Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes

The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.     

NuMA is a component of an insoluble matrix at mitotic spindle poles

NuMA associates with microtubule motors during mitosis to perform an essential role in organizing microtubule minus ends at spindle poles. Using immunogold electron microscopy, we show that NuMA is a component of an electron-dense material concentrated at both mitotic spindle poles in PtK1 cells and the core of microtubule asters formed through a centrosome-independent mechanism in cell-free mitotic extracts. This NuMA-containing material is distinct from the peri-centriolar material and forms a matrix that appears to anchor microtubule ends at the spindle pole. In stark contrast to conventional microtubule-associated proteins whose solubility is directly dependent on microtubules, we find that once NuMA is incorporated into this matrix either in vivo or in vitro, it becomes insoluble and this insolubility is no longer dependent on microtubules. NuMA is essential for the formation of this insoluble matrix at the core of mitotic asters assembled in vitro because the matrix is absent from mitotic asters assembled in a cell-free mitotic extract that is specifically depleted of NuMA. These physical properties are consistent with NuMA being a component of the putative mitotic spindle matrix in vertebrate cells. Furthermore, given that NuMA is essential for spindle pole organization in vertebrate systems, it is likely that this insoluble matrix plays an essential structural function in anchoring and/or stabilizing microtubule minus ends at spindle poles in mitotic cells.     

Force on spindle microtubule minus ends moves chromosomes

The spindle is a dynamic self-assembling machine that coordinates mitosis. The spindle’s function depends on its ability to organize microtubules into poles and maintain pole structure despite mechanical challenges and component turnover. Although we know that dynein and NuMA mediate pole formation, our understanding of the forces dynamically maintaining poles is limited: we do not know where and how quickly they act or their strength and structural impact. Using laser ablation to cut spindle microtubules, we identify a force that rapidly and robustly pulls severed microtubules and chromosomes poleward, overpowering opposing forces and repairing spindle architecture. Molecular imaging and biophysical analysis suggest that transport is powered by dynein pulling on minus ends of severed microtubules. NuMA and dynein/dynactin are specifically enriched at new minus ends within seconds, reanchoring minus ends to the spindle and delivering them to poles. This force on minus ends represents a newly uncovered chromosome transport mechanism that is independent of plus end forces at kinetochores and is well suited to robustly maintain spindle mechanical integrity.     

Chromosome movement in mitosis requires microtubule anchorage at spindle poles

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.     

Spindle pole mechanics studied in mitotic asters: dynamic distribution of spindle forces through compliant linkages

During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored. To examine the mechanical properties of microtubule cross-links we applied optical trapping to mitotic asters that form in mammalian mitotic extracts. These asters are foci of microtubules, motors, and microtubule-associated proteins that reflect many of the functional properties of spindle poles and represent centrosome-independent spindle-pole analogs. We observed bidirectional motor-driven microtubule movements, showing that microtubule linkages within asters are remarkably compliant (mean stiffness 0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motor Eg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical properties of microtubule asters in a manner consistent with its localization to spindle poles in cells. We propose that compliant linkages among microtubules provide a mechanical architecture capable of accommodating microtubule movements and distributing force among microtubules without loss of pole integrity—a mechanical paradigm that may be important throughout the spindle.     

K-fibre minus ends are stabilized by a RanGTP-dependent mechanism essential for functional spindle assembly

Chromosome segregation requires the formation of K-fibres, microtubule bundles that attach sister kinetochores to spindle poles. Most K-fibre microtubules originate around the chromosomes through a non-centrosomal RanGTP-dependent pathway and become oriented with the plus ends attached to the kinetochore and the minus ends focused at the spindle poles. The capture and stabilization of microtubule plus ends at the kinetochore has been extensively studied but very little is known on how their minus-end dynamics are controlled. Here we show that MCRS1 is a RanGTP-regulated factor essential for non-centrosomal microtubule assembly. MCRS1 localizes to the minus ends of chromosomal microtubules and K-fibres, where it protects them from depolymerization. Our data reveal the existence of a mechanism that stabilizes the minus ends of chromosomal microtubules and K-fibres, and is essential for the assembly of a functional bipolar spindle.     

Requirements for NuMA in maintenance and establishment of mammalian spindle poles

Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes.     

Mechanism and function of poleward flux in Xenopus extract meiotic spindles

In Xenopus extract meiotic spindles, microtubules slide continuously towards their minus ends, a process called poleward flux. This article discusses recent progress in determining the mechanism of poleward flux, and its functions in spindle organization and generating force on chromosomes. Bipolar organization is required for flux and inhibition of the mitotic kinesin Eg5 inhibits flux, suggesting the sliding force for flux is generated by Eg5 pushing anti-parallel microtubules apart. An important function of flux in spindle organization may be to transport minus ends nucleated at chromatin towards the pole. By pulling microtubules through attachment sites at kinetochores, flux may generate poleward force on metaphase chromosomes.     

Micromanipulation of Chromosomes in Mitotic Vertebrate Tissue Cells: Tension Controls the State of Kinetochore Movement

In mitotic vertebrate tissue cells, chromosome congression to the spindle equator in prometaphase and segregation to the poles in anaphase depend on the movements of kinetochores at their kinetochore microtubule attachment sites. To test if kinetochores sense tension to control their states of movement poleward (P) and away from the pole (AP), we applied an external force to the spindle in preanaphase newt epithelial cells by stretching chromosome arms with microneedles. For monooriented chromosomes (only one kinetochore fiber), an abrupt stretch of an arm away from the attached pole induced the single attached kinetochore to persist in AP movement at about 2 μm/min velocity, resulting in chromosome movement away from the pole. When the stretch was reduced or the needle removed, the kinetochore switched to P movement at about 2 μm/min and pulled the chromosome back to near the premanipulation position within the spindle. For bioriented chromosomes (sister kinetochores attached to opposite poles) near the spindle equator, stretching one arm toward a pole placed the kinetochore facing away from the direction of stretch under tension and the sister facing toward the stretch under reduced tension or compression. Kinetochores under increased tension exhibited prolonged AP movement while kinetochores under reduced tension or compression exhibited prolonged P movement, moving the centromeres at about 2 μm/min velocities off the metaphase plate in the direction of stretch. Removing the needle resulted in centromere movement back to near the spindle equator at similar velocities. These results show that tension controls the direction of kinetochore movement and associated kinetochore microtubule assembly/disassembly to position centromeres within the spindle of vertebrate tissue cells. High tension induces persistent AP movement while low tension induces persistent P movement. The velocity of P and AP movement appears to be load independent and governed by the molecular mechanisms which attach kinetochores to the dynamic ends of kinetochore microtubules.     

Kinetochore capture and bi-orientation on the mitotic spindle

Kinetochores are large protein complexes that are formed on chromosome regions known as centromeres. For high-fidelity chromosome segregation, kinetochores must be correctly captured on the mitotic spindle before anaphase onset. During prometaphase, kinetochores are initially captured by a single microtubule that extends from a spindle pole and are then transported poleward along the microtubule. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles - this is known as bi-orientation. Here we discuss the molecular mechanisms of these processes, by focusing on budding yeast and drawing comparisons with other organisms.     

Bi-orienting chromosomes: acrobatics on the mitotic spindle

To maintain their genetic integrity, eukaryotic cells must segregate their chromosomes properly to opposite poles during mitosis. This process mainly depends on the forces generated by microtubules that attach to kinetochores. During prometaphase, kinetochores initially interact with a single microtubule that extends from a spindle pole and then move towards a spindle pole. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles (sister kinetochore bi-orientation). If sister kinetochores interact with microtubules in wrong orientation, this must be corrected before the onset of anaphase. Here, I discuss the processes leading to bi-orientation and the mechanisms ensuring this pivotal state that is required for proper chromosome segregation.     

The drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.     

Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly

During spindle assembly, microtubules may attach to kinetochores or pair to form antiparallel pairs or interpolar microtubules, which span the two spindle poles and contribute to mitotic pole separation and chromosome segregation. Events in the specification of the interpolar microtubules are poorly understood. Using three-dimensional electron tomography and analysis of spindle dynamical behavior in living cells, we investigated the process of spindle assembly. Unexpectedly, we found that the phosphorylation state of an evolutionarily conserved Cdk1 site (S360) in γ-tubulin is correlated with the number and organization of interpolar microtubules. Mimicking S360 phosphorylation (S360D) results in bipolar spindles with a normal number of microtubules but lacking interpolar microtubules. Inhibiting S360 phosphorylation (S360A) results in spindles with interpolar microtubules and high-angle, antiparallel microtubule pairs. The latter are also detected in wild-type spindles <1 μm in length, suggesting that high-angle microtubule pairing represents an intermediate step in interpolar microtubule formation. Correlation of spindle architecture with dynamical behavior suggests that microtubule pairing is sufficient to separate the spindle poles, whereas interpolar microtubules maintain the velocity of pole displacement during early spindle assembly. Our findings suggest that the number of interpolar microtubules formed during spindle assembly is controlled in part through activities at the spindle poles.     

Correcting improper chromosome-spindle attachments during cell division

For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.     

hTPX2 is required for normal spindle morphology and centrosome integrity during vertebrate cell division

Bipolar spindle formation is essential for the accurate segregation of genetic material during cell division. Although centrosomes influence the number of spindle poles during mitosis, motor and non-motor microtubule-associated proteins (MAPs) also play key roles in determining spindle morphology. TPX2 is a novel MAP also characterized in Xenopus cell-free extracts. To examine hTPX2 (human TPX2) function in human cells, we used siRNA to knock-down its expression and found that cells lacking hTPX2 arrest in mitosis with multipolar spindles. NuMA, gamma-tubulin, and centrin localize to each pole, and nocodazole treatment of cells lacking hTPX2 demonstrates that the localization of gamma-tubulin to multiple spindle poles requires intact microtubules. Furthermore, we show that the formation of monopolar microtubule arrays in human cell extracts does not require hTPX2, demonstrating that the mechanism by which hTPX2 promotes spindle bipolarity is independent of activities focusing microtubule minus ends at spindle poles. Finally, inhibition of the kinesin Eg5 in hTPX2-depleted cells leads to monopolar spindles, indicating that Eg5 function is necessary for multipolar spindle formation in the absence of hTPX2. Our observations reveal a structural role for hTPX2 in spindles and provide evidence for a balance between microtubule-based motor forces and structural spindle components.     

Constitutive dynein activity in She1 mutants reveals differences in microtubule attachment at the yeast spindle pole body

The organization of microtubules is determined in most cells by a microtubule-organizing center, which nucleates microtubule assembly and anchors their minus ends. In Saccharomyces cerevisiae cells lacking She1, cytoplasmic microtubules detach from the spindle pole body at high rates. Increased rates of detachment depend on dynein activity, supporting previous evidence that She1 inhibits dynein. Detachment rates are higher in G1 than in metaphase cells, and we show that this is primarily due to differences in the strengths of microtubule attachment to the spindle pole body during these stages of the cell cycle. The minus ends of detached microtubules are stabilized by the presence of γ-tubulin and Spc72, a protein that tethers the γ-tubulin complex to the spindle pole body. A Spc72-Kar1 fusion protein suppresses detachment in G1 cells, indicating that the interaction between these two proteins is critical to microtubule anchoring. Overexpression of She1 inhibits the loading of dynactin components, but not dynein, onto microtubule plus ends. In addition, She1 binds directly to microtubules in vitro, so it may compete with dynactin for access to microtubules. Overall, these results indicate that inhibition of dynein activity by She1 is important to prevent excessive detachment of cytoplasmic microtubules, particularly in G1 cells.     

TPX2, A novel xenopus MAP involved in spindle pole organization

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673-685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein-dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.     

Interpolar spindle microtubules in PTK cells

Spindle microtubules (MTs) in PtK1 cells, fixed at stages from metaphase to telophase, have been reconstructed using serial sections, electron microscopy, and computer image processing. We have studied the class of MTs that form an interdigitating system connecting the two spindle poles (interpolar MTs or ipMTs) and their relationship to the spindle MTs that attach to kinetochores (kMTs). Viewed in cross section, the ipMTs cluster with antiparallel near neighbors throughout mitosis; this bundling becomes much more pronounced as anaphase proceeds. While the minus ends of most kMTs are near the poles, those of the ipMTs are spread over half of the spindle length, with at least 50% lying > 1.5 microns from the poles. Longitudinal views of the ipMT bundles demonstrate a major rearrangement of their plus ends between mid- and late anaphase B. However, the minus ends of these MTs do not move appreciably farther from the spindle midplane, suggesting that sliding of these MTs contributes little to anaphase B. The minus ends of ipMTs are markedly clustered in the bundles of kMTs throughout anaphase A. These ends lie close to kMTs much more frequently than would be expected by chance, suggesting a specific interaction. As sister kinetochores separate and kMTs shorten, the minus ends of the kMTs remain associated with the spindle poles, but the minus ends of many ipMTs are released from the kMT bundles, allowing the spindle pole and the kMTs to move away from the ipMTs as the spindle elongates.     

The microtubule catastrophe promoter Sentin delays stable kinetochore–microtubule attachment in oocytes

The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore–microtubule attachment in oocytes.