© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.
211383
REVIEW
Mitotic spindles revisited – new insights from 3D electron
microscopy
Thomas Mü ller-Reichert‡, Robert Kiewisz and Stefanie Redemann*,‡
ABSTRACT
The mitotic spindle is a complex three-dimensional (3D) apparatus
that functions to ensure the faithful segregation of chromosomes
during cell division. Our current understanding of spindle architecture
is mainly based on a plethora of information derived from light
microscopy with rather few insights about spindle ultrastructure
obtained from electron microscopy. In this Review, we will provide
insights into the history of imaging of mitotic spindles and highlight
recent technological advances in electron tomography and data
processing, which have delivered detailed 3D reconstructions of
mitotic spindles in the early embryo of the nematode Caenorhabditis
elegans. Tomographic reconstructions provide novel views on
spindles and will enable us to revisit and address long-standing
questions in the field of mitosis.
KEY WORDS: 3D reconstruction, Electron tomography, Microtubule
segmentation, Microtubule, Mitosis, Spindle
Introduction
During mitosis, chromosomes are segregated with high precision to
generate two identical daughter cells. The process of segregation is
driven by a dynamic bipolar spindle apparatus (Helmke et al., 2013;
McIntosh, 2017). Microtubules are the main building blocks of such
spindles. They polymerize from heterodimers consisting of α- and
β-tubulin and display a characteristic stochastic switch from slow
growth to fast shrinkage, described as dynamic instability
(Mitchison and Kirschner, 1984).
Microtubules show a distinct polarity with a relatively stable
minus-end and a dynamic plus-end (Helmke et al., 2013). Most
microtubule minus-ends are associated with the centrosome (Wu and
Akhmanova, 2017). This non-membrane-bound organelle is the
major site of microtubule nucleation in animal cells, although other
sites of microtubule nucleation have been reported. In general,
microtubules can also be formed within the spindle itself, a
phenomenon called microtubule branching (Goshima et al., 2008;
Petry et al., 2013), or nucleated around chromosomes as observed in
Xenopus extracts (Heald et al., 1996). Microtubule nucleation at the
mitotic centrosome, however, causes a distinct orientation of
microtubules within the bipolar spindle, in that the microtubule
minus-ends are located within the pericentriolar material (PCM) of
the centrosome and the microtubule plus-ends are growing away from
the centrosome. According to the direction of microtuble plus-end
growth and interaction with a particular cellular target site,
Technische Universitä t Dresden, Experimental Center, Medical Faculty Carl Gustav
Carus, Fiedlerstraße 42, 01307 Dresden, Germany.
*Present address: Center for Membrane and Cell Physiology and Department of
Molecular Physiology and Biological Physics, University of Virginia, School of
Medicine, Charlottesville, VA 22908-0886, USA.
‡
Authors for correspondence (mueller-reichert@tu-dresden.de; SZ5j@virginia.edu)
R.K., 0000-0003-2733-4978; S.R., 0000-0003-2334-7309
ARTICLE SERIES: IMAGING
microtubules of bipolar spindles are grouped into different classes.
A canonical view of mitotic spindle structure in metaphase (Fig. 1)
shows the following classes of microtubules (MTs): astral-MTs
(AMTs), kinetochore-MTs (KMTs), interdigitating-MTs (IMTs) and
spindle MTs (SMTs). AMTs are those microtubules that grow away
from centrosomes towards the cellular cortex, thus mainly playing a
role in positioning the spindle apparatus (Grill et al., 2001). The plus-
ends of KMTs are directly connected to the kinetochores (i.e. to
specific centromeric microtubule-binding sites on the chromosomes)
(Musacchio and Desai, 2017). IMTs are thought to interact with each
other in the midzone of the spindle. This interaction is supposed to
build a direct pole-to-pole connection through microtubules of
opposite polarity (Mastronarde et al., 1993). SMTs are microtubules
in the body of the spindles that are neither overlapping with other
microtubules (IMTs), nor connecting to the kinetochore (KMTs)
(Redemann et al., 2017). In addition, numerous microtubule-
associated proteins and molecular motors function during spindle
formation, positioning and chromosome segregation (Helmke et al.,
2013; McIntosh et al., 2012).
This general scheme of spindle organization is used in many
textbooks to describe the ‘basic organization’ of mitotic spindles.
However, despite the evolutionary conservation of essential
proteins and regulatory factors, there is a remarkable variability in
the structure and organization of mitotic spindles between
organisms and within cells from a single organism. In addition,
the process of chromosome segregation appears to be variable, in
that organisms show differences in the mechanism of anaphase
(Scholey et al., 2016).
So, where does our current understanding of mitotic spindle
structure and organization come from and what technology was used
to increase this body of knowledge? Clearly, our understanding of
mitosis is intimately linked to improvements in light and electron
microscopy, as well as in advances in specimen preparation and
image processing. Key steps in specimen preparation and imaging are
briefly summarized in the following paragraphs.
Shedding light on mitotic spindles – highlights of mitosis
research
The vast majority of information we currently have about mitotic
spindles is derived from light microscopy. The first investigators to
Journal of Cell Science
describe the process of mitosis were the Polish scientist Wacław
Mayzel (Mayzel, 1875) and the German scientist Otto Bütschli
(Bütschli, 1875, 1876). About 3 years later, the term ‘mitosis’,
derived from the Greek word for ‘thread’, was coined by the
German scientist Walther Flemming (Flemming, 1878, 1965).
Flemming investigated cell division and used aniline dyes to stain
cells and observe chromosome distribution in the fins and gills of
salamanders (Fig. 2). Even though Flemming also observed living
cells, the groundbreaking findings of mitotic chromosome
segregation were made from analysis of fixed and stained
samples, providing a static view about dynamic spindles. The
1
REVIEW
Key
KMTs AMTs SMTs IMTs Centrosome Chromosome
Fig. 1. Canonical view of mitotic spindle structure in metaphase. The
bipolar spindle is organized from two centrosomes (light green spheres, with
centriole pair). Microtubule minus-ends (−) are anchored at the centrosome
and plus-ends (+) are growing out towards target sites. Kinetochore
microtubules (KMTs, red) are attached to chromosomes (gray), astral
microtubules (AMTs, dark green) are growing towards the cell periphery.
Spindle microtubules (SMTs, light green) are growing towards the
chromosomes but are not connected to the kinetochores; some SMTs will
eventually turn into KMTs. Interdigitating microtubules (IMTs, orange) are
thought to interact with each other in the middle of the spindle.
hand-drawn images of mitotic spindles produced by Flemming
provided the first detailed images of spindles. It is important to point
out that chromosomes, because they were stained, were easy to
identify. In contrast, the microtubule cytoskeleton could not be
observed, and appeared as empty regions in the cytoplasm.
Nevertheless, the formation of spindle fibers and astral arrays was
proposed (Flemming, 1878). The nature and composition of such
fibers and arrays, however, was unknown at the time and many ideas
were postulated. For example, fibers and arrays were thought to
crystallize out of the protoplasm around a spindle pole, or to form
through a morphological re-arrangement of the pre-existing
Fig. 2. Spindle structures as observed and drawn by Walther Flemming in
1882. The images show mitotic steps of epithelial cells of salamander larvae.
The terminology as used by Flemming is: (A) “Umordnungsstadium:
Metakinese” (remodeling state: metakinesis). (B) “Endform der Metakinese”
(end stage of metakinesis). (C) “Folgendes Stadium: Auseinanderweichen,
Anfang der Tochtersternform” (following stage: moving apart, beginning of
daughter star formation). Reproduced from Flemming, 1882.
Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383
protoplasm. Alternatively, lamellae were also proposed to form
the wall of elongated chambers. The theories of mitosis discussed at
the time were excellently covered by E. B. Wilson (Wilson, 1902).
The discovery of mitotic cell division in 1875 was followed by
decades of research, in which the existence of the so-called spindle
fibers and astral arrays was vigorously debated. Opponents of the
theories suspected the fibers were nothing else but artifacts of the
fixation and staining procedure (Wilson, 1902).
The development of polarized light microscopy was key to
establishing the existence of spindle fibers, as spindles could be
observed in living cells. Using this technique, the first observation
of sea urchin spindles was performed by W. J. Schmidt in the 1930s
(Schmidt, 1937, 1939) and provided evidence for the existence of
such spindle fibers, even though distinct fibers could not be
resolved. It still took several years before Hughes and Swann
(Hughes and Swann, 1947) and Shinya Inoué (Inoué, 1951)
demonstrated of the existence of spindle fibers. The application of
polarized light microscopy, culminating in the development of the
Pole-Scope, also opened an entire new field of research, in that
mitotic spindles could be analyzed in living cells after specific
perturbations and manipulations (Inoue and Oldenbourg, 1998). As
early as in 1953, Chaetopterus eggs were treated with colchicine or
exposed to cold temperatures; this resulted in the first description of
what we know today to be the kinetochore fiber (k-fiber) (Inoué,
1953). In the end, it was the use of osmium tetroxide (Roth and
Daniels, 1962) and glutaraldehyde (Sabatini et al., 1963) for
specimen fixation for electron microscopy that finally proved the
existence of spindle fibers, which were described and named
‘microtubules’ in 1963 (Ledbetter and Porter, 1963).
The composition of microtubules was only resolved in 1968
when Edwin Taylor and colleagues succeeded in the purification of
tubulin by co-purifying it with colchicine, a drug known to destroy
mitotic spindles (Weisenberg et al., 1968). The purification of
tubulin and the development of assays for test-tube polymerization
of microtubules eventually opened up the field of in vitro
microtubule research, thereby significantly increasing our
understanding of microtubule dynamics (Johnson and Borisy,
1975; Mitchison and Kirschner, 1984; Weisenberg, 1972). The
application of different optical methods, such as fluorescence
recovery after photobleaching (FRAP) (Axelrod et al., 1976) and the
use of fluorescein-labeled tubulin, together with the injection of
fluorescently labeled dyes (Salmon et al., 1984), allowed the
characterization of both microtubule and spindle dynamics in vivo.
Microinjection with fluorescent tubulin was extensively used for
mammalian tissue cells because of the advantages of the
fluorophores and the ease of the injection procedure (Waterman-
Storer and Salmon, 1997).
The discovery of green fluorescent protein (GFP) in the 1960s
(Shimomura et al., 1962) and the successful cloning of GFP in the
early 1990s (Prasher et al., 1992) resulted in the first expression of
GFP-labeled β-tubulin in the touch receptor neurons in
Journal of Cell Science
Caenorhabditis elegans (Chalfie et al., 1994); this opened up the
field of light microscopy to protein localization in living cells.
Subsequently, GFP was expressed in distinct positions or organs in
animals and at specific time points during the development of a
number of model organisms, thus expanding the possibilities to
observe and characterize mitotic spindles in vivo. Today a number of
GFP derivatives, such as yellow fluorescent protein (YFP) and
others are available to simultaneously label distinct spindle
components in living cells (Rodriguez et al., 2017).
Over the past 20 years, advances in light microscopy, such as
stimulated emission depletion (STED) microscopy (Klar et al., 2000)
2
REVIEW
or saturated structural illumination microscopy (SSIM) (Gustafsson,
2005) have resulted in an improvement of resolution from ∼500 nm
to 100 nm. Moreover, the development of super-resolution
technology, such as photoactivated localization (PALM) (Betzig
et al., 2006) and stochastic optical reconstruction microscopy
(STORM) (Rust et al., 2006), has further pushed the resolution
limit to ∼40 nm. So far, superresolution microscopy has been applied
to analyze spindle components, such as centrosomes (Mennella et al.,
2014, 2012) and kinetochores (Ribeiro et al., 2010).
In summary, over the past 50 years, remarkable improvements in
reagents and technology for light microscopy enabled an explosion
of knowledge about the structure and function of mitotic spindles.
Although providing great tools to analyze spindle dynamics in
living cells, light microscopy today cannot offer sufficient
resolution to visualize each microtubule in a given spindle, to
determine the length of individual microtubules and assign each
microtubule to a distinct functional class according to their position
within the spindle (Fig. 3A). Such a detailed analysis is only
possible using 3D electron microscopy (Fig. 3B) and the major
advances in this field for mitosis research are briefly summarized in
the next section.
Making use of electrons – ultrastructural views on spindle
architecture
Electron microscopy (EM) of in vitro assembled microtubules
stimulated investigations on the structure of microtubules and the
Fig. 3. Organization of the first mitotic spindle at metaphase in the early
C. elegans embryo. (A) Level of resolution as expected from light microscopy.
(B) Level of information obtained from electron microscopy. The density map of
image A was extracted from the data shown in B. The data is based on a 3D
reconstruction of microtubules obtained from electron tomography, with
microtubules simplified as straight lines. A was convolved with a two-
dimensional Gaussian point-spread function with a full-width at half maximum
(FWHM) of 0.45 µm. A quantitative analysis of microtubule number and length
can only be obtained from electron microscopy data, as light microscopy can
not deliver single-microtubule resolution. Scale bar: 5 µm.
Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383
interaction of molecular motors with microtubule walls. Both
negative staining and immobilization of microtubules in frozen-
hydrated samples revealed information about protofilament numbers,
functional end-morphologies of individual microtubules, and the
mode of interaction of specific minus- and plus-end-directed motors
(Downing and Nogales, 2010). It was the discovery of vitrification of
water (Dubochet et al., 1982; Dubochet and McDowall, 1981) that
laid the groundwork for the presentation of atomic maps of tubulin
and culminated in the 2017 Nobel prize for chemistry for the
development of cryo-EM for the high-resolution determination of
macromolecular structure (Beck and Baumeister, 2016; Irobalieva
et al., 2016). However, what about the structural analysis of
microtubules within the cellular context?
Early EM studies used glutaraldehyde to routinely fix whole
cells. After dehydration at room temperature and plastic embedding,
thin (60–80 nm) sections were then imaged in a transmission
electron microscope. Spindle components from a number of
specimens were thus visualized in two dimensions. From the
beginning, the aim was to combine the contextual information of
light microscopy with the ultrastructural data as obtained from EM
to gain information about specific mitotic stages and spindle
components (Brinkley et al., 1967; Webster et al., 1978). Correlative
light and electron microscopic (CLEM) approaches are still used in
current cell biological research related to mitosis (McDonald, 2009;
Müller-Reichert et al., 2007).
The next level of solving the ultrastructure of mitotic spindles was
reached by the stacking of serial thin sections to quantify microtubule
numbers as a function of their position along the spindle axis (Fuge,
1973; McIntosh and Landis, 1971). Later on, such serial-section
analysis allowed the reconstruction of whole diatom spindles
(McDonald et al., 1977, 1979), entire yeast spindles (Ding et al.,
1993; Winey et al., 1995, 2005) and kinetochore fibers (McDonald
et al., 1992), as well as of interpolar microtubules in PtK2 cells grown
in culture (Mastronarde et al., 1993). The EM laboratory in Boulder
(University of Colorado at Boulder, USA) pioneered the development
of software tools (IMOD) to computationally stack the individual
images in order to present 3D models of segmented microtubules and
organelles (Kremer et al., 1996).
Glutaraldehyde is a standard fixative for EM, but its use is fraught
with a number of problems. The biggest issues are the slow
diffusion rate of chemical fixation and the subsequent dehydration
procedure at room temperature for plastic embedding. A major step
forward towards quantitative analysis of spindle structure was
therefore the introduction of high-pressure freezing for biological
samples (Moor, 1987). High-pressure freezing overcomes the
thickness limitation of plunge freezing, where the ability to cryo-
immobilize samples with a thickness of up to 0.2 mm is possible.
The combination of high-pressure freezing with subsequent freeze
substitution has significantly contributed to achieving superb
ultrastructural preservation for EM. This approach is now a
routine preparation method for ultrastructural investigations in
Journal of Cell Science
current model systems, such as yeasts, worms, flies and mammalian
samples (Müller-Reichert, 2010).
An in-depth analysis of the mitotic spindle architecture in 3D
requires a high resolution in the z direction. Obviously, the
z-resolution in serial-section reconstructions is rather low (as it is
limited by the section thickness, typically 60–80 nm) and certainly a
major limiting factor of this approach. This limitation is evident in
early 3D reconstructions of serial sections of mitotic spindles in
budding yeast (Winey et al., 1995), and it was one of the major
motivations to turn to electron tomography to reconstruct
microtubules at the yeast spindle poles (O’Toole et al., 1999). A
3
REVIEW
major advantage of this method is that this technology allows a
resolution of 5–6 nm in 3D. It was again the Boulder laboratory that
pioneered the development of software tools for electron
tomography, such as SerialEM for automatic data acquisition and
the calculation of double-tilt tomograms (Mastronarde, 1997, 2005;
O’Toole et al., 2017). In combination with high-pressure freezing
and freeze substitution, electron tomography was initially applied
to reconstruct small spindle components, such as centrosomes
(O’Toole et al., 2003), centrioles in C. elegans (Pelletier et al., 2006)
and mammalian kinetochores (Dong et al., 2007; McIntosh et al.,
2008; VandenBeldt et al., 2006), as well as whole spindles of
budding yeast (O’Toole et al., 1999). However, analyzing larger
volumes by electron tomography has been a difficult and time-
consuming task, and it took several years to develop tools to
montage individual tomograms of a given section and to stack a
number of consecutive tomograms to increase the reconstructed
volume (Höög et al., 2007; Ladinsky et al., 1999; Noske et al.,
2008). Last but not least, microtubules had to be segmented
manually and the joining of data over several consecutive sections
was difficult and also needed to be done manually. Therefore, 3D
reconstruction of complete spindles containing hundreds to
thousands of microtubules was a massive undertaking and fraught
with numerous technical difficulties.
Keeping these technical hurdles in mind, it is not surprising that
we currently have only very limited ultrastructural 3D data on
mitotic spindles. In fact, most of the data had already been obtained
in the 1970s to 1990s in just a few model systems (Ding et al., 1993;
Mastronarde et al., 1993; McDonald et al., 1992; Winey et al.,
1995). However, recent advances in software tools for electron
tomography (see below), have allowed us to obtain large-scale
reconstructions of mitotic spindles in C. elegans and we will present
the unexpected findings of this study in the next paragraphs.
Going further on the 3D route – current advances in electron
tomography and image processing
Several advances in software development have led to automated
approaches to segment microtubules in electron tomograms
Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383
(Redemann et al., 2014; Weber et al., 2012), to stitch segmented
microtubule across consecutive tomograms (Weber et al., 2014),
and to quantify spindle structure by taking each individual
reconstructed microtubule into account. This, in combination with
developments in visualization technology, significantly pushed the
3D reconstruction of large spindles and enabled the first large-scale
spindle reconstruction from the early embryo of the nematode
C. elegans (Fig. 3B), a well established model system in mitosis
research (Müller-Reichert et al., 2010).
In order to generate large-scale 3D reconstructions of mitotic
spindles, we cryo-immobilized single-cell C. elegans embryos by
high-pressure freezing, followed by freeze substitution and thin-
layer embedding (Müller-Reichert et al., 2007). Such plastic-
embedded embryos are then cut into 300-nm-thick serial sections.
To cover the pole-to-pole volume of a metaphase spindle, for
instance, we acquired 12 double-tilt tomograms per section, with ∼5
GB of tomographic data per section (Redemann et al., 2017).
Applying the same procedure, we then acquired 24 of these sections
to cover the z-axis, thus recording a significant volume of the mitotic
metaphase spindle (Fig. 4). Following image acquisition for
electron tomography, dual-axis tomograms were then computed
for each montage panel.
The C. elegans spindles were modeled in two steps. First,
microtubules were automatically segmented in each section, using a
template-matching approach, followed by an automatic tracing
algorithm (Redemann et al., 2014; Weber et al., 2012). Second, the
segmented microtubules were then used to automatically stitch the
serial sections to a single stack (Weber et al., 2014). However, we
found that this automatic workflow for stitching was often
problematic owing to the large size of the data, deformation of
individual sections and missing visual validation steps. Therefore,
we developed a novel stitching tool that allows users to achieve an
alignment and matching of microtubules across a stack of serial
sections by applying a combination of automatic steps, visual
validation and interactive correction. This approach supports quality
assessment by highlighting regions that have been checked for
stitching errors and verified (Redemann et al., 2017).
Fig. 4. Application of serial-section
electron microscopy for the 3D
reconstruction of spindle
architecture. (A) Principle of electron
tomography. A semi-thick section is tilted
in the electron beam. A series of tilted
views is then used for an in silico
calculation of tomograms. (B) Montaging
of individual sections to cover the pole-
to-pole region of a metaphase spindle of
the early C. elegans embryo. Two
montages of 3x2 tomograms (each 5.5
μm in length and width) are joined and
the overlap region of the two montages is
indicated. (C) Individual tomograms are
Journal of Cell Science
stacked to increase the volume of the
reconstruction. This example shows the
stacking of 11 individual serial
tomograms. (D) 3D model
corresponding to the stacked
tomograms as seen in C. The
microtubule centerlines from adjacent
sections have to be stitched together to
obtain the full reconstruction.
Reproduced from Redemann et al.,
2017, where it was published under a
CC-BY license (https://
creativecommons.org/licenses/by/4.0/).
4
REVIEW Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383

In addition to the automatic segmentation and the stitching of
microtubules across multiple sections, we also developed a large
set of tools in collaboration with the Zuse Institute in Berlin
(http://www.zib.de/projects/reconstruction-and-analysis-microtubule-
spindle-using-electron-tomography) to analyze the obtained 3D data
(Redemann et al., 2017). Our goal was to have a standardized set of
quantitative tools to automatically analyze microtubule position and
length, and the interaction of microtubules with each other, as well
as with chromosomes. We are convinced that detailed 3D data, as
obtained by the analysis of complete spindle reconstructions, is a
huge leap forward in our efforts to gain a deeper understanding of
spindle assembly and the mechanics of chromosome segregation.
We set out to reconstruct the complex spatial arrangements of
microtubules in C. elegans mitotic spindles in order to understand
how KMTs are assembled and interact with the holocentric
kinetochore of C. elegans by combining electron tomography of
serial sections with light microscopy and mathematical modeling
(Redemann et al., 2017). Based on the large-scale reconstructions,
we first classified individual microtubules according to their
position within the spindle and the interaction of their minus-ends
with target complexes. We thus identified AMTs, KMTs and SMTs.
It is worth noting that such a classification required the
reconstruction of complete microtubules (end-to-end) for at least
KMTs and SMTs.
This classification of microtubules enabled us to further quantify
the distinct properties of each microtubule class (Redemann et al.,
2017). In fact, data of such high resolution in combination with light
microscopy is a very powerful tool to investigate the properties of
specific microtubules within the spindle. As an example, while
our light microscopy data and mutant studies strongly suggested
that microtubules are nucleated from the mitotic centrosome in
C. elegans, our structural data showed that only a few KMTs are
directly connected to the centrosomes (Fig. 5A) (Redemann et al.,
2017). Our quantitative analysis suggested that minus-ends of
KMTs selectively detach and depolymerize from the centrosome
(Fig. 5B). Thus, our results show that the connection between
chromosomes and centrosomes is mediated by an anchoring of KMTs
into the entire spindle network and that there are only few direct
connections through KMTs. In addition, these indirect connections are
likely highly transient (Redemann et al., 2017). In general, this finding
raised the question about the necessity of a direct chromosome-to-
centrosome connection for the segregation process in anaphase.
Another surprising outcome of our 3D study was that the ‘classic’
interdigitating microtubules were not detected in our reconstructions.
Ultimately, we are interested in whether our findings are specific to the
nematode system, or whether they point to general implications that
are also applicable to mammalian spindles.
Diversity of spindle architecture: a focus on model systems
Certainly, there is no simple generic scheme that can be used to
explain the diversity of the functional organization of the numerous
mitotic spindles that have been described in the literature (Helmke
et al., 2013). Differences in spindle structure are mainly related to
the type of mitosis, the organization of the kinetochore and the
preferred mechanism of chromosome segregation. First, mitotic
systems differ in the degree and timing of nuclear envelope
breakdown (NEBD). A closed mitosis with an intact envelope
during the entire course of mitosis is found, for instance, in budding
and fission yeast. In these systems, the spindle pole bodies, the
functional equivalents of the animal centrosome, are embedded in
the intact nuclear envelope (Ding et al., 1993; Winey et al., 1995).
An open mitosis is found in the ‘typical’ mammalian cell with a

Journal of Cell Science

Key
KMTs AMTs SMTs Centrosome
Centrosome Growing
Growing end
end Shrinking
Shrin king end

Fig. 5. Metaphase spindle in the early C. elegans embryo. (A) Schematic representation showing growth of microtubules (AMTs, dark green; KMTs, red; SMTs,
light green) from the centrosomes (light green spheres with centrioles). KMTs are attached with their plus-ends to the holocentric kinetochores of the chromosomes
(gray). As schematically shown, the majority of KMT plus-ends in this mitotic spindle are not directly attached to the centrosomes. (B) Microtubules grow out from
the centrosome (upper panel) and eventually attach to the holocentric kinetochore, thus converting into KMTs (mid panel). An attachment of the KMT plus-ends at
the kinetochore (lower panel) causes a selective detachment of the KMT minus-ends from the centrosome, possibly because of mechanical stress. As a
consequence, most of the KMT minus-ends are not directly attached to the centrosomes. Green arrows indicate microtubule growth, red arrowheads microtubule
depolymerization. Reproduced from Redemann et al., 2017, where it was published under a CC-BY license (https://creativecommons.org/licenses/by/4.0/).

5
REVIEW
complete NEBD occurring at mitotic prophase (McDonald et al.,
1992). A semi-closed (or semi-open) mitosis is observed in
C. elegans (Albertson, 1984) and Drosophila embryos (Harel
et al., 1989). In the first embryonic division in C. elegans, the
nuclear envelope is opened at the position of the two opposite
centrosomes, a phenomenon called ‘polar fenestration’ (Rahman
et al., 2015).
Second, embryonic or cellular systems differ in the organization of
the kinetochore. The monocentric kinetochore of mammalian cells is
the most common type and characterized by a distinct region on
chromosomes (i.e. the centromere) to which KMTs are physically
attached (Musacchio and Desai, 2017). In addition, KMTs in
mammalian systems are organized into k-fibers (McDonald et al.,
1992). In budding yeast, only a single microtubule is attached to a
‘point’ kinetochore (Winey et al., 1995). In contrast, chromosomes
with a holocentric kinetochore show an attachment of KMTs along
their entire length. This phenomenon of a dispersed kinetochore,
however, is fairly common in nature and can be observed in
nematodes, hemipteran insects and in a number of monocot plants
(Melters et al., 2012). Interestingly, both types of kinetochores appear
to have a similar protein composition, despite the difference in their
functional organization (Dernburg, 2001).
Third, cellular systems show differences in their anaphase A
(shortening of the pole-to-chromosome distance) and anaphase B
(increase in the pole-to-pole distance) patterns. While some show
either anaphase A or B, others show both phases, with either
anaphase A or B occurring first (Oegema et al., 2001; von Dassow
et al., 2009).
It is the diversity in microtubule dynamics and spindle organization
that makes it difficult to draw general conclusions about spindle
organization and raises a number of important questions. For
instance, are there similarities in the length distribution of KMTs in
C. elegans and k-fibers in mammalian cells? Is it possible to draw
general conclusions about spindle structure and to work out
underlying principles that are operating behind the different
systems? What needs to be done next to answer these questions?
Perspectives
While we have succeeded in the reconstruction of a number of
C. elegans mitotic spindles, there are still a number of technical
issues that need to be solved. One demanding task is the further
development of computational tools for image acquisition and
processing, and quantitative analysis of the large-scale tomographic
data. The bottleneck of serial electron tomography is still the
tremendous amount of time needed to join numerous tomograms for
montaging and stitching.
Automation of data processing is an important aspect for high-
throughput analysis, and we have already pushed automation of
several steps. In addition, the use of machine learning, or deep
learning, is very attractive for future data analysis. As an example,
this could be implemented to automatically group microtubule ends
according to their morphology (either closed or open), giving us
insights into their dynamic state, as has been achieved for
microtubules assembled in vitro (Chretien et al., 1995;
Mandelkow et al., 1991; Müller-Reichert et al., 1998).
Furthermore, there are also alternative microscopy techniques for
spindle reconstruction. Recent advances in serial block face
scanning electron microscopy (SBF-SEM) now allow the
reconstruction of large areas and volumes of cells and tissues
(Nixon et al., 2017). Serial block-face imaging does not rely on the
production and collection of serial sections to produce
reconstruction of large volumes, and its biggest advantage is that
Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383
deformations of the sample can be largely avoided. Unfortunately,
the biggest advantage of the SBF-SEM, the sample being contained
within a single block, is also its biggest disadvantage. In order to
visualize cellular structures by EM, those structures need to be post-
stained by heavy metals to increase the contrast of the sample.
However, if the sample is contained in a big volume of resin, then
the staining solution will not be able to penetrate the entire volume,
resulting in a relatively low contrast. Therefore, at this point, the use
of this technology is largely limited by a low contrast when using
cryo-immobilized plastic sections for spindle reconstruction.
Despite encouraging work, there is a long way to go before the
same precision in the quantification of microtubule architecture can
be achieved by this method as is currently obtained by tomographic
reconstruction of semi-thick sections.
One future goal would be to apply cryo-electron tomography to
image the mitotic spindle in a frozen-hydrated state, thus avoiding
dehydration of the sample by freeze substitution. Such work has
been carried out on the small spindles of Ostreococcus tauri (Gan
et al., 2011), but applying these methods to larger more complex
spindles would be a major technical challenge as sectioning of
frozen samples is difficult due to the brittle nature of the ice. An
alternative means to apply cryo-electron tomography could
certainly be the production of cryo-lamellae as obtained by cryo-
focused ion beam-scanning electron microscopy (cryo-FIB-SEM)
(Mahamid et al., 2016; Villa et al., 2013). This technology uses a
focused ion beam to produce a section (or cryo-lamella) of a frozen-
hydrated cell, avoiding sectioning with an ultramicrotome.
However, it is not possible to create serial sections with this
technology as the regions above and below the lamella are milled
away by the focused ion beam in the production process. Although
this technique is technically highly demanding, cryo-FIB-SEM
makes it possible to obtain high-resolution information of selected
regions of spindles. Cryo-electron tomography of frozen-hydrated
cells would push the level of resolution further and would be an
excellent approach to resolve any motor proteins located in-between
microtubules in the spindle, given that an averaging during steps of
image processing would be possible.
In the future, the availability of large reconstructions with a
single-microtubule resolution from different systems should make it
possible to also revisit the mammalian spindle architecture and
question any conclusions and hypothesis that were made on the
basis of light microscopy observations. An important question to
revisit, for example, would be the origin of microtubules in the
spindle [i.e. centrosome-based nucleation (Wu and Akhmanova,
2017), nucleation on pre-existing microtubules (Goshima et al.,
2008; Petry et al., 2013) and nucleation on or around chromosomes
(Heald et al., 1996)]. Our understanding of the role of each
nucleation mechanism and its contribution to spindle formation is
certainly limited. By a combination of dynamic and ultrastructural
data as obtained from both light and electron microscopy, it will be
possible to further investigate the origin of microtubules within
Journal of Cell Science
spindles. Another issue to analyze is the re-occuring question of the
presence of microtubule bundles within spindles. In order to detect
bundles within the spindles, however, one first has to define the
properties of a bundle. How many microtubules make a bundle?
How close or distant can those microtubules be? Do the
microtubules have to be arranged in parallel and if so, over what
distance does this parallelism have to persist? Although there are
certainly ongoing discussions about this issue in the field (Muscat
et al., 2015), a definite answer is currently lacking. In addition, we
have also no clear understanding of how forces might be generated
and transmitted within and outside of the spindle. What are the
6
REVIEW
interactions of microtubules with each other as well as with the cell
cortex or other components of the cells, such as organelles or
membranes? It will also be very interesting to follow up on the
structure of k-fibers in mammalian cells. Are all microtubules in
those k-fibers directly connected to the centrosomes? Moreover,
recently published light microscopy data presented evidence for a
role of bridging fibers that interact with k-fibers in the segregation of
mitotic chromosomes in mammalian cells (Vukusic et al., 2017). At
this point, it is unclear how k-fibers and bridging fibers interact, and
how this observation fits with a possible role of IMTs in mammalian
spindles. We anticipate that serial-section electron tomography will
offer the resolution necessary to analyze the fine structure of the
microtubules in such bridging fibers. Now that we have the tools to
answer these and other interesting questions in the field, we look
forward to exciting new insights in the near future.
Acknowledgements
The authors would like to thank Dr Johannes Baumgart (MPI-PKS, Dresden,
Germany) for extracting the density maps in Fig. 3 and Drs Sebastian Fü rthauer,
Ehssan Nazockdast and Michael Shelley (Flatiron Institute, Center for
Computational Biology, New York, USA) for collaborating on mathematical modeling
of spindle structure. The authors are grateful to Drs Eileen O’Toole and Richard
McIntosh (Boulder, USA) for a critical reading of the manuscript.
Competing interests
The authors declare no competing or financial interests.
Funding
Work in the Mü ller-Reichert laboratory is supported by funds from the Deutsche
Foschungsgemeinschaft (MU 1423/8-1 and 1423/10-1) and by a Marie Skłodowska-
Curie Innovative Training Network Grant (DivIDE), a training network of the
European Commission’s H2020 Programme. S. Redemann received funding from
the Medical Faculty Carl Gustav Carus (Frauenhabilitationsstipendium) of the TU
Dresden.
References
Albertson, D. G. (1984). Formation of the first cleavage spindle in nematode
embryos. Dev. Biol. 101, 61-72.
Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E. and Webb, W. W. (1976).
Mobility measurement by analysis of fluorescence photobleaching recovery
kinetics. Biophys. J. 16, 1055-1069.
Beck, M. and Baumeister, W. (2016). Cryo-electron tomography: can it reveal the
molecular sociology of cells in atomic detail? Trends Cell Biol. 26, 825-837.
Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S.,
Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J. and Hess, H. F.
(2006). Imaging intracellular fluorescent proteins at nanometer resolution.
Science 313, 1642-1645.
Brinkley, B. R., Murphy, P. and Richardson, L. C. (1967). Procedure for
embedding in situ selected cells cultured in vitro. J. Cell Biol. 35, 279-283.
Bü tschli, O. (1875). Vorlä ufige Mittheilung ü ber Untersuchungen, betreffend die
ersten Entwickelungsvorgä nge im befruchteten Ei von Nematoden und
Schnecken. Z. Wiss. Zool. 25, 201-213.
Bü tschli, O. (1876). Studien ü ber die ersten Entwicklungsvorgä nge der Eizelle, die
Zelltheilung und die Conjugation der Infusorien. Abhandlungen der
Senckenbergischen Naturforschenden Gesellschaft Frankfurt am Main 10, 1-250.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green
fluorescent protein as a marker for gene expression. Science 263, 802-805.
Chretien, D., Fuller, S. D. and Karsenti, E. (1995). Structure of growing microtubule
ends: two-dimensional sheets close into tubes at variable rates. J. Cell Biol. 129,
1311-1328.
Dernburg, A. F. (2001). Here, there, and everywhere: kinetochore function on
holocentric chromosomes. J. Cell Biol. 153, F33-F38.
Ding, R., McDonald, K. L. and McIntosh, J. R. (1993). Three-dimensional
reconstruction and analysis of mitotic spindles from the yeast,
Schizosaccharomyces pombe. J. Cell Biol. 120, 141-151.
Dong, Y., Vanden Beldt, K. J., Meng, X., Khodjakov, A. and McEwen, B. F.
(2007). The outer plate in vertebrate kinetochores is a flexible network with
multiple microtubule interactions. Nat. Cell Biol. 9, 516-522.
Downing, K. H. and Nogales, E. (2010). Cryoelectron microscopy applications in
the study of tubulin structure, microtubule architecture, dynamics and assemblies,
and interaction of microtubules with motors. Methods Enzymol. 483, 121-142.
Dubochet, J. and McDowall, A. W. (1981). Vitrification of pure water for electron
microscopy. J. Microsc. 124, RP3-RP4.
Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383
Dubochet, J., Lepault, J., Freeman, R., Berriman, J. A. and Homo, J.-C. (1982).
Electron microscopy of frozen water and aqueous solutions. J. Microsc. 128,
219-237.
Flemming, W. (1878). Zur Kenntniss der Zelle und ihrer Theilungs-Erscheinungen.
Schriften des Naturwissenschaftlichen Vereins fü r Schleswig-Holstein 3.
Flemming, W. (1882). Zellsubstanz, Kern und Zelltheilung. Leipzig: Verlag von
F.C.W. Vogel.
Flemming, W. (1965). Contributions to the knowledge of the cell and its vital
processes. J. Cell Biol. 25, 3-69.
Fuge, H. (1973). Microtubule distribution in metaphase and anaphase spindles of
the spermatocytes of Pales ferruginea. A quantitative analysis of serial cross-
sections. Chromosoma 43, 109-143.
Gan, L., Ladinsky, M. S. and Jensen, G. J. (2011). Organization of the smallest
eukaryotic spindle. Curr. Biol. 21, 1578-1583.
Goshima, G., Mayer, M., Zhang, N., Stuurman, N. and Vale, R. D. (2008). Augmin:
a protein complex required for centrosome-independent microtubule generation
within the spindle. J. Cell Biol. 181, 421-429.
Grill, S. W., Gö nczy, P., Stelzer, E. H. K. and Hyman, A. A. (2001). Polarity controls
forces governing asymmetric spindle positioning in the Caenorhabditis elegans
embryo. Nature 409, 630-633.
Gustafsson, M. G. L. (2005). Nonlinear structured-illumination microscopy: wide-
field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad.
Sci. USA 102, 13081-13086.
Harel, A., Zlotkin, E., Nainudel-Epszteyn, S., Feinstein, N., Fisher, P. A. and
Gruenbaum, Y. (1989). Persistence of major nuclear envelope antigens in an
envelope-like structure during mitosis in Drosophila melanogaster embryos.
J. Cell Sci. 94, 463-470.
Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman, A.
and Karsenti, E. (1996). Self-organization of microtubules into bipolar spindles
around artificial chromosomes in Xenopus egg extracts. Nature 382, 420-425.
Helmke, K. J., Heald, R. and Wilbur, J. D. (2013). Interplay between spindle
architecture and function. Int. Rev. Cell Mol. Biol. 306, 83-125.
Hö ö g, J. L., Schwartz, C., Noon, A. T., O’Toole, E. T., Mastronarde, D. N.,
McIntosh, J. R. and Antony, C. (2007). Organization of interphase microtubules
in fission yeast analyzed by electron tomography. Dev. Cell 12, 349-361.
Hughes, A. F. and Swann, M. M. (1947). Anaphase movements in the living cell.
J. Exp. Biol. 25, 45-72.
Inoué , S. (1951). A method for measuring small retardations of structures in living
cells. Exp. Cell Res. 2, 513-517.
Inoué , S. (1953). Polarization optical studies of the mitotic spindle. I. The
demonstration of spindle fibers in living cells. Chromosoma 5, 487-500.
Inoue, S. and Oldenbourg, R. (1998). Microtubule dynamics in mitotic spindle
displayed by polarized light microscopy. Mol. Biol. Cell 9, 1603-1607.
Irobalieva, R. N., Martins, B. and Medalia, O. (2016). Cellular structural biology as
revealed by cryo-electron tomography. J. Cell Sci. 129, 469-476.
Johnson, K. A. and Borisy, G. G. (1975). The equilibrium assembly of microtubules
in vitro. Soc. Gen. Physiol. Ser. 30, 119-141.
Klar, T. A., Jakobs, S., Dyba, M., Egner, A. and Hell, S. W. (2000). Fluorescence
microscopy with diffraction resolution barrier broken by stimulated emission. Proc.
Natl. Acad. Sci. USA 97, 8206-8210.
Kremer, J. R., Mastronarde, D. N. and McIntosh, J. R. (1996). Computer
visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116,
71-76.
Ladinsky, M. S., Mastronarde, D. N., McIntosh, J. R., Howell, K. E. and
Staehelin, L. A. (1999). Golgi structure in three dimensions: functional insights
from the normal rat kidney cell. J. Cell Biol. 144, 1135-1149.
Ledbetter, M. C. and Porter, K. R. (1963). A “Microtubule” in plant cell fine structure.
J. Cell Biol. 19, 239-250.
Mahamid, J., Pfeffer, S., Schaffer, M., Villa, E., Danev, R., Cuellar, L. K., Forster,
F., Hyman, A. A., Plitzko, J. M. and Baumeister, W. (2016). Visualizing the
molecular sociology at the HeLa cell nuclear periphery. Science 351, 969-972.
Mandelkow, E. M., Mandelkow, E. and Milligan, R. A. (1991). Microtubule
dynamics and microtubule caps: a time-resolved cryo-electron microscopy study.
J. Cell Biol. 114, 977-991.
Journal of Cell Science
Mastronarde, D. N. (1997). Dual-axis tomography: an approach with alignment
methods that preserve resolution. J. Struct. Biol. 120, 343-352.
Mastronarde, D. N. (2005). Automated electron microscope tomography using
robust prediction of specimen movements. J. Struct. Biol. 152, 36-51.
Mastronarde, D. N., McDonald, K. L., Ding, R. and McIntosh, J. R. (1993).
Interpolar spindle microtubules in PTK cells. J. Cell Biol. 123, 1475-1489.
Mayzel, W. (1875). Ueber eigenthü mliche Vorgä nge bei der Theilung der Kerne in
Epithelialzellen. Zentralblatt fü r die Medizinischen Wissenschaften. 849-850.
McDonald, K. L. (2009). A review of high-pressure freezing preparation techniques
for correlative light and electron microscopy of the same cells and tissues.
J. Microsc. 235, 273-281.
McDonald, K., Pickett-Heaps, J. D., McIntosh, J. R. and Tippit, D. H. (1977). On
the mechanism of anaphase spindle elongation in Diatoma vulgare. J. Cell Biol.
74, 377-388.
7
REVIEW
McDonald, K. L., Edwards, M. K. and McIntosh, J. R. (1979). Cross-sectional
structure of the central mitotic spindle of Diatoma vulgare. Evidence for specific
interactions between antiparallel microtubules. J. Cell Biol. 83, 443-461.
McDonald, K. L., O’Toole, E. T., Mastronarde, D. N. and McIntosh, J. R. (1992).
Kinetochore microtubules in PTK cells. J. Cell Biol. 118, 369-383.
McIntosh, J. R. (2017). Mechanisms of Mitotic Chromosome Segregation. Basel,
Switzerland: MDPI AG.
McIntosh, J. R. and Landis, S. C. (1971). The distribution of spindle microtubules
during mitosis in cultured human cells. J. Cell Biol. 49, 468-497.
McIntosh, J. R., Grishchuk, E. L., Morphew, M. K., Efremov, A. K., Zhudenkov,
K., Volkov, V. A., Cheeseman, I. M., Desai, A., Mastronarde, D. N. and
Ataullakhanov, F. I. (2008). Fibrils connect microtubule tips with kinetochores: a
mechanism to couple tubulin dynamics to chromosome motion. Cell 135,
322-333.
McIntosh, J. R., Molodtsov, M. I. and Ataullakhanov, F. I. (2012). Biophysics of
mitosis. Q. Rev. Biophys. 45, 147-207.
Melters, D. P., Paliulis, L. V., Korf, I. F. and Chan, S. W. L. (2012). Holocentric
chromosomes: convergent evolution, meiotic adaptations, and genomic analysis.
Chromosome Res. 20, 579-593.
Mennella, V., Keszthelyi, B., McDonald, K. L., Chhun, B., Kan, F., Rogers, G. C.,
Huang, B. and Agard, D. A. (2012). Subdiffraction-resolution fluorescence
microscopy reveals a domain of the centrosome critical for pericentriolar material
organization. Nat. Cell Biol. 14, 1159-1168.
Mennella, V., Agard, D. A., Huang, B. and Pelletier, L. (2014). Amorphous no
more: subdiffraction view of the pericentriolar material architecture. Trends Cell
Biol. 24, 188-197.
Mitchison, T. and Kirschner, M. (1984). Dynamic instability of microtubule growth.
Nature 312, 237-242.
Moor, H. (1987). Theory and practice of high-pressure freezing. In Cryotechniques
in Biological Electron Microscopy (ed. R.A. Steinbrecht and K. Zierold), pp.
175-191. Berlin: Springer Verlag.
Mü ller-Reichert, T. (2010). Electron Microscopy of Model Systems. Academic
Press; Burlington, MA, USA.
Mü ller-Reichert, T., Chretien, D., Severin, F. and Hyman, A. A. (1998).
Structural changes at microtubule ends accompanying GTP hydrolysis:
information from a slowly hydrolyzable analogue of GTP, guanylyl (alpha,
beta)methylenediphosphonate. Proc. Natl. Acad. Sci. USA 95, 3661-3666.
Mü ller-Reichert, T., Srayko, M., Hyman, A., O’Toole, E. T. and McDonald, K.
(2007). Correlative light and electron microscopy of early Caenorhabditis elegans
embryos in mitosis. Methods Cell Biol. 79, 101-119.
Mü ller-Reichert, T., Greenan, G., O’Toole, E. and Srayko, M. (2010). The elegans
of spindle assembly. Cell. Mol. Life Sci. 67, 2195-2213.
Musacchio, A. and Desai, A. (2017). A molecular view of kinetochore assembly
and function. Biology 6, e5.
Muscat, C. C., Torre-Santiago, K. M., Tran, M. V., Powers, J. A. and Wignall,
S. M. (2015). Kinetochore-independent chromosome segregation driven by lateral
microtubule bundles. Elife 4, e06462.
Nixon, F. M., Honnor, T. R., Clarke, N. I., Starling, G. P., Beckett, A. J., Johansen,
A. M., Brettschneider, J. A., Prior, I. A. and Royle, S. J. (2017). Microtubule
organization within mitotic spindles revealed by serial block face scanning
electron microscopy and image analysis. J. Cell Sci. 130, 1845-1855.
Noske, A. B., Costin, A. J., Morgan, G. P. and Marsh, B. J. (2008). Expedited
approaches to whole cell electron tomography and organelle mark-up in situ in
high-pressure frozen pancreatic islets. J. Struct. Biol. 161, 298-313.
Oegema, K., Desai, A., Rybina, S., Kirkham, M. and Hyman, A. A. (2001).
Functional analysis of kinetochore assembly in Caenorhabditis elegans. J. Cell
Biol. 153, 1209-1226.
O’Toole, E. T., Winey, M. and McIntosh, J. R. (1999). High-voltage electron
tomography of spindle pole bodies and early mitotic spindles in the yeast
Saccharomyces cerevisiae. Mol. Biol. Cell 10, 2017-2031.
O’Toole, E. T., McDonald, K. L., Mä ntler, J., McIntosh, J. R., Hyman, A. A. and
Mü ller-Reichert, T. (2003). Morphologically distinct microtubule ends in the
mitotic centrosome of Caenorhabditis elegans. J. Cell Biol. 163, 451-456.
O’Toole, E., van der Heide, P., McIntosh, J. R. and Mastronarde, D. (2017).
Large-scale electron tomography of cells using SerialEM and IMOD. In Cellular
Imaging: Electron Tomography and Related Techniques (ed. E. Hanssen).
Heidelberg, Berlin: Springer. (In press).
Pelletier, L., O’Toole, E., Schwager, A., Hyman, A. A. and Mü ller-Reichert, T.
(2006). Centriole assembly in Caenorhabditis elegans. Nature 444, 619-623.
Petry, S., Groen, A. C., Ishihara, K., Mitchison, T. J. and Vale, R. D. (2013).
Branching microtubule nucleation in Xenopus egg extracts mediated by augmin
and TPX2. Cell 152, 768-777.
Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. and Cormier,
M. J. (1992). Primary structure of the Aequorea victoria green-fluorescent protein.
Gene 111, 229-233.
Rahman, M. M., Munzig, M., Kaneshiro, K., Lee, B., Strome, S., Muller-Reichert,
T. and Cohen-Fix, O. (2015). Caenorhabditis elegans polo-like kinase PLK-1 is
Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383
required for merging parental genomes into a single nucleus. Mol. Biol. Cell 26,
4718-4735.
Redemann, S., Weber, B., Mö ller, M., Verbavatz, J.-M., Hyman, A. A., Baum, D.,
Prohaska, S. and Mü ller-Reichert, T. (2014). The segmentation of microtubules
in electron tomograms using Amira. Methods Mol. Biol. 1136, 261-278.
Redemann, S., Baumgart, J., Lindow, N., Shelley, M., Nazockdast, E., Kratz, A.,
Prohaska, S., Brugues, J., Furthauer, S. and Muller-Reichert, T. (2017). C.
elegans chromosomes connect to centrosomes by anchoring into the spindle
network. Nat. Commun. 8, 15288.
Ribeiro, S. A., Vagnarelli, P., Dong, Y., Hori, T., McEwen, B. F., Fukagawa, T.,
Flors, C. and Earnshaw, W. C. (2010). A super-resolution map of the vertebrate
kinetochore. Proc. Natl. Acad. Sci. USA 107, 10484-10489.
Rodriguez, E. A., Campbell, R. E., Lin, J. Y., Lin, M. Z., Miyawaki, A., Palmer,
A. E., Shu, X., Zhang, J. and Tsien, R. Y. (2017). The growing and glowing
toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci. 42,
111-129.
Roth, L. E. and Daniels, E. W. (1962). Electron microscopic studies of mitosis in
amebae: II The Giant Ameba Pelomyxa carolinensis. J. Cell Biol. 12, 57-78.
Rust, M. J., Bates, M. and Zhuang, X. (2006). Sub-diffraction-limit imaging by
stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793-795.
Sabatini, D. D., Bensch, K. and Barrnett, R. J. (1963). Cytochemistry and electron
microscopy. The preservation of cellular ultrastructure and enzymatic activity by
aldehyde fixation. J. Cell Biol. 17, 19-58.
Salmon, E. D., Leslie, R. J., Saxton, W. M., Karow, M. L. and McIntosh, J. R.
(1984). Spindle microtubule dynamics in sea urchin embryos: analysis using a
fluorescein-labeled tubulin and measurements of fluorescence redistribution after
laser photobleaching. J. Cell Biol. 99, 2165-2174.
Schmidt, W. J. (1937). Doppelbrechung von Karyoplasma, Metaplasma und
Zytoplasma. Berlin: Gebrueder Borntraeger.
Schmidt, W. J. (1939). Doppelbrechung der Kernspindel und Zugfasertheorie der
Chromosomenbewegung. Chromosoma 1, 253-264.
Scholey, J. M., Civelekoglu-Scholey, G. and Brust-Mascher, I. (2016). Anaphase
B. Biology 5, 51.
Shimomura, O., Johnson, F. H. and Saiga, Y. (1962). Extraction, purification and
properties of aequorin, a bioluminescent protein from the luminous
hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59, 223-239.
VandenBeldt, K. J., Barnard, R. M., Hergert, P. J., Meng, X., Maiato, H. and
McEwen, B. F. (2006). Kinetochores use a novel mechanism for coordinating the
dynamics of individual microtubules. Curr. Biol. 16, 1217-1223.
Villa, E., Schaffer, M., Plitzko, J. M. and Baumeister, W. (2013). Opening windows
into the cell: focused-ion-beam milling for cryo-electron tomography. Curr. Opin.
Struct. Biol. 23, 771-777.
von Dassow, G., Verbrugghe, K. J., Miller, A. L., Sider, J. R. and Bement, W. M.
(2009). Action at a distance during cytokinesis. J. Cell Biol. 187, 831-845.
Vukusic, K., Buda, R., Bosilj, A., Milas, A., Pavin, N. and Tolic, I. M. (2017).
Microtubule sliding within the bridging fiber pushes kinetochore fibers apart to
segregate chromosomes. Dev. Cell 43, 11-23 e16.
Waterman-Storer, C. M. and Salmon, E. D. (1997). Actomyosin-based retrograde
flow of microtubules in the lamella of migrating epithelial cells influences
microtubule dynamic instability and turnover and is associated with microtubule
breakage and treadmilling. J. Cell Biol. 139, 417-434.
Weber, B., Greenan, G., Prohaska, S., Baum, D., Hege, H.-C., Mü ller-Reichert,
T., Hyman, A. A. and Verbavatz, J.-M. (2012). Automated tracing of microtubules
in electron tomograms of plastic embedded samples of C. elegans embryos.
J. Struct. Biol. 178, 129-138.
Weber, B., Tranfield, E. M., Hö ö g, J. L., Baum, D., Antony, C., Hyman, T.,
Verbavatz, J. M. and Prohaska, S. (2014). Automated stitching of microtubule
centerlines across serial electron tomograms. PLoS ONE 9, e113222.
Webster, R. E., Osborn, M. and Weber, K. (1978). Visualization of the same PtK2
cytoskeletons by both immunofluorescence and low power electron microscopy.
Exp. Cell Res. 117, 47-61.
Weisenberg, R. C. (1972). Microtubule formation in vitro in solutions containing low
calcium concentrations. Science 177, 1104-1105.
Weisenberg, R. C., Borisy, G. G. and Taylor, E. W. (1968). The colchicine-binding
protein of mammalian brain and its relation to microtubules. Biochemistry 7,
4466-4479.
Journal of Cell Science
Wilson, E. B. (1902). The Cell in Development and Inheritance. New York: The
Macmillan Company.
Winey, M., Mamay, C. L., O’Toole, E. T., Mastronarde, D. N., Giddings, T. H., Jr,
McDonald, K. L. and McIntosh, J. R. (1995). Three-dimensional ultrastructural
analysis of the Saccharomyces cerevisiae mitotic spindle. J. Cell Biol. 129,
1601-1615.
Winey, M., Morgan, G. P., Straight, P. D., Giddings, T. H., Jr. and Mastronarde,
D. N. (2005). Three-dimensional ultrastructure of Saccharomyces cerevisiae
meiotic spindles. Mol. Biol. Cell 16, 1178-1188.
Wu, J. and Akhmanova, A. (2017). Microtubule-organizing centers. Annu. Rev.
Cell Dev. Biol. 33, 51-75.