© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs211383. doi:10.1242/jcs.211383 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 1 REVIEW 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 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