Poleward Transport of TPX2 in the Mammalian Mitotic Spindle Requires Dynein,Eg5, and Microtubule Flux

TPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.     

Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts

TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.     

Aurora B and Kif2A control microtubule length for assembly of a functional central spindle during anaphase

The central spindle is built during anaphase by coupling antiparallel microtubules (MTs) at a central overlap zone, which provides a signaling scaffold for the regulation of cytokinesis. The mechanisms underlying central spindle morphogenesis are still poorly understood. In this paper, we show that the MT depolymerase Kif2A controls the length and alignment of central spindle MTs through depolymerization at their minus ends. The distribution of Kif2A was limited to the distal ends of the central spindle through Aurora B–dependent phosphorylation and exclusion from the spindle midzone. Overactivation or inhibition of Kif2A affected interchromosomal MT length and disorganized the central spindle, resulting in uncoordinated cell division. Experimental data and model simulations suggest that the steady-state length of the central spindle and its symmetric position between segregating chromosomes are predominantly determined by the Aurora B activity gradient. On the basis of these results, we propose a robust self-organization mechanism for central spindle formation.     

Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A

To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome-based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A-independent function of TPX2 is required to bipolarize spindles.     

Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP regulates CPC-spindle interaction to ensure proper microtubule dynamics

Dynamic microtubules facilitate chromosome arrangement before anaphase, whereas during anaphase microtubule stability assists chromosome separation. Changes in microtubule dynamics at the metaphase-anaphase transition are regulated by Cdk1. Cdk1-mediated phosphorylation of Sli15/INCENP promotes preanaphase microtubule dynamics by preventing chromosomal passenger complex (CPC; Sli15/INCENP, Bir1/Survivin, Nbl1/Borealin, Ipl1/Aurora) association with spindles. However, whether Cdk1 has sole control over microtubule dynamics, and how CPC-microtubule association influences microtubule behavior, are unclear. Here, we show that Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP modulates microtubule dynamics by preventing CPC binding to the preanaphase spindle and to the central spindle until late anaphase, facilitating spatiotemporal control of microtubule dynamics required for proper metaphase centromere positioning and anaphase spindle elongation. Decreased Ipl1-dependent Sli15 phosphorylation drives direct CPC binding to microtubules, revealing how the CPC influences microtubule dynamics. We propose that Cdk1 and Ipl1/Aurora cooperatively modulate microtubule dynamics and that Ipl1/Aurora-dependent phosphorylation of Sli15 controls spindle function by excluding the CPC from spindle regions engaged in microtubule polymerization.     

Identification of a TPX2-like microtubule-associated protein in Drosophila

Chromosome segregation during mitosis and meiosis relies on the spindle and the functions of numerous microtubule-associated proteins (MAPs). One of the best-studied spindle MAPs is the highly conserved TPX2, which has been reported to have characteristic intracellular dynamics and molecular activities, such as nuclear localisation in interphase, poleward movement in the metaphase spindle, microtubule nucleation, microtubule stabilisation, microtubule bundling, Aurora A kinase activation, kinesin-5 binding, and kinesin-12 recruitment. This protein has been shown to be essential for spindle formation in every cell type analysed so far. However, as yet, TPX2 homologues have not been found in the Drosophila genome. In this study, I found that the Drosophila protein Ssp1/Mei-38 has significant homology to TPX2. Sequence conservation was limited to the putative spindle microtubule-associated region of TPX2, and intriguingly, D-TPX2 (Ssp1/Mei-38) lacks Aurora A- and kinesin-5-binding domains, which are highly conserved in other animal and plant species, including many insects such as ants and bees. D-TPX2 uniformly localised to kinetochore microtubule-enriched regions of the metaphase spindle in the S2 cell line, and it had microtubule binding and bundling activities in vitro. In comparison with other systems, the contribution of D-TPX2 to cell division seems to be minor; live cell imaging of microtubules and chromosomes after RNAi knockdown identified significant delay in chromosome congression in only 18% of the cells. Thus, while this conserved spindle protein is present in Drosophila, other mechanisms may largely compensate for its spindle assembly and chromosome segregation functions.     

Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2

Construction of a mitotic spindle requires biochemical pathways to assemble spindle microtubules and structural proteins to organize these microtubules into a bipolar array. Through a complex with dynein, the receptor for hyaluronan-mediated motility (RHAMM) cross-links mitotic microtubules to provide structural support, maintain spindle integrity, and correctly orient the mitotic spindle. Here, we locate RHAMM to sites of microtubule assembly at centrosomes and non-centrosome sites near kinetochores and demonstrate that RHAMM is required for the activation of Aurora kinase A. Silencing of RHAMM delays the kinetics of spindle assembly, mislocalizes targeting protein for XKlp2 (TPX2), and attenuates the localized activation of Aurora kinase A with a consequent reduction in mitotic spindle length. The RHAMM–TPX2 complex requires a C-terminal basic leucine zipper in RHAMM and a domain that includes the nuclear localization signal in TPX2. Together, our findings identify RHAMM as a critical regulator for Aurora kinase A signaling and suggest that RHAMM ensures bipolar spindle assembly and mitotic progression through the integration of biochemical and structural pathways.     

An essential function of the C. elegans ortholog of TPX2 is to localize activated aurora A kinase to mitotic spindles

In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. Here, we identify TPXL-1, a C. elegans protein that is the first characterized invertebrate ortholog of TPX2. We demonstrate that an essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. Our data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly.     

A novel mechanism for activation of the protein kinase Aurora A

Segregation of chromosomes during mitosis requires interplay between several classes of protein on the spindle, including protein kinases, protein phosphatases, and microtubule binding motor proteins [1-4]. Aurora A is an oncogenic cell cycle-regulated protein kinase that is subject to phosphorylation-dependent activation [5-11]. Aurora A localization to the mitotic spindle depends on the motor binding protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2), but the protein(s) involved in Aurora A activation are unknown [11-13]. Here, we purify an activator of Aurora A from Xenopus eggs and identify it as TPX2. Remarkably, Aurora A that has been fully deactivated by Protein Phosphatase 2A (PP2A) becomes phosphorylated and reactivated by recombinant TPX2 in an ATP-dependent manner. Increased phosphorylation and activation of Aurora A requires its own kinase activity, suggesting that TPX2 stimulates autophosphorylation and autoactivation of the enzyme. Consistently, wild-type Aurora A, but not a kinase inactive mutant, becomes autophosphorylated on the regulatory T loop residue (Thr 295) after TPX2 treatment. Active Aurora A from bacteria is further activated at least 7-fold by recombinant TPX2, and TPX2 also impairs the ability of protein phosphatases to inactivate Aurora A in vitro. This concerted mechanism of stimulation of activation and inhibition of deactivation implies that TPX2 is the likely regulator of Aurora A activity at the mitotic spindle and may explain why loss of TPX2 in model systems perturbs spindle assembly [14-16]. Our finding that a known binding protein, and not a conventional protein kinase, is the relevant activator for Aurora A suggests a biochemical model in which the dynamic localization of TPX2 on mitotic structures directly modulates the activity of Aurora A for spindle assembly.     

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly

The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.     

Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr⁷²) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr⁷², we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr⁷² does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr⁷² regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.     

Human CLASP1 is an outer kinetochore component that regulates spindle microtubule dynamics

One of the most intriguing aspects of mitosis is the ability of kinetochores to hold onto plus ends of microtubules that are actively gaining or losing tubulin subunits. Here, we show that CLASP1, a microtubule-associated protein, localizes preferentially near the plus ends of growing spindle microtubules and is also a component of a kinetochore region that we term the outer corona. A truncated form of CLASP1 lacking the kinetochore binding domain behaves as a dominant negative, leading to the formation of radial arrays of microtubule bundles that are highly resistant to depolymerization. Microinjection of CLASP1-specific antibodies suppresses microtubule dynamics at kinetochores and throughout the spindle, resulting in the formation of monopolar asters with chromosomes buried in the interior. Incubation with microtubule-stabilizing drugs rescues the kinetochore association with microtubule plus ends at the periphery of the asters. Our data suggest that CLASP1 is required at kinetochores for attached microtubules to exhibit normal dynamic behavior.     

Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

Anaphase central spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the central spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the central spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1. In the presence of phosphorylated KIF4A, microtubules grew more slowly and showed long pauses in growth, resulting in the generation of shorter PRC1-stabilized microtubule overlaps in vitro. Cells expressing only mutant forms of KIF4A lacking the Aurora B phosphorylation site overextended the anaphase central spindle, demonstrating that this regulation is crucial for microtubule length control in vivo. Aurora B therefore ensures that suppression of microtubule dynamic instability by KIF4A is restricted to a specific subset of microtubules and thereby contributes to central spindle size control in anaphase.     

A switch in microtubule dynamics at the onset of anaphase B in the mitotic spindle of Schizosaccharomyces pombe

Microtubule dynamics have key roles in mitotic spindle assembly and chromosome movement [1]. Fast turnover of spindle microtubules at metaphase and polewards flux of microtubules (polewards movement of the microtubule lattice with depolymerization at the poles) at both metaphase and anaphase have been observed in mammalian cells [2]. Imaging spindle dynamics in genetically tractable yeasts is now possible using green fluorescent protein (GFP)-tagging of tubulin and sites on chromosomes [3] [4] [5] [6] [7] [8]. We used photobleaching of GFP-labeled tubulin to observe microtubule dynamics in the fission yeast Schizosaccharomyces pombe. Photobleaching did not perturb progress through mitosis. Bleached marks made on the spindle during metaphase recovered their fluorescence rapidly, indicating fast microtubule turnover. Recovery was spatially non-uniform, but we found no evidence for polewards flux. Marks made during anaphase B did not recover fluorescence, and were observed to slide away from each other at the same rate as spindle elongation. Fast microtubule turnover at metaphase and a switch to stable microtubules at anaphase suggest the existence of a cell-cycle-regulated molecular switch that controls microtubule dynamics and that may be conserved in evolution. Unlike the situation for vertebrate spindles, microtubule depolymerization at poles and polewards flux may not occur in S. pombe mitosis. We conclude that GFP-tubulin photobleaching in conjunction with mutant cells should aid research on molecular mechanisms causing and regulating dynamics.     

A unique kinesin-8 surface loop provides specificity for chromosome alignment

Microtubule length control is essential for the assembly and function of the mitotic spindle. Kinesin-like motor proteins that directly attenuate microtubule dynamics make key contributions to this control, but the specificity of these motors for different subpopulations of spindle microtubules is not understood. Kif18A (kinesin-8) localizes to the plus ends of the relatively slowly growing kinetochore fibers (K-fibers) and attenuates their dynamics, whereas Kif4A (kinesin-4) localizes to mitotic chromatin and suppresses the growth of highly dynamic, nonkinetochore microtubules. Although Kif18A and Kif4A similarly suppress microtubule growth in vitro, it remains unclear whether microtubule-attenuating motors control the lengths of K-fibers and nonkinetochore microtubules through a common mechanism. To address this question, we engineered chimeric kinesins that contain the Kif4A, Kif18B (kinesin-8), or Kif5B (kinesin-1) motor domain fused to the C-terminal tail of Kif18A. Each of these chimeric kinesins localizes to K-fibers; however, K-fiber length control requires an activity specific to kinesin-8s. Mutational studies of Kif18A indicate that this control depends on both its C-terminus and a unique, positively charged surface loop, called loop2, within the motor domain. These data support a model in which microtubule-attenuating kinesins are molecularly “tuned” to control the dynamics of specific subsets of spindle microtubules.     

The N-terminal domain of DDA3 regulates the spindle-association of the microtubule depolymerase Kif2a and controls the mitotic function of DDA3

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the dynamics of the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. DDA3 interacts with the microtubule depolymerase Kif2a and controls the association of Kif2a to the mitotic spindle and the dynamic turnover of microtubules in the spindle. To understand the function and regulation of DDA3, we analyzed its domain structure and found that the C-terminal domain of DDA3 directly binds to microtubules in vitro and associates with the mitotic spindle in vivo. The N-terminal domain of DDA3 does not interact with microtubules, but acts dominant negatively over the wild-type protein. Ectopic expression of this domain prevents the endogenous DDA3 from association with the spindle and results in a high frequency of unaligned chromosomes in metaphase cells, a phenotype similar to that in metaphase cells depleted of DDA3. Mechanistically, expression of N-terminal DDA3 reduces the amount of spindle-associated Kif2a and increases the spindle microtubule density, pheno-copying those in DDA3-depleted cells. We conclude that DDA3 has a distinct domain structure. The C-terminal domain confers its ability to associate with the mitotic spindle, while the regulatory N-terminal domain controls the microtubule-binding by the C-terminal domain and determines the cellular activity of the DDA3 protein.     

The distribution of TPX2 in dividing leaf cells of the fern Asplenium nidus

Plant cell division requires the dynamic organisation of several microtubule arrays. The mechanisms of regulation of the above arrays are under rigorous research. Among several factors that are involved in plant microtubule dynamics, the Targeting Protein for Xklp2 (TPX2) has been found to play a role in spindle organisation, in combination with Aurora kinases, in dividing cells of angiosperms. Microtubule organisation in dividing cells of ferns exhibits certain peculiarities. Accordingly, the presence and distribution of a TPX2 homologue might be helpful in understanding the patterns and regulatory mechanisms of microtubule arrays in this plant group. In this study, a putative TPX2 homologue was identified using Western blotting in the fern Asplenium nidus. It was found, using immunostaining and CLSM, that it is co‐localised with perinuclear preprophase microtubules and the prophase spindle, and follows the microtubule pattern during metaphase/anaphase and telophase. During cytokinesis, while in angiosperms TPX2 is degraded, in A. nidus the TPX2 signal persists, co‐localising with the phragmoplast. In early post‐cytokinetic cells, a TPX2 signal is present on the nuclear surface facing the daughter cell wall and, thereafter it is co‐localised with the fern‐specific microtubule aggregation that lines the new wall, which is possibly involved in cortical microtubule assembly.     

Prophase microtubule arrays undergo flux-like behavior in mammalian cells

In higher eukaryotic cells, microtubules within metaphase and anaphase spindles undergo poleward flux, the slow, poleward movement of tubulin subunits through the spindle microtubule lattice. Although a number of studies have documented this phenomenon across a wide range of model systems, the possibility of poleward flux before nuclear envelope breakdown (NEB) has not been examined. Using a mammalian cell line expressing photoactivatable green fluorescent protein (GFP)-tubulin, we observe microtubule motion, both toward and away from centrosomes, at a wide range of rates (0.5–4.5 μm/min) in prophase cells. Rapid microtubule motion in both directions is dynein dependent. In contrast, slow microtubule motion, which occurs at rates consistent with metaphase flux, is insensitive to inhibition of dynein but sensitive to perturbation of Eg5 and Kif2a, two proteins with previously documented roles in flux. Our results demonstrate that microtubules in prophase cells are unexpectedly dynamic and that a subpopulation of these microtubules shows motion that is consistent with flux. We propose that the marked reduction in rate and directionality of microtubule motion from prophase to metaphase results from changes in microtubule organization during spindle formation.     

Poleward tubulin flux in spindles: regulation and function in mitotic cells

The poleward flux of tubulin subunits through spindle microtubules is a striking and conserved phenomenon whose function and molecular components remain poorly understood. To screen for novel components of the flux machinery, we utilized RNA interference to deplete regulators of microtubule dynamics, individually and in various combinations, from S2 cells and examined the resulting impact on flux rate. This led to the identification of two previously unknown flux inhibitors, KLP59C and KLP67A, and a flux promoter, Mini-spindles. Furthermore, we find that flux rate is regulated by functional antagonism among microtubule stabilizers and destabilizers specifically at plus ends. Finally, by examining mitosis on spindles in which flux has been up- or down-regulated or restored after the codepletion of antagonistic flux regulators, we show that flux is an integral contributor to anaphase A but is not responsible for chromosome congression, interkinetochore tension, or the establishment of normal spindle length during prometaphase/metaphase.     

CLASP1, astrin and Kif2b form a molecular switch that regulates kinetochore‐microtubule dynamics to promote mitotic progression and fidelity

Accurate chromosome segregation during mitosis requires precise coordination of various processes, such as chromosome alignment, maturation of proper kinetochore–microtubule (kMT) attachments, correction of erroneous attachments, and silencing of the spindle assembly checkpoint (SAC). How these fundamental aspects of mitosis are coordinately and temporally regulated is poorly understood. In this study, we show that the temporal regulation of kMT attachments by CLASP1, astrin and Kif2b is central to mitotic progression and chromosome segregation fidelity. In early mitosis, a Kif2b–CLASP1 complex is recruited to kinetochores to promote chromosome movement, kMT turnover, correction of attachment errors, and maintenance of SAC signalling. However, during metaphase, this complex is replaced by an astrin–CLASP1 complex, which promotes kMT stability, chromosome alignment, and silencing of the SAC. We show that these two complexes are differentially recruited to kinetochores and are mutually exclusive. We also show that other kinetochore proteins, such as Kif18a, affect kMT attachments and chromosome movement through these proteins. Thus, CLASP1–astrin–Kif2b complex act as a central switch at kinetochores that defines mitotic progression and promotes fidelity by temporally regulating kMT attachments.