Mitotic Spindles Revisited – New Insights from 3D Electron Microscopy Thomas Müller-Reichert‡, Robert Kiewisz and Stefanie Redemann*,‡

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REVIEW ARTICLE SERIES: IMAGING Mitotic spindles revisited – new insights from 3D electron microscopy Thomas Müller-Reichert‡, Robert Kiewisz and Stefanie Redemann*,‡

ABSTRACT microtubules of bipolar spindles are grouped into different classes. The mitotic spindle is a complex three-dimensional (3D) apparatus A canonical view of mitotic spindle structure in metaphase (Fig. 1) that functions to ensure the faithful segregation of chromosomes shows the following classes of microtubules (MTs): astral-MTs during cell division. Our current understanding of spindle architecture (AMTs), kinetochore-MTs (KMTs), interdigitating-MTs (IMTs) and is mainly based on a plethora of information derived from light spindle MTs (SMTs). AMTs are those microtubules that grow away microscopy with rather few insights about spindle ultrastructure from centrosomes towards the cellular cortex, thus mainly playing a obtained from electron microscopy. In this Review, we will provide role in positioning the spindle apparatus (Grill et al., 2001). The plus- insights into the history of imaging of mitotic spindles and highlight ends of KMTs are directly connected to the kinetochores (i.e. to recent technological advances in electron tomography and data specific centromeric microtubule-binding sites on the chromosomes) processing, which have delivered detailed 3D reconstructions of (Musacchio and Desai, 2017). IMTs are thought to interact with each mitotic spindles in the early embryo of the nematode Caenorhabditis other in the midzone of the spindle. This interaction is supposed to elegans. Tomographic reconstructions provide novel views on build a direct pole-to-pole connection through microtubules of spindles and will enable us to revisit and address long-standing opposite polarity (Mastronarde et al., 1993). SMTs are microtubules questions in the field of mitosis. in the body of the spindles that are neither overlapping with other microtubules (IMTs), nor connecting to the kinetochore (KMTs) KEY WORDS: 3D reconstruction, Electron tomography, Microtubule (Redemann et al., 2017). In addition, numerous microtubule- segmentation, Microtubule, Mitosis, Spindle associated proteins and molecular motors function during spindle formation, positioning and chromosome segregation (Helmke et al., Introduction 2013; McIntosh et al., 2012). During mitosis, chromosomes are segregated with high precision to This general scheme of spindle organization is used in many generate two identical daughter cells. The process of segregation is textbooks to describe the ‘basic organization’ of mitotic spindles. driven by a dynamic bipolar spindle apparatus (Helmke et al., 2013; However, despite the evolutionary conservation of essential McIntosh, 2017). Microtubules are the main building blocks of such proteins and regulatory factors, there is a remarkable variability in spindles. They polymerize from heterodimers consisting of α- and the structure and organization of mitotic spindles between β-tubulin and display a characteristic stochastic switch from slow organisms and within cells from a single organism. In addition, growth to fast shrinkage, described as dynamic instability the process of chromosome segregation appears to be variable, in (Mitchison and Kirschner, 1984). that organisms show differences in the mechanism of anaphase Microtubules show a distinct polarity with a relatively stable (Scholey et al., 2016). minus-end and a dynamic plus-end (Helmke et al., 2013). Most So, where does our current understanding of mitotic spindle microtubule minus-ends are associated with the centrosome (Wu and structure and organization come from and what technology was used Akhmanova, 2017). This non-membrane-bound organelle is the to increase this body of knowledge? Clearly, our understanding of major site of microtubule nucleation in animal cells, although other mitosis is intimately linked to improvements in light and electron sites of microtubule nucleation have been reported. In general, microscopy, as well as in advances in specimen preparation and microtubules can also be formed within the spindle itself, a image processing. Key steps in specimen preparation and imaging are phenomenon called microtubule branching (Goshima et al., 2008; briefly summarized in the following paragraphs. Petry et al., 2013), or nucleated around chromosomes as observed in Xenopus extracts (Heald et al., 1996). Microtubule nucleation at the Shedding light on mitotic spindles – highlights of mitosis mitotic centrosome, however, causes a distinct orientation of research microtubules within the bipolar spindle, in that the microtubule The vast majority of information we currently have about mitotic minus-ends are located within the pericentriolar material (PCM) of spindles is derived from light microscopy. The first investigators to the centrosome and the microtubule plus-ends are growing away from describe the process of mitosis were the Polish scientist Wacław the centrosome. According to the direction of microtuble plus-end Mayzel (Mayzel, 1875) and the German scientist Otto Bütschli growth and interaction with a particular cellular target site, (Bütschli, 1875, 1876). About 3 years later, the term ‘mitosis’, derived from the Greek word for ‘thread’, was coined by the Technische Universität Dresden, Experimental Center, Medical Faculty Carl Gustav German scientist Walther Flemming (Flemming, 1878, 1965). Carus, Fiedlerstraße 42, 01307 Dresden, Germany. Flemming investigated cell division and used aniline dyes to stain *Present address: Center for Membrane and Cell Physiology and Department of Molecular Physiology and Biological Physics, University of Virginia, School of cells and observe chromosome distribution in the fins and gills of Medicine, Charlottesville, VA 22908-0886, USA. salamanders (Fig. 2). Even though Flemming also observed living

‡ cells, the groundbreaking findings of mitotic chromosome Authors for correspondence ([email protected]; [email protected]) segregation were made from analysis of fixed and stained

R.K., 0000-0003-2733-4978; S.R., 0000-0003-2334-7309 samples, providing a static view about dynamic spindles. The Journal of Cell Science

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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

Key early as in 1953, Chaetopterus eggs were treated with colchicine or KMTs AMTs SMTs IMTs Centrosome Chromosome 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 Fig. 1. Canonical view of mitotic spindle structure in metaphase. The bipolar spindle is organized from two centrosomes (light green spheres, with Daniels, 1962) and glutaraldehyde (Sabatini et al., 1963) for centriole pair). Microtubule minus-ends (−) are anchored at the centrosome specimen fixation for electron microscopy that finally proved the and plus-ends (+) are growing out towards target sites. Kinetochore existence of spindle fibers, which were described and named microtubules (KMTs, red) are attached to chromosomes (gray), astral ‘microtubules’ in 1963 (Ledbetter and Porter, 1963). microtubules (AMTs, dark green) are growing towards the cell periphery. The composition of microtubules was only resolved in 1968 Spindle microtubules (SMTs, light green) are growing towards the when Edwin Taylor and colleagues succeeded in the purification of chromosomes but are not connected to the kinetochores; some SMTs will eventually turn into KMTs. Interdigitating microtubules (IMTs, orange) are tubulin by co-purifying it with colchicine, a drug known to destroy thought to interact with each other in the middle of the spindle. 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 hand-drawn images of mitotic spindles produced by Flemming microtubule research, thereby significantly increasing our provided the first detailed images of spindles. It is important to point understanding of microtubule dynamics (Johnson and Borisy, out that chromosomes, because they were stained, were easy to 1975; Mitchison and Kirschner, 1984; Weisenberg, 1972). The identify. In contrast, the microtubule cytoskeleton could not be application of different optical methods, such as fluorescence observed, and appeared as empty regions in the cytoplasm. recovery after photobleaching (FRAP) (Axelrod et al., 1976) and the Nevertheless, the formation of spindle fibers and astral arrays was use of fluorescein-labeled tubulin, together with the injection of proposed (Flemming, 1878). The nature and composition of such fluorescently labeled dyes (Salmon et al., 1984), allowed the fibers and arrays, however, was unknown at the time and many ideas characterization of both microtubule and spindle dynamics in vivo. were postulated. For example, fibers and arrays were thought to Microinjection with fluorescent tubulin was extensively used for crystallize out of the protoplasm around a spindle pole, or to form mammalian tissue cells because of the advantages of the through a morphological re-arrangement of the pre-existing 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 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 Fig. 2. Spindle structures as observed and drawn by Walther Flemming in observe and characterize mitotic spindles in vivo. Today a number of 1882. The images show mitotic steps of epithelial cells of salamander larvae. GFP derivatives, such as yellow fluorescent protein (YFP) and “ The terminology as used by Flemming is: (A) Umordnungsstadium: others are available to simultaneously label distinct spindle Metakinese” (remodeling state: metakinesis). (B) “Endform der Metakinese” (end stage of metakinesis). (C) “Folgendes Stadium: Auseinanderweichen, components in living cells (Rodriguez et al., 2017). Anfang der Tochtersternform” (following stage: moving apart, beginning of Over the past 20 years, advances in light microscopy, such as daughter star formation). Reproduced from Flemming, 1882. stimulated emission depletion (STED) microscopy (Klar et al., 2000) Journal of Cell Science

2 REVIEW Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383 or saturated structural illumination microscopy (SSIM) (Gustafsson, interaction of molecular motors with microtubule walls. Both 2005) have resulted in an improvement of resolution from ∼500 nm negative staining and immobilization of microtubules in frozen- to 100 nm. Moreover, the development of super-resolution hydrated samples revealed information about protofilament numbers, technology, such as photoactivated localization (PALM) (Betzig functional end-morphologies of individual microtubules, and the et al., 2006) and stochastic optical reconstruction microscopy mode of interaction of specific minus- and plus-end-directed motors (STORM) (Rust et al., 2006), has further pushed the resolution (Downing and Nogales, 2010). It was the discovery of vitrification of limit to ∼40 nm. So far, superresolution microscopy has been applied water (Dubochet et al., 1982; Dubochet and McDowall, 1981) that to analyze spindle components, such as centrosomes (Mennella et al., laid the groundwork for the presentation of atomic maps of tubulin 2014, 2012) and kinetochores (Ribeiro et al., 2010). and culminated in the 2017 Nobel prize for chemistry for the In summary, over the past 50 years, remarkable improvements in development of cryo-EM for the high-resolution determination of reagents and technology for light microscopy enabled an explosion macromolecular structure (Beck and Baumeister, 2016; Irobalieva of knowledge about the structure and function of mitotic spindles. et al., 2016). However, what about the structural analysis of Although providing great tools to analyze spindle dynamics in microtubules within the cellular context? living cells, light microscopy today cannot offer sufficient Early EM studies used glutaraldehyde to routinely fix whole resolution to visualize each microtubule in a given spindle, to cells. After dehydration at room temperature and plastic embedding, determine the length of individual microtubules and assign each thin (60–80 nm) sections were then imaged in a transmission microtubule to a distinct functional class according to their position electron microscope. Spindle components from a number of within the spindle (Fig. 3A). Such a detailed analysis is only specimens were thus visualized in two dimensions. From the possible using 3D electron microscopy (Fig. 3B) and the major beginning, the aim was to combine the contextual information of advances in this field for mitosis research are briefly summarized in light microscopy with the ultrastructural data as obtained from EM the next section. to gain information about specific mitotic stages and spindle components (Brinkley et al., 1967; Webster et al., 1978). Correlative Making use of electrons – ultrastructural views on spindle light and electron microscopic (CLEM) approaches are still used in architecture current cell biological research related to mitosis (McDonald, 2009; Electron microscopy (EM) of in vitro assembled microtubules Müller-Reichert et al., 2007). stimulated investigations on the structure of microtubules and the 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 current model systems, such as yeasts, worms, flies and mammalian samples (Müller-Reichert, 2010). Fig. 3. Organization of the first mitotic spindle at metaphase in the early An in-depth analysis of the mitotic spindle architecture in 3D C. elegans embryo. (A) Level of resolution as expected from light microscopy. requires a high resolution in the z direction. Obviously, the (B) Level of information obtained from electron microscopy. The density map of z-resolution in serial-section reconstructions is rather low (as it is image A was extracted from the data shown in B. The data is based on a 3D limited by the section thickness, typically 60–80 nm) and certainly a reconstruction of microtubules obtained from electron tomography, with major limiting factor of this approach. This limitation is evident in microtubules simplified as straight lines. A was convolved with a two- early 3D reconstructions of serial sections of mitotic spindles in 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 budding yeast (Winey et al., 1995), and it was one of the major can only be obtained from electron microscopy data, as light microscopy can motivations to turn to electron tomography to reconstruct not deliver single-microtubule resolution. Scale bar: 5 µm. microtubules at the yeast spindle poles (O’Toole et al., 1999). A Journal of Cell Science

3 REVIEW Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383 major advantage of this method is that this technology allows a (Redemann et al., 2014; Weber et al., 2012), to stitch segmented resolution of 5–6 nm in 3D. It was again the Boulder laboratory that microtubule across consecutive tomograms (Weber et al., 2014), pioneered the development of software tools for electron and to quantify spindle structure by taking each individual tomography, such as SerialEM for automatic data acquisition and reconstructed microtubule into account. This, in combination with the calculation of double-tilt tomograms (Mastronarde, 1997, 2005; developments in visualization technology, significantly pushed the O’Toole et al., 2017). In combination with high-pressure freezing 3D reconstruction of large spindles and enabled the first large-scale and freeze substitution, electron tomography was initially applied spindle reconstruction from the early embryo of the nematode to reconstruct small spindle components, such as centrosomes C. elegans (Fig. 3B), a well established model system in mitosis (O’Toole et al., 2003), centrioles in C. elegans (Pelletier et al., 2006) research (Müller-Reichert et al., 2010). and mammalian kinetochores (Dong et al., 2007; McIntosh et al., In order to generate large-scale 3D reconstructions of mitotic 2008; VandenBeldt et al., 2006), as well as whole spindles of spindles, we cryo-immobilized single-cell C. elegans embryos by budding yeast (O’Toole et al., 1999). However, analyzing larger high-pressure freezing, followed by freeze substitution and thin- volumes by electron tomography has been a difficult and time- layer embedding (Müller-Reichert et al., 2007). Such plastic- consuming task, and it took several years to develop tools to embedded embryos are then cut into 300-nm-thick serial sections. montage individual tomograms of a given section and to stack a To cover the pole-to-pole volume of a metaphase spindle, for number of consecutive tomograms to increase the reconstructed instance, we acquired 12 double-tilt tomograms per section, with ∼5 volume (Höög et al., 2007; Ladinsky et al., 1999; Noske et al., GB of tomographic data per section (Redemann et al., 2017). 2008). Last but not least, microtubules had to be segmented Applying the same procedure, we then acquired 24 of these sections manually and the joining of data over several consecutive sections to cover the z-axis, thus recording a significant volume of the mitotic was difficult and also needed to be done manually. Therefore, 3D metaphase spindle (Fig. 4). Following image acquisition for reconstruction of complete spindles containing hundreds to electron tomography, dual-axis tomograms were then computed thousands of microtubules was a massive undertaking and fraught for each montage panel. with numerous technical difficulties. The C. elegans spindles were modeled in two steps. First, Keeping these technical hurdles in mind, it is not surprising that microtubules were automatically segmented in each section, using a we currently have only very limited ultrastructural 3D data on template-matching approach, followed by an automatic tracing mitotic spindles. In fact, most of the data had already been obtained algorithm (Redemann et al., 2014; Weber et al., 2012). Second, the in the 1970s to 1990s in just a few model systems (Ding et al., 1993; segmented microtubules were then used to automatically stitch the Mastronarde et al., 1993; McDonald et al., 1992; Winey et al., serial sections to a single stack (Weber et al., 2014). However, we 1995). However, recent advances in software tools for electron found that this automatic workflow for stitching was often tomography (see below), have allowed us to obtain large-scale problematic owing to the large size of the data, deformation of reconstructions of mitotic spindles in C. elegans and we will present individual sections and missing visual validation steps. Therefore, the unexpected findings of this study in the next paragraphs. we developed a novel stitching tool that allows users to achieve an alignment and matching of microtubules across a stack of serial Going further on the 3D route – current advances in electron sections by applying a combination of automatic steps, visual tomography and image processing validation and interactive correction. This approach supports quality Several advances in software development have led to automated assessment by highlighting regions that have been checked for approaches to segment microtubules in electron tomograms 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 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/). Journal of Cell Science

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

Key KMTs AMTs SMTs CCentrosomeentrosome GrowiGrowingng enendd Shrin Shrinkingking 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/). Journal of Cell Science

5 REVIEW Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383 complete NEBD occurring at mitotic prophase (McDonald et al., deformations of the sample can be largely avoided. Unfortunately, 1992). A semi-closed (or semi-open) mitosis is observed in the biggest advantage of the SBF-SEM, the sample being contained C. elegans (Albertson, 1984) and Drosophila embryos (Harel within a single block, is also its biggest disadvantage. In order to et al., 1989). In the first embryonic division in C. elegans, the visualize cellular structures by EM, those structures need to be post- nuclear envelope is opened at the position of the two opposite stained by heavy metals to increase the contrast of the sample. centrosomes, a phenomenon called ‘polar fenestration’ (Rahman However, if the sample is contained in a big volume of resin, then et al., 2015). the staining solution will not be able to penetrate the entire volume, Second, embryonic or cellular systems differ in the organization of resulting in a relatively low contrast. Therefore, at this point, the use the kinetochore. The monocentric kinetochore of mammalian cells is of this technology is largely limited by a low contrast when using the most common type and characterized by a distinct region on cryo-immobilized plastic sections for spindle reconstruction. chromosomes (i.e. the centromere) to which KMTs are physically Despite encouraging work, there is a long way to go before the attached (Musacchio and Desai, 2017). In addition, KMTs in same precision in the quantification of microtubule architecture can mammalian systems are organized into k-fibers (McDonald et al., be achieved by this method as is currently obtained by tomographic 1992). In budding yeast, only a single microtubule is attached to a reconstruction of semi-thick sections. ‘point’ kinetochore (Winey et al., 1995). In contrast, chromosomes One future goal would be to apply cryo-electron tomography to with a holocentric kinetochore show an attachment of KMTs along image the mitotic spindle in a frozen-hydrated state, thus avoiding their entire length. This phenomenon of a dispersed kinetochore, dehydration of the sample by freeze substitution. Such work has however, is fairly common in nature and can be observed in been carried out on the small spindles of Ostreococcus tauri (Gan nematodes, hemipteran insects and in a number of monocot plants et al., 2011), but applying these methods to larger more complex (Melters et al., 2012). Interestingly, both types of kinetochores appear spindles would be a major technical challenge as sectioning of to have a similar protein composition, despite the difference in their frozen samples is difficult due to the brittle nature of the ice. An functional organization (Dernburg, 2001). alternative means to apply cryo-electron tomography could Third, cellular systems show differences in their anaphase A certainly be the production of cryo-lamellae as obtained by cryo- (shortening of the pole-to-chromosome distance) and anaphase B focused ion beam-scanning electron microscopy (cryo-FIB-SEM) (increase in the pole-to-pole distance) patterns. While some show (Mahamid et al., 2016; Villa et al., 2013). This technology uses a either anaphase A or B, others show both phases, with either focused ion beam to produce a section (or cryo-lamella) of a frozen- anaphase A or B occurring first (Oegema et al., 2001; von Dassow hydrated cell, avoiding sectioning with an ultramicrotome. et al., 2009). However, it is not possible to create serial sections with this It is the diversity in microtubule dynamics and spindle organization technology as the regions above and below the lamella are milled that makes it difficult to draw general conclusions about spindle away by the focused ion beam in the production process. Although organization and raises a number of important questions. For this technique is technically highly demanding, cryo-FIB-SEM instance, are there similarities in the length distribution of KMTs in makes it possible to obtain high-resolution information of selected C. elegans and k-fibers in mammalian cells? Is it possible to draw regions of spindles. Cryo-electron tomography of frozen-hydrated general conclusions about spindle structure and to work out cells would push the level of resolution further and would be an underlying principles that are operating behind the different excellent approach to resolve any motor proteins located in-between systems? What needs to be done next to answer these questions? microtubules in the spindle, given that an averaging during steps of image processing would be possible. Perspectives In the future, the availability of large reconstructions with a While we have succeeded in the reconstruction of a number of single-microtubule resolution from different systems should make it C. elegans mitotic spindles, there are still a number of technical possible to also revisit the mammalian spindle architecture and issues that need to be solved. One demanding task is the further question any conclusions and hypothesis that were made on the development of computational tools for image acquisition and basis of light microscopy observations. An important question to processing, and quantitative analysis of the large-scale tomographic revisit, for example, would be the origin of microtubules in the data. The bottleneck of serial electron tomography is still the spindle [i.e. centrosome-based nucleation (Wu and Akhmanova, tremendous amount of time needed to join numerous tomograms for 2017), nucleation on pre-existing microtubules (Goshima et al., montaging and stitching. 2008; Petry et al., 2013) and nucleation on or around chromosomes Automation of data processing is an important aspect for high- (Heald et al., 1996)]. Our understanding of the role of each throughput analysis, and we have already pushed automation of nucleation mechanism and its contribution to spindle formation is several steps. In addition, the use of machine learning, or deep certainly limited. By a combination of dynamic and ultrastructural learning, is very attractive for future data analysis. As an example, data as obtained from both light and electron microscopy, it will be this could be implemented to automatically group microtubule ends possible to further investigate the origin of microtubules within according to their morphology (either closed or open), giving us spindles. Another issue to analyze is the re-occuring question of the insights into their dynamic state, as has been achieved for presence of microtubule bundles within spindles. In order to detect microtubules assembled in vitro (Chretien et al., 1995; bundles within the spindles, however, one first has to define the Mandelkow et al., 1991; Müller-Reichert et al., 1998). properties of a bundle. How many microtubules make a bundle? Furthermore, there are also alternative microscopy techniques for How close or distant can those microtubules be? Do the spindle reconstruction. Recent advances in serial block face microtubules have to be arranged in parallel and if so, over what scanning electron microscopy (SBF-SEM) now allow the distance does this parallelism have to persist? Although there are reconstruction of large areas and volumes of cells and tissues certainly ongoing discussions about this issue in the field (Muscat (Nixon et al., 2017). Serial block-face imaging does not rely on the et al., 2015), a definite answer is currently lacking. In addition, we production and collection of serial sections to produce have also no clear understanding of how forces might be generated reconstruction of large volumes, and its biggest advantage is that and transmitted within and outside of the spindle. What are the Journal of Cell Science

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Contributions to the knowledge of the cell and its vital role of bridging fibers that interact with k-fibers in the segregation of processes. J. Cell Biol. 25, 3-69. mitotic chromosomes in mammalian cells (Vukusic et al., 2017). At Fuge, H. (1973). Microtubule distribution in metaphase and anaphase spindles of this point, it is unclear how k-fibers and bridging fibers interact, and the spermatocytes of Pales ferruginea. A quantitative analysis of serial cross- sections. Chromosoma 43, 109-143. how this observation fits with a possible role of IMTs in mammalian Gan, L., Ladinsky, M. S. and Jensen, G. J. (2011). Organization of the smallest spindles. We anticipate that serial-section electron tomography will eukaryotic spindle. Curr. Biol. 21, 1578-1583. offer the resolution necessary to analyze the fine structure of the Goshima, G., Mayer, M., Zhang, N., Stuurman, N. and Vale, R. D. (2008). Augmin: microtubules in such bridging fibers. Now that we have the tools to a protein complex required for centrosome-independent microtubule generation within the spindle. J. Cell Biol. 181, 421-429. answer these and other interesting questions in the field, we look Grill, S. W., Gönczy, P., Stelzer, E. H. K. and Hyman, A. A. (2001). Polarity controls forward to exciting new insights in the near future. forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo. Nature 409, 630-633. Acknowledgements Gustafsson, M. G. L. (2005). Nonlinear structured-illumination microscopy: wide- The authors would like to thank Dr Johannes Baumgart (MPI-PKS, Dresden, field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Germany) for extracting the density maps in Fig. 3 and Drs Sebastian Fürthauer, Sci. USA 102, 13081-13086. Ehssan Nazockdast and Michael Shelley (Flatiron Institute, Center for Harel, A., Zlotkin, E., Nainudel-Epszteyn, S., Feinstein, N., Fisher, P. 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