Kinetochores, microtubules and the metaphase checkpoint

A decade ago, kinetochores were generally regarded as rather uninteresting structures that served only to attach mitotic chromosomes to microtubules. In the past few years, however, a number of experiments have belied this view and demonstrated that kinetochores are actively involved in moving chromosomes along the microtubules of the mitotic spindle. Now it appears that in addition to their function in motility, kinetochores act as dynamic and adaptable centres for regulating cell cycle progression through mitosis.     

Kinetochores use a novel mechanism for coordinating the dynamics of individual microtubules

Chromosome alignment during mitosis is frequently accompanied by a dynamic switching between elongation and shortening of kinetochore fibers (K-fibers) that connect kinetochores and spindle poles . In higher eukaryotes, mature K-fibers consist of 10-30 kinetochore microtubules (kMTs) whose plus ends are embedded in the kinetochore . A critical and long-standing question is how the dynamics of individual kMTs within the K-fiber are coordinated . We have addressed this question by using electron tomography to determine the polymerization/depolymerization status of individual kMTs in the K-fibers of PtK1 and Drosophila S2 cells. Surprisingly, we find that the plus ends of two-thirds of kMTs are in a depolymerizing state, even when the K-fiber exhibits net tubulin incorporation at the plus end . Furthermore, almost all individual K-fibers examined had a mixture of kMTs in the polymerizing and depolymerizing states. Therefore, although K-fibers elongate and shrink as a unit, the dynamics of individual kMTs within a K-fiber are not coordinated at any given moment. Our results suggest a novel control mechanism through which attachment to the kinetochore outer plate prevents shrinkage of kMTs. We discuss the ramifications of this new model on the regulation of chromosome movement and the stability of K-fibers.     

Centrosomes and microtubules: wedded with a ring

How centrosomes nucleate microtubule growth is a question that has puzzled cell biologists for decades. It has been suspected for some time that a centrosome contains multiple copies of a basic microtubule-nucleating structure, each of which is responsible for nucleating a single microtubule. This suspicion has now been confirmed. A ring of gamma-tubulin molecules, associated with a large protein complex, apparently serves as the long-sought-after microtubule-nucleating structure.     

The role of microtubules in contractile ring function.

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.     

Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells

When viewed by light microscopy the mitotic spindle in newt pneumocytes assembles in an optically clear area of cytoplasm, virtually devoid of mitochondria and other organelles, which can be much larger than the forming spindle. This unique optical property has allowed us to examine the behavior of individual microtubules, at the periphery of asters in highly flattened living prometaphase cells, by video-enhanced differential interference-contrast light microscopy and digital image processing. As in interphase newt pneumocytes (Cassimeris, L., N. K. Pryer, and E. D. Salmon. 1988. J. Cell Biol. 107:2223-2231), centrosomal (i.e., astral) microtubules in prometaphase cells appear to exhibit dynamic instability, elongating at a mean rate of 14.3 +/- 5.1 microns/min (N = 19) and shortening at approximately 16 microns/min. Under favorable conditions the initial interaction between a kinetochore and the forming spindle can be directly observed. During this process the unattached chromosome is repeatedly probed by microtubules projecting from one of the polar regions. When one of these microtubules contacts the primary constriction the chromosome rapidly undergoes poleward translocation. Our observations on living mitotic cells directly demonstrate, for the first time, that chromosome attachment results from an interaction between astral microtubules and the kinetochore.     

Making microtubules and mitotic spindles in cells without functional centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.     

Subunit organization in the Dam1 kinetochore complex and its ring around microtubules

All eukaryotic cells must segregate their chromosomes equally between two daughter cells at each division. This process needs to be robust, as errors in the form of loss or gain of genetic material have catastrophic effects on viability. Chromosomes are captured, aligned, and segregated to daughter cells via interaction with spindle microtubules mediated by the kinetochore. In Saccharomyces cerevisiae one microtubule attaches to each kinetochore, requiring extreme processivity from this single connection. The yeast Dam1 complex, an essential component of the outer kinetochore, forms rings around microtubules and in vitro recapitulates much of the functionality of a kinetochore-microtubule attachment. To understand the mechanism of the Dam1 complex at the kinetochore, we must know how it binds to microtubules, how it assembles into rings, and how assembly is regulated. We used electron microscopy to map several subunits within the structure of the Dam1 complex and identify the organization of Dam1 complexes within the ring. Of importance, new data strongly support a more passive role for the microtubule in Dam1 ring formation. Integrating this information with previously published data, we generated a structural model for the Dam1 complex assembly that advances our understanding of its function and will direct future experiments.     

Under Tension: Kinetochores and Basic Research

The Genetics Society of America’s Edward Novitski Prize recognizes an extraordinary level of creativity and intellectual ingenuity in the solution of significant problems in genetics research. The 2015 winner, Sue Biggins, has made significant contributions to our understanding of how chromosomes attach to the mitotic spindle, a process essential for cell division and frequently impaired in cancer. Among other achievements, Biggins was the first to demonstrate that the Aurora B protein kinase is a key regulator of kinetochore function and that chromatin composition and centromere identity can be regulated by histone proteolysis. In 2010, Biggins and her colleagues were the first to purify kinetochores and, using this system, have already made several groundbreaking discoveries about the function and structure of these crucial components of the segregation machinery.Open in a separate windowIt is easy to forget that basic research on a simple model organism has led to many fundamental insights about how cells work and what goes wrong in disease, especially with the continual pressure from funding agencies and institutions to perform translational research. It is also easy to make the mistake of thinking that all major discoveries using model organisms have already been made.In his Novitski prize essay last year, Charlie Boone noted that the yeast Saccharomyces cerevisiae is better understood than any other cell. This year, I am honored to receive the same award for work that exploited yeast’s powerful combination of relative simplicity and strong conservation of function. In a collaborative effort with Chip Asbury’s lab, we reconstituted kinetochore–microtubule attachments that withstand tension in vitro for the first time (Akiyoshi et al. 2010). Our work is an example of how yeast can provide unexpected insights into conserved processes, and why it is important to support scientists in exploring new directions.Many key discoveries about cell division were initially made using budding yeast. Centromeres were first identified and cloned from yeast, and this information was critical to constructing the first artificial chromosome (Clarke and Carbon 1980; Murray and Szostak 1983; Bloom 2015). Over the years, yeast genetic screens have identified most kinetochore components as well as the key pathways that regulate chromosome segregation (for reviews, see Biggins 2013; Malvezzi and Westermann 2014). Cell-cycle checkpoints were first demonstrated in this organism (Weinert and Hartwell 1988), and the majority of conserved spindle checkpoint genes were identified in two seminal genetic yeast screens (Hoyt et al. 1991; Li and Murray 1991).

Isolating intact kinetochores was an intellectual and technical tour-de-force that laid the groundwork for mechanistic and proteomic analysis of kinetochore proteins. Sue Biggins’ perseverance and intellectual creativity in pursuing this question produced extraordinary insights into how kinetochores interact with microtubules.— Needhi Bhalla, University of California, Santa Cruz

It has been known for decades that chromosome segregation in all organisms relies on the tension-dependent stabilization of proper kinetochore–microtubule attachments (for review, see Nicklas and Ward 1994). This behavior was attributed to a protein kinase-mediated error correction mechanism that destabilizes incorrect attachments because they lack tension (for review, see van der Horst and Lens 2014). To ultimately understand how tension regulates the kinase, I decided that we needed a system for directly manipulating tension on kinetochore–microtubule attachments in vitro. However, kinetochores had never been isolated from any organism; we were far from testing the regulation of error correction in vitro.I was trained as a geneticist, not a biochemist. However, the supportive culture at the “Hutch” (Fred Hutchinson Cancer Research Center) helped our lab to take a risk on something new. Bungo Akiyoshi (now at the University of Oxford) developed the first technique with which to purify the core yeast kinetochore (Akiyoshi et al. 2010). We got a lot of advice and support from colleagues at the Hutch with biochemistry expertise, especially from Toshi Tsukiyama. Once Bungo had optimized a protocol by which to purify kinetochores, our next step was to develop a technique for binding these kinetochores to microtubules and putting them under tension. Fortunately, we are located near Chip Asbury’s lab at the University of Washington, which pioneered laser-trapping techniques to study kinetochore proteins (Asbury et al. 2006; Franck et al. 2007; Powers et al. 2009). Together, our labs used the purified kinetochores to reconstitute kinetochore–microtubule attachments under tension. Our reconstitution system does not include the error correction kinase or any additional cellular factors, so we were surprised to find that the kinetochore–microtubule attachments were stabilized directly by tension (Akiyoshi et al. 2010). Discoveries come from unexpected places—this was preliminary work intended as the foundation for analyzing how tension regulates the error-correction kinase.This finding helps to explain one aspect of the long-standing observation that attachments under tension in vivo are stable. We do not yet know whether and how often the aneuploidy that is a hallmark of so many cancers is due to alterations in kinetochore function, but this reconstitution system can now be applied to understanding the properties of kinetochores in other organisms and in cancer cells. We have also started to use our purification technique to address other aspects of kinetochore function and structure (Gonen et al. 2012; London et al. 2012; Sarangapani et al. 2013; London and Biggins 2014; Sarangapani et al. 2014).

It is […] easy to make the mistake of thinking that all major discoveries using model organisms have already been made. —S.B.

When I first started this work, my grant renewal application did not get a fundable score because of the risky nature of the project and the lack of convincing preliminary data. Luckily, I had colleagues at the Hutch who supported our attempts to do something new despite our lack of expertise. Funding agencies often dismiss applications when the investigator isn’t well versed in the necessary skills, and it is difficult for investigators to obtain money to initiate pilot projects. The current movement of the National Institute of General Medical Sciences and other institutes at the National Institutes of Health to fund investigator-initiated research as well as project-based research is a step in the right direction. We also need to promote collaborations that can help move fields forward, to integrate genetics with other disciplines, and to foster an environment where scientists can try something new. Research in model organisms will continue to provide unpredictable insights into biological processes, especially if we stay open minded to the research we fund and we continue to support investigators who take on new endeavors.     

The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules

Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring–derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.     

Kinetochores and chromatin diminution in early embryos of Parascaris univalens

In Parascaris the mitotic chromosomes of gonial germline cells are holocentric and possess a continuous kinetochore along their entire length. By contrast, in meiotic cells, the centromeric activity is restricted to the heterochromatic tips where direct insertion of spindle microtubules into chromatin without any kinetochore plate is seen. In the presomatic cells of early embryos, which undergo heterochromatin elimination, only euchromatin shows kinetic activity. After developing a technique to separate the very resistant egg shell from the embryos, we studied the cell divisions during early embryogenesis by immunochemical and EM approaches. The results reported here show that in presomatic cells microtubules bind only the euchromatin where a continuous kinetochore plate is present. We also report observations suggesting that the binding of the long kinetochores to the mitotic spindle initiates to a limited number of sites and extends along the entire length, during chromosome condensation. The existence of different centromere stages in different cell types, rends Parascaris chromosomes a very good model to study centromere organization.     

Inherited ring chromosome 8 without loss of subtelomeric sequences

We report the first case of inherited ring chromosome 8 syndrome without loss of subtelomeric sequences. The proband is a 6 1/2-year-old boy with short stature, microcephaly, mild mental retardation, and behavioral problems including hyperactivity and attention deficit. His mother presented the same physical features but intelligence was normal. Family history also revealed an uncle and a grandmother, with short stature and microcephaly. Moderate mental retardation was reported in the uncle. Karyotypes and fluorescence in situ hybridization (FISH) analyses were performed on peripheral blood lymphocytes for both child and mother. The child's karyotype was reported as 46,XY,r(8)(p23q24.3)[24]/45,XY,-8[2] and the mother's karyotype 46,XX,r(8)(p23q24.3)[22]/45,XX,-8[2]/47,XX,r(8)(p23q24.3), +r(8)(p23q24.3)[1]. FISH studies showed no deletion of subtelomeric sequences for both child and mother indicating that no or little chromosomal euchromatic material has been deleted. These findings indicate that ring chromosome 8 without loss of subtelomeric sequences can be inherited and that carriers in a same family present with cognitive function ranging from mild mental retardation to normal intelligence.     

Kinetochores and disease: keeping microtubule dynamics in check!

The essential role of microtubules in cell division has long been known. Yet the mechanism by which microtubule attachment to chromosomes at kinetochores is regulated has only been recently revealed. Here, we review the role of kinetochore-microtubule (kMT) attachment dynamics in the cell cycle as well as emerging evidence linking deregulation of kMT attachments to diseases where chromosome mis-segregation and aneuploidy play a central role. Evidence indicates that the dynamic behavior of kMTs must fall within narrow permissible boundaries, which simultaneously allow a level of stability sufficient to establish and maintain chromosome-microtubule attachments and a degree of instability that permits error correction required for accurate chromosome segregation.     

Tau protein binding forms a 1 nm thick layer along protofilaments without affecting the radial elasticity of microtubules

Tau is one of the most abundant microtubule-associated proteins involved in kinetic stabilization and bundling of axonal microtubules. Although intense research has revealed much about tau function and its involvement in Alzheimer's disease during the past years, it still remains unclear how exactly tau binds on microtubules and if the kinetic stabilization of microtubules by tau is accompanied, at least in part, by a mechanical reinforcement of microtubules. In this paper, we have used atomic force microscopy to address both aspects by visualizing and mechanically analyzing microtubules in the presence of native tau isoforms. We could show that tau at saturating concentrations forms a 1 nm thick layer around the microtubule, but leaves the protofilament structure well visible. The latter observation argues for tau binding mainly along and not across the protofilaments. The radial elasticity of microtubules was almost unaffected by tau, consistent with tau binding along the tops of the protofilaments. Tau did increase the resistance of microtubules against rupture. Finite-element calculations confirmed our findings.     

Intra-nuclear microtubules and a mitotic spindle orientation checkpoint

Cells of the fission yeast Schizosaccharomyces pombe have a checkpoint mechanism that reportedly monitors the orientation of the mitotic spindle. Astral microtubules in pre-anaphase spindles are thought to contact the contractile actin ring at the plasma membrane in order to rotate the spindle and to sense spindle orientation. Here, we show that these microtubules are actually inside the nuclear envelope.     

Telophase-interphase transition in taxol-treatedTriticum root cells: cortical microtubules appear without the prior presence of a radial perinuclear array

Summary Taxol stabilizes phragmoplast microtubules (Mts) in cytokinetic root cells ofTriticum, causing a delay in the rate of cytokinesis. As a result, the daughter nuclei acquire interphase appearance in mid- to late-cytokinetic taxol-affected cells much earlier than in control cells. Cortical Mts in such cells appear directly in the cell cortex, without the prior organization of a radial perinuclear Mt array as in control cells. These observations suggest that: (a) Whether perinuclear Mt assembly occurs or not in post-telophase cells is a matter of timing between the nuclear cycle and cytokinesis, (b) Mt organizing activity on the daughter nuclei surface is temporal, (c) Cortical Mts can be in situ assembled in the cortex of post-telophase cells of flowering plants without any participation of perinuclear Mts.Abbreviations Mt microtubules - MTOC microtubule organizing centre - DMSO dimethyl sulfoxide - EM electron microscope