
(Figure 13B), we conclude that there were very roughly about 1250
ribosomes per cell. In comparison, exponentially growing Saccharo-
myces cerevisiae cells have ,200,000 ribosomes each [47]. Since S.
cerevisiae cells have up to 150 times more cytoplasm than O. tauri
[48,49], the concentration of ribosomes in these cells is approx-
imately the same. In O. tauri, the ribosomes appeared to be uniformly
distributed within the cytoplasm (Figure 13C).

Plasma Membrane Extensions and Surface Proteins

Three cells (6%) exhibited a bulge containing multiple 5-nm
diameter filaments (Figure 14A) near sheets of ER. Two other
much longer plasma membrane extensions were observed at
Figure 12. Microtubule. (A) 9.6-nm slice through one particular opposite poles of an unusual cell (Figure 2E–F). One extension was
microtubule (arrowheads) along most of its length, showing its uniform at least 585 nm long (continued to the edge of the reconstructed
diameter and hollow nature. (B) 16.8-nm slice through a microtubule in area) and contained two internal filaments while the other was much
its cellular context, showing its open, blunt ends (arrowheads) shorter and contained a ‘‘Y’’ shaped inner vesicle protruding into the
terminating between the NE (arrows) and plasma membrane. The
extension. Large macromolecular complexes were also seen decorat-
insert shows a 2-fold enlarged, 38.4-nm thick slice through the
microtubule perpendicular to its long axis, emphasizing its tubular ing the outer surface of cells (Figure 14B). While these various
shape (arrowheads). Scale bar 100 nm. specializations likely mediate contacts with other cells or substrates,
doi:10.1371/journal.pone.0000749.g012 their functions and mechanisms are of course at this point unclear.

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 3-D Ultrastructure of O. tauri

Figure 13. Ribosome-like complexes. (A) Three cells (shown in panels D–F of Figure 3) were searched for ribosome-like particles, and the resulting
local-normalized cross-correlation coefficients are plotted from best to worst from left to right. There were fewer total positions ranked in cell ‘‘3D’’
because its cytoplasm was smaller. (B) 24-nm thick slice through a cell with the 500 (red) or 2000 (green) most ribosome-resembling objects in the
cytoplasm circled, showing that these are reasonable under- and over-estimates of the total number. (C) 3-D positions of the 1250 most ribosome-
resembling objects in the cytoplasm (light purple spheres), showing their close and even distribution. Scale bar 250 nm.
doi:10.1371/journal.pone.0000749.g013

Conclusion specific states and interesting mutants) will likely provide key
Despite application of all the best imaging technologies available, insights into the organization of cells and the molecular
many fundamentally important aspects of eukaryotic ultrastructure mechanisms responsible. As examples, here we have seen that in
remain elusive (including for instance, as a single example, the G1, the nucleus and chloroplast sit opposite one another, and this
higher-order structure of chromatin). This study demonstrated may simply be because they are the largest bodies within the size-
that the emerging technique ECT can reveal the detailed minimizing cell. While the mitochondrion seems to position itself
ultrastructure of at least the smallest known eukaryotic cell, O. randomly, the other major organelles appear to be organized by
tauri, in an intact and near-native state. While ECT has already their relationship to the NE. The chloroplast exhibited its own
been applied to purified mitochondria and nuclei [7,27], here they division plane, and thus must possess its own segregation
were seen in their cellular context along with structures that machinery for its internal features.
cannot be isolated (like the Golgi). Because O. tauri is becoming an Nevertheless the resolution was limited in both space and time.
increasingly well-developed model system as a simple plant and As one benchmark example, while microtubules were easily
‘‘minimal’’ eukaryotic cell, systematic high-resolution ultrastruc- resolved here, actin filaments were not, though that has been
tural analysis by ECT as done here (but also of cells stalled in possible in other thinner specimens [8,50–54]. Thus it remained

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 3-D Ultrastructure of O. tauri

TEC, Liechtenstein). The samples were freeze substituted with 1%

osmium tetroxide and 0.1% uranyl acetate in acetone using an
EM AFS (Leica, Vienna). Samples were held at 290uC for
18 hours, warmed to 225uC at a rate of 5uC/hr, held at 225uC
for 12 hours, and then warmed to room temperature at a rate of
5uC/hr. The samples were then rinsed in pure acetone, infiltrated
and embedded in Epon-Araldite resin.

Plunge Freezing
Cells were harvested by centrifugation as above, then resuspended,
and combined with 10 nm gold fiducial markers. This sample was
applied to glow-discharged R 2/2 Quantifoil grids (SPI Supplies),
excess media was removed by a single 1 s blot (24 mm offset, 1 s
drain time), and vitrified by plunging into liquid ethane in
a Vitrobot (FEI) [56].
Figure 14. External macromolecular complexes. (A) 12-nm thick slice
showing multiple filaments (black arrowheads) emerging from a cellular Electron cryotomography
protuberance. Black arrows point to ER. (B) 12-nm thick slice showing Plunge-frozen grids were loaded into ‘‘flip-flop’’ tilt rotation
macromolecular complexes (white arrowheads) on the outer surface of holders [19] and imaged in a 300 keV, FEG, G2 Polara
the plasma membrane. Scale bar 100 nm.
doi:10.1371/journal.pone.0000749.g014 transmission electron microscope (FEI) equipped with an energy
filter (Gatan). Throughout loading and imaging, samples were
cooled with liquid nitrogen [57]. Sequentially tilted, energy-filtered
(slit width 20 eV) images of individual cells were collected
unclear, for instance, what the molecular mechanisms were that automatically using either the UCSF tomography [58] or Leginon
established the division plane or segregated the different copies of software packages [59] in 2u increments up to +/2,56–60u along
each organelle. While future technological advances in both either one (36 cells) or two (16 cells) axes [19] at 18,0006
instrumentation and specimen preparation can be expected to magnification, resulting in a CCD pixel size of 1.2 nm. The
improve the situation soon [6], it is unclear now which of O. tauri’s cumulative dose for each tilt series was 165 e2/Å2.
eukaryotic secrets will be the most interesting! Our original hope of
visualizing undisturbed chromatin and mitosis in molecular detail,
for instance, has been replaced by intrigue over the possibility that
Image processing
Images were aligned using the gold fiducials and three-dimen-
O. tauri may have remarkable and unique adaptations, since the
sional reconstructions were calculated and visualized using the
nuclear envelope was wide open throughout most of the cell cycle
IMOD software package [60]. Reconstructions were denoised
and neither condensed chromosomes nor a mitotic spindle were
using thirty cycles of non-linear anisotropic diffusion with
ever seen. While it is possible that these expected features exist so
parameter lambda = 0.3 [61] using Bsoft [62] and the Peach
briefly that we simply missed them in our sampling of 52 frozen
distributed-computing system [63]. Denoised 3-D reconstructions
cells, it is also possible that O. tauri will reveal significantly
were manually segmented using the Amira software package
simplified mitotic mechanisms. (Mercury Computer System, Inc.) on a Wacom Cintiq 21UX
display. Volume and surface area estimates of the segmented
MATERIALS AND METHODS volumes were also made using Amira. Cross-correlation searches
Cell growth were done using the MolMatch software [64] and an appropriately
Ostreococcus tauri strain OTH95 (RCC 745 from the Roscoff scaled and low pass-filtered cryo-EM reconstruction of a ribosome
Culture Collection) was grown in f/2 medium [55] made with [65] as template. Cytoplasmic peaks were ranked based upon
Sigma Sea Salt to 36% salinity. Cells were grown at a constant cross-correlation coefficients and then displayed using Amira.
20uC with gentle, 60-RPM agitation on a rotary shaker in a cycle
of twelve hours of light and twelve hours of dark. SUPPORTING INFORMATION
Video S1 Electron cryotomographic reconstruction of an
Traditional Fixation Ostreococcus tauri cell. The three-dimensional reconstruction of
O. tauri cells were harvested during early and mid exponential a single cell is shown slice-by-slice, from which the segmented
growth phase, and concentrated by 5,000 RCF centrifugation. model emerges in a second pass. After the various subcellular
The cell pellet was then mixed with a 1% glutaraldehyde made up organelles are identified, the substructure of first the Golgi body
in filtered ocean water. The cells were re-centrifuged, washed and then the mitochondrion are shown in isolation, including the
twice with cacodylate buffer, and fixed in 1% OsO4 for position and shape of the cristae junctions.
30 minutes. The cells were re-washed twice with cacodylate Found at: doi:10.1371/journal.pone.0000749.s001 (5.06 MB
buffer, washed with 30% ethanol for 10 minutes, and then mixed MOV)
with 30% ethanol and 4% uranyl acetate for 20 minutes. Finally
cells were then successively dehydrated with a series of ethanol ACKNOWLEDGMENTS
washes and embedded in Epon. We thank Hervé Moreau (Observatoire Océanologique, Laboratoire
Arago) and Brian Palenik (Scripps Institution of Oceanography) for
helping us obtain and work with O. tauri cultures and for reading the
High-pressure fixation and freeze substitution manuscript, Kent L. McDonald (U.C. Berkeley) for help high-pressure
Cells were harvested by centrifugation as above. The cell pellet freezing, Wolfgang Baumeister’s group (Max Planck Institute for Bio-
was high pressure-frozen in a BAL-TEC HPM 010 freezer (BAL- chemistry) for the most current version of the MolMatch software, Andrea

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 3-D Ultrastructure of O. tauri

Manuell for discussions on the chloroplast, Christian Suloway (Caltech) for

assistance with Leginon and Jane Ding (Caltech) for help running the cross-
Author Contributions
correlation search on the supercomputer. Conceived and designed the experiments: GJ GH LG. Performed the
experiments: GH LG. Analyzed the data: GH LG. Contributed reagents/
materials/analysis tools: GJ. Wrote the paper: GJ GH LG.

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