MCAK-independent functions of ch-Tog/XMAP215 in microtubule plus-end dynamics

The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells.     

Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK

During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere-associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.     

CaMKIIgamma-mediated inactivation of the Kin I kinesin MCAK is essential for bipolar spindle formation

MCAK, a member of the kinesin-13 family, is a microtubule (MT) depolymerase that is necessary to ensure proper kinetochore MT attachment during spindle formation. Regulation of MCAK activity and localization is controlled in part by Aurora B kinase at the centromere. Here we analyzed human cells depleted of the ubiquitous Ca(2+)/calmodulin-dependent protein kinase IIgamma isoform (CaMKIIgamma) by RNA interference and found that CaMKIIgamma was necessary to suppress MCAK depolymerase activity in vivo. A functional overlap with TOGp, a MT regulator known to counteract MCAK, was suggested by similar CaMKIIgamma- and TOGp-depletion phenotypes, namely disorganized multipolar spindles. A replicating vector system, which permits inducible overexpression in cells that simultaneously synthesize interfering short hairpin RNAs, was used to dissect the functional interplay between CaMKIIgamma, TOGp, and MCAK. Our results revealed two distinct but functionally overlapping mechanisms for negative regulation of the cytosolic/centrosomal pool of MCAK. These two mechanisms, involving CaMKIIgamma and TOGp, respectively, are both essential for spindle bipolarity in a normal physiological context, but not in MCAK-depleted cells.     

PLK1 phosphorylates mitotic centromere-associated kinesin and promotes its depolymerase activity

During cell division, interaction between kinetochores and dynamic spindle microtubules governs chromosome movements. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator of mitotic spindle assembly and dynamics. However, the regulatory mechanisms underlying its depolymerase activity during the cell cycle remain elusive. Here, we showed that PLK1 is a novel regulator of MCAK in mammalian cells. MCAK interacts with PLK1 in vitro and in vivo. The neck and motor domain of MCAK associates with the kinase domain of PLK1. MCAK is a novel substrate of PLK1, and the phosphorylation stimulates its microtubule depolymerization activity of MCAK in vivo. Overexpression of a polo-like kinase 1 phosphomimetic mutant MCAK causes a dramatic increase in misaligned chromosomes and in multipolar spindles in mitotic cells, whereas overexpression of a nonphosphorylatable MCAK mutant results in aberrant anaphase with sister chromatid bridges, suggesting that precise regulation of the MCAK activity by PLK1 phosphorylation is critical for proper microtubule dynamics and essential for the faithful chromosome segregation. We reasoned that dynamic regulation of MCAK phosphorylation by PLK1 is required to orchestrate faithful cell division, whereas the high levels of PLK1 and MCAK activities seen in cancer cells may account for a mechanism underlying the pathogenesis of genomic instability.     

The far C-terminus of MCAK regulates its conformation and spindle pole focusing

To ensure proper spindle assembly, microtubule (MT) dynamics needs to be spatially regulated within the cell. The kinesin-13 MCAK is a potent MT depolymerase with a complex subcellular localization, yet how MCAK spatial regulation contributes to spindle assembly is not understood. Here we show that the far C-terminus of MCAK plays a critical role in regulating MCAK conformation, subspindle localization, and spindle assembly in Xenopus egg extracts. Alteration of MCAK conformation by the point mutation E715A/E716A in the far C-terminus increased MCAK targeting to the poles and reduced MT lifetimes, which induced spindles with unfocused poles. These effects were phenocopied by the Aurora A phosphomimetic mutation, S719E. Furthermore, addition of the kinesin-14 XCTK2 to spindle assembly reactions rescued the unfocused-pole phenotype. Collectively our work shows how the regional targeting of MCAK regulates MT dynamics, highlighting the idea that multiple phosphorylation pathways of MCAK cooperate to spatially control MT dynamics to maintain spindle architecture.     

TOGp, the human homolog of XMAP215/Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly

The XMAP215/Dis1 MAP family is thought to regulate microtubule plus-end assembly in part by antagonizing the catastrophe-promoting function of kin I kinesins, yet XMAP215/Dis1 proteins localize to centrosomes. We probed the mitotic function of TOGp (human homolog of XMAP215/Dis1) using siRNA. Cells lacking TOGp assembled multipolar spindles, confirming results of Gergely et al. (2003. Genes Dev. 17, 336-341). Eg5 motor activity was necessary to maintain the multipolar morphology. Depletion of TOGp decreased microtubule length and density in the spindle by approximately 20%. Depletion of MCAK, a kin I kinesin, increased MT lengths and density by approximately 20%, but did not disrupt spindle morphology. Mitotic cells lacking both TOGp and MCAK formed bipolar and monopolar spindles, indicating that TOGp and MCAK contribute to spindle bipolarity, without major effects on MT stability. TOGp localized to centrosomes in the absence of MTs and depletion of TOGp resulted in centrosome fragmentation. TOGp depletion also disrupted MT minus-end focus at the spindle poles, detected by localizations of NuMA and the p150 component of dynactin. The major functions of TOGp during mitosis are to focus MT minus ends at spindle poles, maintain centrosome integrity, and contribute to spindle bipolarity.     

KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly

During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere–associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(−) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(−) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore–microtubule (k–MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(−) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k–MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly.     

Aurora B phosphorylates centromeric MCAK and regulates its localization and microtubule depolymerization activity

BACKGROUND: Sister kinetochores must bind microtubules in a bipolar fashion to equally segregate chromosomes during mitosis. The molecular mechanisms underlying this process remain unclear. Aurora B likely promotes chromosome biorientation by regulating kinetochore-microtubule attachments. MCAK (mitotic centromere-associated kinesin) is a Kin I kinesin that can depolymerize microtubules. These two proteins both localize to mitotic centromeres and have overlapping mitotic functions, including regulation of microtubule dynamics, proper chromosome congression, and correction of improper kinetochore-microtubule attachments. RESULTS: We show that Aurora B phosphorylates and regulates MCAK both in vitro and in vivo. Specifically, we mapped six Aurora B phosphorylation sites on MCAK in both the centromere-targeting domain and the neck region. Aurora B activity was required to localize MCAK to centromeres, but not to spindle poles. Aurora B phosphorylation of serine 196 in the neck region of MCAK inhibited its microtubule depolymerization activity. We found that this key site was phosphorylated at centromeres and anaphase spindle midzones in vivo. However, within the inner centromere there were pockets of both phosphorylated and unphosphorylated MCAK protein, suggesting that phosphate turnover is crucial in the regulation of MCAK activity. Addition of alpha-p-S196 antibodies to Xenopus egg extracts or injection of alpha-p-S196 antibodies into cells caused defects in chromosome positioning and/or segregation. CONCLUSIONS: We have established a direct link between the microtubule depolymerase MCAK and Aurora B kinase. Our data suggest that Aurora B both positively and negatively regulates MCAK during mitosis. We propose that Aurora B biorients chromosomes by directing MCAK to depolymerize incorrectly oriented kinetochore microtubules.     

The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore-microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity.     

MCAK activity at microtubule tips regulates spindle microtubule length to promote robust kinetochore attachment

Mitotic centromere-associated kinesin (MCAK) is a microtubule-depolymerizing kinesin-13 member that can track with polymerizing microtubule tips (hereafter referred to as tip tracking) during both interphase and mitosis. MCAK tracks with microtubule tips by binding to end-binding proteins (EBs) through the microtubule tip localization signal SKIP, which lies N terminal to MCAK's neck and motor domain. The functional significance of MCAK's tip-tracking behavior during mitosis has never been explained. In this paper, we identify and define a mitotic function specific to the microtubule tip-associated population of MCAK: negative regulation of microtubule length within the assembling bipolar spindle. This function depends on MCAK's ability to bind EBs and track with polymerizing nonkinetochore microtubule tips. Although this activity antagonizes centrosome separation during bipolarization, it ultimately benefits the dividing cell by promoting robust kinetochore attachments to the spindle microtubules.     

The Ncd tail domain promotes microtubule assembly and stability

Non-claret disjunctional (Ncd) is a Drosophila kinesin-like motor required for spindle assembly and maintenance in oocytes and early embryos. Ncd has an ATP-independent microtubule binding site in the N-terminal tail domain as well as an ATP-dependent microtubule binding site in the C-terminal motor domain. The Ncd tail domain shares many properties with the microtubule-associated proteins that regulate microtubule assembly, including microtubule binding and bundling activity and an abundance of basic and proline residues. Given these similarities, we examined the ability of Ncd tail domain proteins to promote MT assembly and stability. The results indicate that the Ncd tail domain can promote MT assembly and stabilize MTs against conditions that induce MT disassembly, and suggest that Ncd may influence MT dynamics within the spindle.     

Aurora A,MCAK, and Kif18b promote Eg5-independent spindle formation

Inhibition of the microtubule (MT) motor protein Eg5 results in a mitotic arrest due to the formation of monopolar spindles, making Eg5 an attractive target for anti-cancer therapies. However, Eg5-independent pathways for bipolar spindle formation exist, which might promote resistance to treatment with Eg5 inhibitors. To identify essential components for Eg5-independent bipolar spindle formation, we performed a genome-wide siRNA screen in Eg5-independent cells (EICs). We find that the kinase Aurora A and two kinesins, MCAK and Kif18b, are essential for bipolar spindle assembly in EICs and in cells with reduced Eg5 activity. Aurora A promotes bipolar spindle assembly by phosphorylating Kif15, hereby promoting Kif15 localization to the spindle. In turn, MCAK and Kif18b promote bipolar spindle assembly by destabilizing the astral MTs. One attractive way to interpret our data is that, in the absence of MCAK and Kif18b, excessive astral MTs generate inward pushing forces on centrosomes at the cortex that inhibit centrosome separation. Together, these data suggest a novel function for astral MTs in force generation on spindle poles and how proteins involved in regulating microtubule length can contribute to bipolar spindle assembly.     

Aurora B regulates spindle bipolarity in meiosis in vertebrate oocytes

Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine¹²² is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.     

Molecular dissection of the microtubule depolymerizing activity of mitotic centromere-associated kinesin

Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerizer that is consistent with its role in promoting chromosome segregation during mitosis. Here we show that the conserved motor domain of MCAK is necessary but not sufficient for microtubule depolymerization in cells or in vitro. The addition of only 30 amino acids N-terminal to the motor restores depolymerization activity. Furthermore, dimerization studies revealed that the smallest functional MCAK deletion constructs are monomers. These results define a highly conserved domain within MCAK and related (KIN I) kinesins that is critical for depolymerization activity and show that this depolymerization is not dependent on MCAK dimerization.     

Aurora B regulates spindle bipolarity in meiosis in vertebrate oocytes

Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine¹²² is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.     

Full-length dimeric MCAK is a more efficient microtubule depolymerase than minimal domain monomeric MCAK

MCAK belongs to the Kinesin-13 family, whose members depolymerize microtubules rather than translocate along them. We defined the minimal functional unit of MCAK as the catalytic domain plus the class specific neck (MD-MCAK), which is consistent with previous reports. We used steady-state ATPase kinetics, microtubule depolymerization assays, and microtubule.MCAK cosedimentation assays to compare the activity of full-length MCAK, which is a dimer, with MD-MCAK, which is a monomer. Full-length MCAK exhibits higher ATPase activity, more efficient microtubule end binding, and reduced affinity for the tubulin heterodimer. Our studies suggest that MCAK dimerization is important for its catalytic cycle by promoting MCAK binding to microtubule ends, enhancing the ability of MCAK to recycle for multiple rounds of microtubule depolymerization, and preventing MCAK from being sequestered by tubulin heterodimers.     

Effects of anti-microtubule agents on microtubule organization in cells lacking the kinesin-13 MCAK

Dynamic microtubules are necessary for proper mitotic spindle assembly and chromosome segregation during mitosis. Members of the kinesin superfamily of molecular motor proteins are important to spindle function. Of particular interest is the Kinesin-13 family member MCAK, which acts to regulate microtubule dynamics during spindle assembly and to ensure proper attachments of chromosomes to spindle microtubules. The unique ability of MCAK to regulate microtubule dynamics makes it a potential target for development of new drugs that alter spindle function. Here, we knocked down MCAK via RNAi in normal and malignant cell lines and found that the two tested malignant cell lines were acutely sensitive to MCAK knockdown, while the tested normal cells were less sensitive. In addition, we looked at the effect of combining MCAK knockdown and drug treatment with paclitaxel or vinblastine to identify spindle assembly defects. We found that MCAK knockdown increased the morphological defects of the microtubule cytoskeleton in HeLa cells caused by anti-microtubule drugs. Our studies support the idea that MCAK would be a good target for new chemotherapeutic development and may be particular useful in combination therapies with currently available anti-microtubule agents.     

Localized Aurora B activity spatially controls non-kinetochore microtubules during spindle assembly

Efficient spindle assembly involves the generation of spatial cues around chromosomes that locally stabilize microtubule (MT) plus-ends. In addition to the small GTPase Ran, there is evidence that Aurora B kinase might also generate a spatial cue around chromosomes but direct proof for this is still lacking. Here, we find that the Aurora B substrate MCAK localizes to MT plus-ends throughout the mitotic spindle, but its accumulation is strongly reduced on MT plus-ends near chromatin, suggesting that a signal emanating from chromosomes negatively regulates MCAK plus-end binding. Indeed, we show that Aurora B is the kinase responsible for producing this chromosome-derived signal. These results are the first to visualize spatially restricted Aurora B kinase activity around chromosomes on an endogenous substrate and explain how Aurora B could spatially control the dynamics of non-kinetochore MTs during spindle assembly.     

K-loop insertion restores microtubule depolymerizing activity of a "neckless" MCAK mutant

Unlike most kinesins, mitotic centromere-associated kinesin (MCAK) does not translocate along the surface of microtubules (MTs), but instead depolymerizes them. Among the motile kinesins, refinements that are unique for specific cellular functions, such as directionality and processivity, are under the control of a "neck" domain adjacent to the ATP-hydrolyzing motor domain. Despite its apparent lack of motility, MCAK also contains a neck domain. We found that deletions and alanine substitutions of highly conserved positively charged residues in the MCAK neck domain significantly reduced MT depolymerization activity. Furthermore, substitution of MCAK's neck domain with either the positively charged KIF1A K-loop or poly-lysine rescues the loss of MT-depolymerizing activity observed in the neckless MCAK mutant. We propose that the neck, analogously to the K-loop, interacts electrostatically with the tubulin COOH terminus to permit diffusional translocation of MCAK along the surface of MTs. This weak-binding interaction may also play an important role in processivity of MCAK-induced MT depolymerization.     

Differentiation of cytoplasmic and meiotic spindle assembly MCAK functions by Aurora B-dependent phosphorylation

The KinI kinesin MCAK is a microtubule depolymerase important for governing spindle microtubule dynamics during chromosome segregation. The dynamic nature of spindle assembly and chromosome-microtubule interactions suggest that mechanisms must exist that modulate the activity of MCAK, both spatially and temporally. In Xenopus extracts, MCAK associates with and is stimulated by the inner centromere protein ICIS. The inner centromere kinase Aurora B also interacts with ICIS and MCAK raising the possibility that Aurora B may regulate MCAK activity as well. Herein, we demonstrate that recombinant Aurora B-INCENP inhibits Xenopus MCAK activity in vitro in a phosphorylation-dependent manner. Substituting endogenous MCAK in Xenopus extracts with the alanine mutant XMCAK-4A, which is resistant to inhibition by Aurora B-INCENP, led to assembly of mono-astral and monopolar structures instead of bipolar spindles. The size of these structures and extent of tubulin polymerization in XMCAK-4A extracts indicate that XM-CAK-4A is not defective for microtubule dynamics regulation throughout the cytoplasm. We further demonstrate that the ability of XMCAK-4A to localize to inner centromeres is abolished. Our results show that MCAK regulation of cytoplasmic and spindle-associated microtubules can be differentiated by Aurora B-dependent phosphorylation, and they further demonstrate that this regulation is required for bipolar meiotic spindle assembly.