Post-slippage multinucleation renders cytotoxic variation in anti-mitotic drugs that target the microtubules or mitotic spindle

One common cancer chemotherapeutic strategy is to perturb cell division with anti-mitotic drugs. Paclitaxel, the classic microtubule-targeting anti-mitotic drug, so far still outperforms the newer, more spindle-specific anti-mitotics in the clinic, but the underlying cellular mechanism is poorly understood. In this study we identified post-slippage multinucleation, which triggered extensive DNA damage and apoptosis after drug-induced mitotic slippage, contributes to the extra cytotoxicity of paclitaxel in comparison to the spindle-targeting drug, Kinesin-5 inhibitor. Based on quantitative single-cell microscopy assays, we showed that attenuation of the degree of post-slippage multinucleation significantly reduced DNA damage and apoptosis in response to paclitaxel, and that post-slippage apoptosis was likely mediated by the p53-dependent DNA damage response pathway. Paclitaxel appeared to act as a double-edge sword, capable of killing proliferating cancer cells both during mitotic arrest and after mitotic slippage by inducing DNA damage. Our results thus suggest that to predict drug response to paclitaxel and anti-mitotics in general, 2 distinct sets of bio-markers, which regulate mitotic and post-slippage cytotoxicity, respectively, may need to be considered. Our findings provide important new insight not only for elucidating the cytotoxic mechanisms of paclitaxel, but also for understanding the variable efficacy of different anti-mitotic chemotherapeutics.     

NuSAP, a mitotic RanGTP target that stabilizes and cross-links microtubules

Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) alpha, Impbeta, and Imp7. While Impalpha and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impbeta specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.     

The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly

Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.     

Importin beta is a mitotic target of the small GTPase Ran in spindle assembly

The GTPase Ran has recently been shown to stimulate microtubule polymerization in mitotic extracts, but its mode of action is not understood. Here we show that the mitotic role of Ran is largely mediated by the nuclear transport factor importin beta. Importin beta inhibits spindle formation in vitro and in vivo and sequesters an aster promoting activity (APA) that consists of multiple, independent factors. One component of APA is the microtubule-associated protein NuMA. NuMA and other APA components are discharged from importin beta by RanGTP and induce spindle-like structures in the absence of centrosomes, chromatin, or Ran. We propose that RanGTP functions in mitosis as in interphase by locally releasing cargoes from transport factors. In mitosis, this promotes spindle assembly by organizing microtubules in the vicinity of chromosomes.     

Detection of natural products that induce aberrations of the mitotic spindle

A number of natural products which inhibit the synthesis and functioning of the mitotic spindle have been shown to be potent antitumour agents. As an aid to the detection and evaluation of these chemicals that produce aberrations of mitotic cell division we have utilized a model screening system based upon the simultaneous visualisation of both the mitotic spindle and the chromosomes.This assay allows the detection of morphological aberrations of both spindle synthesis and function produced by exposure to chemicals active upon the mitotic spindle. This assay has been evaluated for its ability to detect natural spindle damaging agents. Extracts of Catharanthus roseus, which produces the potent spindle toxins vinblastine and vincristine, derived from both leaves and tissue culture samples were assayed for their ability to induce spindle aberrations in human fibroblasts. Samples of extract which lacked natural vinblastine were spiked with pure vinblastine sulphate to estimate the sensitivity of the assay to detect low concentrations of known spindle toxins.This study indicates that spindle toxins may be identified in specific plant extracts at high levels of sensitivity thus providing an effective screening technique for the identification of potentially valuable plant materials for their use as sources of antitumour chemicals.     

Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint

Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18→E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.     

Isolation and partial characterization of a cage of filaments that surrounds the mammalian mitotic spindle

Mitotic cells have been detergent extracted under conditions that support microtubule assembly. When HeLa cells are lysed in the presence of brain tubulin, mitotic-arrested cells nucleate large asters and true metaphase cells yield spindles that remain enclosed within a roughly spherical cage of filamentous material. Detergent-extracted mitotic Chinese hamster ovary (CHO) cells show a similar, insoluble cage but the mitotic apparatus is only occasionally stabilized. In later stages of mitosis, HeLa cages are observed in elongated and furrowed configurations. In the terminal stages of cell division, two daughter filamentous networks are connected by the intercellular bridge. When observed in the electron microscope the cages include fibers 7-11 nm in diameter. The polypeptide composition of cages isolated from mitotic HeLa cells is complex, but the major polypeptides are a group with mol wt ranging from 43,000-60,000 daltons and a high molecular weight polypeptide. CHO cells contain a subset of these proteins which includes a major 58,000-dalton and a high molecular weight polypeptide. Two different antisera directed against the vimentin-containing intermediate filaments bind to polypeptides in the electrophoretic profiles of isolated HeLa and CHO cages and stain the cages, as visualized by indirect immunofluorescence. These results suggest that the HeLa and CHO cages include intermediate filaments of the vimentin type. The polypeptide composition of HeLa cages suggests that they also contain tonofilaments. The cages apparently form as the cells enter mitosis. We propose that these filamentous cages maintain the structural continuity of the cytoplasm while the cell is in mitosis.     

A novel pathway that coordinates mitotic exit with spindle position

In budding yeast, the spindle position checkpoint (SPC) delays mitotic exit until the mitotic spindle moves into the neck between the mother and bud. This checkpoint works by inhibiting the mitotic exit network (MEN), a signaling cascade initiated and controlled by Tem1, a small GTPase. Tem1 is regulated by a putative guanine exchange factor, Lte1, but the function and regulation of Lte1 remains poorly understood. Here, we identify novel components of the checkpoint that operate upstream of Lte1. We present genetic evidence in agreement with existing biochemical evidence for the molecular mechanism of a pathway that links microtubule-cortex interactions with Lte1 and mitotic exit. Each component of this pathway is required for the spindle position checkpoint to delay mitotic exit until the spindle is positioned correctly.     

KL1 is a novel microtubule severing enzyme that regulates mitotic spindle architecture

Katanin is a microtubule severing enzyme with demonstrated roles in a variety of cellular activities including mitosis. Here we identify the closely related, but relatively uncharacterized human protein, Katanin-like 1 (KL1), as a novel mitotic regulator. Over expression of KL1 in tissue culture cells results in the complete disassembly of cellular microtubules strongly suggesting that it is an active microtubule severing protein. During mitosis, the localization of KL1 is restricted to spindle poles and is notably absent from centrosomes. This is in clear contrast to conventional Katanin whose localization extends from centrosomes onto poles. Consistent with its localization, siRNA depletion of KL1 from U2OS cells results in a specific and significant reduction in the density of microtubules at spindle poles and significantly increases spindle length. Depletion of KL1 also alters the distribution of gamma-tubulin at centrosomes/spindle poles. Despite its impact on spindle morphology, we could find no evidence that KL1 influences anaphase chromosome motility. Based on our findings, we propose that KL1-mediated microtubule severing is utilized to generate microtubule seeds within the poles and that loss of this activity alters the normal balance of motor-generated forces that determine spindle length.     

Concentrating on the mitotic spindle

In eukaryotes, the microtubule-based spindle drives chromosome segregation. In this issue, Schweizer et al. (2015; J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506107) find that the spindle area is demarcated by a semipermeable organelle barrier. Molecular crowding, which is microtubule independent, causes the enrichment and/or retention of crucial factors in the spindle region. Their results add an important new feature to the models of how this structure assembles and is regulated.Mitosis is marked by the assembly of the mitotic spindle, a microtubule-based structure that facilitates accurate chromosome segregation. Many biochemical reactions are coupled to spindle assembly, from tubulin polymerization itself to the mitotic checkpoint, which inhibits chromosome disjunction until all the chromosomes are properly attached and aligned (Cleveland et al., 2003). Interestingly, these reactions are virtually unaltered over a broad morphological range; large spindles and small spindles follow roughly the same biochemical rules despite quite distinct geometries (Brown et al., 2007; Wühr et al., 2008). In this issue, Schweizer et al. report that the mitotic spindle area is delineated by membrane-bound organelles, generating a “spindle envelope” with unique molecular constituents compared with the surrounding cytoplasm. Spindle envelope–based molecular crowding provides an enticing hypothetical solution to the broad problem of confining mitotic biochemistry to a specific cellular space irrespective of cell size.Schweizer et al. (2015) used FRAP and fluorescence correlation spectroscopy (FCS) to measure the mobility of specific proteins. The authors found that tubulin and the Mad2 spindle assembly checkpoint protein were enriched in the spindle area in a microtubule polymer-independent manner. Given that the mobility of these proteins outside and within the spindle area was the same, changes in local concentration were likely a result of a barrier effect. In support of this hypothesis, modeling their FCS data with a fenestrated barrier separating the spindle area from the cytoplasm reproduced the measured FCS results. To test if a barrier surrounding the spindle area was important for spindle function, the authors disrupted the envelope area by laser microsurgery and found chromosome segregation errors consistent with defects in spindle assembly and kinetochore attachment monitoring. Thus, spindle envelope–based concentration of basal components in two critical spindle reactions, spindle assembly and mitotic checkpoint signaling, could mechanistically catalyze cell division.Molecular crowding can catalyze reactions and stabilize proteins by altering the local concentration of one or more rate-limiting components and is best characterized by membrane-bound organelles. Crowding by aggregation can greatly increase biochemical reactions without the need for a contiguous membrane (Weber and Brangwynne, 2012; Brangwynne, 2013) and this phenomenon can regulate cell cycle states (Lee et al., 2013). Here, Schweizer et al. (2015) propose that by creating a membrane-bound organelle exclusion zone, a spindle envelope could cause the molecular crowding of important spindle proteins and thereby their enrichment in the spindle area.The mitotic spindle is known to scale with cell size: smaller cells have smaller spindles (Levy and Heald, 2012). Spindle size scaling is prominent during development when repeated cell division without embryonic growth results in cells that can be several orders of magnitude smaller than that of the zygote. Recently, cytoplasm volume and tubulin concentration was shown to be an important factor in spindle size scaling; however, a curious exception to the size scaling rule is that there seems to be an upper limit to spindle size, resulting in stable spindle size when a threshold cell size is reached (Wühr et al., 2008; Good et al., 2013; Hazel et al., 2013). A spindle envelope would provide mechanisms to maintain increased local tubulin concentration independent of the absolute amount available in the cell. The net effect would be that spindle size scales in very large cells to the spindle envelope size rather than cell size in a manner analogous to chromosome size scaling to nuclear size independently of cell size (Fig. 1 A). Clearly this is a more complex problem and factors such as tubulin protein production and polymerization cofactors (such as the Tog family of proteins) clearly play an important role (Slep, 2009). However, spindle envelope–based molecular crowding could provide an elegant solution to a biochemical problem.Open in a separate windowFigure 1.Molecular crowding in the mitotic spindle. Schematic view of how a spindle envelope could mitigate spindle size scaling during development (A) or cell cycle control (B) in a common cytoplasm. (A) A spindle envelope (black dotted line) that excludes large membrane-bound organelles (yellow) could locally increase the concentration of spindle proteins (depicted as a red background) such as tubulin to control spindle size independent of cell size during early development. (B) Neighboring nuclei in a common cytoplasm (e.g., the syncytial mitotic gonad in C. elegans) could have differing mitotic states by restricting the diffusion of important regulatory proteins such as Mad2 (similar coloring as in A).A spindle envelope could also provide a cell biological solution to another developmental problem—independent cell cycle control of separate nuclei within a single cytoplasm (syncytia). For example, the mitotic region of the Caenorhabditis elegans germline contains germ cell precursors that divide independently of one another in a common cytoplasm. In some cases, two neighboring dividing nuclei can have different biochemistry, one arrested in metaphase because of a kinetochore microtubule attachment defect while the other is progressing into anaphase (Gerhold et al., 2015). By restricting the diffusive radius of signaling molecules like Mad2, a steep threshold of checkpoint activity can be maintained, allowing independent cell cycle control even in a common cytoplasm (Fig. 1 B).The mitotic spindle has long been known to exclude large membrane-bound organelles, even in the absence of microtubule polymer, leading to a hypothesized nontubulin-based “spindle matrix.” A spindle matrix would be an excellent candidate to underlie the spindle envelope. The molecular nature of a spindle matrix, however, has never been agreed upon with candidate mechanisms ranging from nonprotein macromolecules to actin (Pickett-Heaps et al., 1984; Chang et al., 2004). A convincing argument can be made that the Skeletor/Megator/Chromator proteins first identified in Drosophila melanogaster constitute a spindle matrix (Walker et al., 2000; Qi et al., 2004; Rath et al., 2004; Schweizer et al., 2014). These proteins are large and are found in the nucleus in interphase and as microtubule-independent fibrous structures in and around the spindle in mitosis. Depletion of these proteins results in mitotic errors; however, these may or may not be caused by a role as the spindle matrix.Schweizer et al. (2015) evaluated Megator (as a representative member of the complex) as a possible basis for generating the spindle envelope. FRAP and FCS showed that, like tubulin and Mad2, Megator is concentrated in the spindle envelope region independent of microtubules. However, Megator in the spindle region had slower diffusive properties compared with that around the cell periphery. Thus, unlike tubulin and Mad2, the mobility of Megator within the spindle was altered, indicating that Megator likely forms a high molecular weight complex with its binding partners Skeletor and Chromator in the spindle area, which may help form the spindle envelope.The sum of these results lead to a possible model whereby the Skeletor/Megator/Chromator proteins complex together and subsequently support a spindle envelope independent of microtubules. The spindle envelope excludes large membrane-bound organelles, leading to increased concentration of mitotic reaction constituents and thus ultimately catalyzing cell division. It will be exciting in the future to determine if the spindle area is indeed subject to molecular crowding in the purest of forms (solvent exclusion) and how this effect drives cell division.     

Types of potentials in a mitotic spindle

The dynamics of a mitotic spindle is very important to understand if the functioning of mitosis has to be understood and defined very accurately. There are a number of forces involved in such a process. Despite of the fact that there have been numerous studies done on the functioning of a mitotic spindle, there is still not a very precise understanding of this system and how it behaves. This study aims at understanding and expressing all the possible potentials which might be responsible in a mitotic spindle and its mechanism.     

The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity

Formation of a bipolar spindle is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, defects in centrosome number and structural organization can lead to a loss of bipolarity. In addition, microtubule-mediated pulling and pushing forces acting on centrosomes and chromosomes are also important for bipolar spindle formation. Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase that has essential roles in the formation of a bipolar spindle with focused poles. However, the mechanism by which Plk1 regulates spindle-pole formation is poorly understood. Here, we identify a novel centrosomal substrate of Plk1, Kizuna (Kiz), depletion of which causes fragmentation and dissociation of the pericentriolar material from centrioles at prometaphase, resulting in multipolar spindles. We demonstrate that Kiz is critical for establishing a robust mitotic centrosome architecture that can endure the forces that converge on the centrosomes during spindle formation, and suggest that Plk1 maintains the integrity of the spindle poles by phosphorylating Kiz.     

Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos

During cell division, eukaryotic cells assemble dynamic microtubule-based spindles to segregate replicated chromosomes. Rapid spindle microtubule turnover, likely derived from dynamic instability, has been documented in yeasts, plants and vertebrates. Less studied is concerted spindle microtubule poleward translocation (flux) coupled to depolymerization at spindle poles. Microtubule flux has been observed only in vertebrates, although there is indirect evidence for it in insect spermatocytes and higher plants. Here we use fluorescent speckle microscopy (FSM) to demonstrate that mitotic spindles of syncytial Drosophila embryos exhibit poleward microtubule flux, indicating that flux is a widely conserved property of spindles. By simultaneously imaging chromosomes (or kinetochores) and flux, we provide evidence that flux is the dominant mechanism driving chromosome-to-pole movement (anaphase A) in these spindles. At 18 degrees C and 24 degrees C, separated sister chromatids moved poleward at average rates (3.6 and 6.6 microm/min, respectively) slightly greater than the mean rates of poleward flux (3.2 and 5.2 microm/min, respectively). However, at 24 degrees C the rate of kinetochore-to-pole movement varied from slower than to twice the mean rate of flux, suggesting that although flux is the dominant mechanism, kinetochore-associated microtubule depolymerization contributes to anaphase A.     

A novel mitotic spindle pole component that originates from the cytoplasm during prophase

Several unique aspects of mitotic spindle formation have been revealed by investigation of an autoantibody present in the serum of a patient with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, schlerodacytyly, and telangiectasias) syndrome. This antibody was previously shown to label at the spindle poles of metaphase and anaphase cells and to be absent from interphase cells. We show here that the serum stained discrete cytoplasmic foci in early prophase cells and only later localized to the spindle poles. The cytoplasmic distribution of the antigen was also seen in nocodazole-arrested cells and prophase cells in populations treated with taxol. In normal and taxol-treated cells, the microtubules appeared to emanate from the cytoplasmic foci and polar stain, and in cells released from nocodazole block, microtubules regrew from antigen-containing centers. This characteristic distribution suggests that the antigen is part of a microtubule organizing center. Thus, we propose that a prophase originating polar antigen functions in spindle pole organization as a coalescing microtubule organizing center that is present only during mitosis. Characterization of the serum showed reactions with multiple proteins at 115, 110, 50, 36, 30, and 28 kD. However, affinity-eluted antibody from the 115/110-kD bands was shown to specifically label the spindle pole and cytosolic foci in prophase cells.     

Emerging molecular mechanisms that power and regulate the anastral mitotic spindle of flowering plants

Flowering plants, lacking centrosomes as well as dynein, assemble their mitotic spindle via a pathway that is distinct visually and molecularly from that of animals and yeast. The molecular components underlying mitotic spindle assembly and function in plants are beginning to be discovered. Here, we review recent evidence suggesting the preprophase band in plants functions analogously to the centrosome in animals in establishing spindle bipolarity, and we review recent progress characterizing the roles of specific motor proteins in plant mitosis. Loss of function of certain minus-end-directed KIN-14 motor proteins causes a broadening of the spindle pole; whereas, loss of function of a KIN-5 causes the formation of monopolar spindles, resembling those formed when the homologous motor protein (e.g., Eg5) is knocked out in animal cells. We present a phylogeny of the kinesin-5 motor domain, which shows deep divergence among plant sequences, highlighting possibilities for specialization. Finally, we review information concerning the roles of selected structural proteins at mitosis as well as recent findings concerning regulation of M-phase in plants. Insight into the mitotic spindle will be obtained through continued comparison of mitotic mechanisms in a diversity of cells.     

Bi-orienting chromosomes on the mitotic spindle

For the proper segregation of sister chromatids before cell division, each sister kinetochore must attach to microtubules that extend to opposite spindle poles. This process is called bipolar microtubule attachment or chromosome bi-orientation. The mechanism for chromosome bi-orientation lies at the heart of chromosome segregation, but is still poorly understood. Recent studies suggest that cells can promote bi-orientation by re-orienting kinetochore-spindle pole connections.