Ariane Abrieu

The proteolysis-dependent metaphase to anaphase transition: calcium/calmodulin-dependent protein kinase II mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs.

It has been shown, using spindles assembled in vitro in extracts containing CSF (the cytostatic factor responsible for arresting unfertilized vertebrate eggs at metaphase), that onset of anaphase requires Ca(2+)‐dependent activation of the ubiquitin‐dependent proteolytic pathway that destroys both mitotic cyclins and an unknown protein responsible for metaphase arrest (Holloway et al., 1993, Cell, 73, 1382‐1402). We showed recently that Ca2+/calmodulin‐dependent protein kinase II (CaM KII) activates the ubiquitin‐dependent cyclin degradation pathway in CSF extracts (Lorca et al., 1993, Nature, 366, 270‐273), but did not investigate its possible effect on sister chromatid segregation. In this work we identify CaM KII as the only target of Ca2+ in inducing anaphase in CSF extracts, and further show that transition to anaphase does not require the direct phosphorylation of metaphase spindle components by CaM KII. A possible interpretation of the above results could have been that the ubiquitin‐dependent degradation pathway is required for onset of anaphase only when spindles are clamped at metaphase due to CSF activity, and not in the regular cell cycle that occurs in the absence of CSF activity. We ruled out this possibility by showing that competitive inhibition of the ubiquitin‐dependent degradation pathway still prevents the onset of anaphase in cycling extracts that lack CSF and do not require Ca2+ for sister chromatid separation.

Phosphorylation Relieves Autoinhibition of the Kinetochore Motor Cenp-E

During mitosis, chromosome alignment depends on the regulated dynamics of microtubules and on motor protein activities. At the kinetochore, the interplay between microtubule-binding proteins, motors, and kinases is poorly understood. Cenp-E is a kinetochore-associated kinesin involved in chromosome congression, but the mechanism by which this is achieved is unclear. Here, we present a study of the regulation of Cenp-E motility by using purified full-length (FL) Xenopus Cenp-E protein, which demonstrates that FL Cenp-E is a genuine plus-end-directed motor. Furthermore, we find that the Cenp-E tail completely blocks the motility of Cenp-E in vitro. This is achieved through direct interaction between its motor and tail domains. Finally, we show that Cenp-E autoinhibition is reversed by MPS1- or CDK1-cyclin B-mediated phosphorylation of the Cenp-E tail. This suggests a model of dynamic control of Cenp-E motility, and hence chromosome congression, dependent upon phosphorylation at the kinetochore.

MAPK inactivation is required for the G2 to M-phase transition of the first mitotic cell cycle.

Down‐regulation of MAP kinase (MAPK) is a universal consequence of fertilization in the animal kingdom, although its role is not known. Here we show that MAPK inactivation is essential for embryos, both vertebrate and invertebrate, to enter first mitosis. Suppressing down‐regulation of MAPK at fertilization, for example by constitutively activating the upstream MAPK cascade, specifically suppresses cyclin B‐cdc2 kinase activation and its consequence, entry into first mitosis. It thus appears that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development. The most general effect of artificially maintaining MAPK activity after fertilization is prevention of the G2 to M‐phase transition in the first mitotic cell cycle, even though inappropriate reactivation of MAPK after fertilization may lead to metaphase arrest in vertebrates. Advancing the time of MAPK inactivation in fertilized eggs does not, however, speed up their entry into first mitosis. Thus, sustained activity of MAPK during part of the first mitotic cell cycle is not responsible for late entry of fertilized eggs into first mitosis.

CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint.

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.

P21-activated kinase 4 (PAK4) is required for metaphase spindle positioning and anchoring

The oncogenic kinase PAK4 was recently found to be involved in the regulation of the G1 phase and the G2/M transition of the cell cycle. We have also identified that PAK4 regulates Ran GTPase activity during mitosis. Here, we show that after entering mitosis, PAK4-depleted cells maintain a prolonged metaphase-like state. In these cells, chromosome congression to the metaphase plate occurs with normal kinetics but is followed by an extended period during which membrane blebbing and spindle rotation are observed. These bipolar PAK4-depleted metaphase-like spindles have a defective astral microtubule (MT) network and are not centered in the cell but are in close contact with the cell cortex. As the metaphase-like state persists, centrosome fragmentation occurs, chromosomes scatter from the metaphase plate and move toward the spindle poles with an active spindle assembly checkpoint, a phenotype that is reminiscent of cohesion fatigue. PAK4 also regulates the acto-myosin cytoskeleton and we report that PAK4 depletion results in the induction of cortical membrane blebbing during prometaphase arrest. However, we show that membrane blebs, which are strongly enriched in phospho-cofilin, are not responsible for the poor anchoring of the spindle. As PAK4 depletion interferes with the localization of components of the dynein/dynactin complexes at the kinetochores and on the astral MTs, we propose that loss of PAK4 could induce a change in the activities of motor proteins.

Natural Loss of Mps1 Kinase in Nematodes Uncovers a Role for Polo-like Kinase 1 in Spindle Checkpoint Initiation

The spindle checkpoint safeguards against chromosome loss during cell division by preventing anaphase onset until all chromosomes are attached to spindle microtubules. Checkpoint signal is generated at kinetochores, the primary attachment site on chromosomes for spindle microtubules. Mps1 kinase initiates checkpoint signaling by phosphorylating the kinetochore-localized scaffold protein Knl1 to create phospho-docking sites for Bub1/Bub3. Mps1 is widely conserved but is surprisingly absent in many nematode species. Here, we show that PLK-1, which targets a substrate motif similar to that of Mps1, functionally substitutes for Mps1 in C. elegans by phosphorylating KNL-1 to direct BUB-1/BUB-3 kinetochore recruitment. This finding led us to re-examine checkpoint initiation in human cells, where we found that Plk1 co-inhibition significantly reduced Knl1 phosphorylation and Bub1 kinetochore recruitment relative to Mps1 inhibition alone. Thus, the finding that PLK-1 functionally substitutes for Mps1 in checkpoint initiation in C. elegans uncovered a role for Plk1 in species that have Mps1.

Probing CENP-E function in chromosome dynamics using small molecule inhibitor syntelin.

Dear Editor, n nChromosome movements during mitosis are orchestrated primarily by the interaction of spindle microtubules with the kinetochore [1], the site for attachment of spindle microtubules to the centromere. The kinetochore has an active function in chromosomal segregation through microtubule-based motors located at or near it [1–2]. CENP-E is a microtubule-based kinesin motor [3], and suppression of CENP-E leads to defects characterized by an intact bipolar spindle with several chromosomes clustered close to the poles [4]. Studies combining electron microscopic analyses with siRNA-mediated suppression indicated that CENP-E deficiency correlated with a failure of mono-oriented chromosomes to congress to the equator [5–6]. In addition, expression of motorless CENP-E [7–8], produced a similar failure in achieving metaphase alignment, suggesting the role of CENP-E motor activity in chromosome congression. However, these studies do not specifically and directly address the function of CENP-E motor in chromosome dynamics. n nTo probe the spatiotemporal dynamics of CENP-E function in mitosis, we screened a chemical library of structurally diversified compounds for lagging chromosome phenotypes and inhibition of CENP-E motility. One identified inhibitor was further modified to produce syntelin (Figure 1A). In in vitro motility assays using purified recombinant proteins, syntelin inhibited CENP-E motility in a dose-dependent manner with an IC50 value of 160 nM (Figure 1B and Supplementary information, Figure S1A). Among an extensive list of mitotic kinesins examined, syntelin was found to be highly selective for CENP-E (Supplementary information, Figure S1B). Importantly, syntelin binds to different sites from those of GSK923295, a recently identified CENP-E ATPase inhibitor [9], as syntelin inhibits CENP-E mutants resistant to GSK923295 in a manner indistinguishable from that of wild type motor (Supplementary information, Figure S1B). Thus, we conclude that syntelin represents a novel class of CENP-E motor inhibitor. n n n nFigure 1 n nSyntelin selectively inhibits CENP-E motor activity. (A) Chemical structure of syntelin. (B) CENP-E motility and syntelin inhibition. Minus-end-marked microtubules were added with 1 mM ATP to a flow chamber containing purified CENP-E tethered to the coverslip ... n n n nSyntelin does not inhibit progression through S and G2 phases of the cell cycle but causes mitotic arrest with lagging chromosomes, a phenotype reminiscent of what was seen in CENP-E-suppressed cells [4]. As expected, inhibition of CENP-E by syntelin did not perturb bipolar spindles but produced misaligned chromosomes near the spindle poles (Figure 1C), similar to those of CENP-E siRNA-treated cells (Supplementary information, Figure S2B). The kinetochore position relative to the pole is an accurate reporter for judging chromosome misalignment (Supplementary information, Figure S2B and S2C; [10]), our quantitative analysis indicated a relatively uniform distribution of kinetochores along the length of the spindle in CENP-E-inhibited and CENP-E-suppressed cells (Supplementary information, Figure S2D). Importantly, inhibition of CENP-E motor activity by syntelin resulted in a significant increase in cells bearing misaligned chromosomes (31.7 ± 6.8%; P < 0.05; Supplementary information, Figure S2D), indicating that CENP-E motor activity is essential for faithful chromosome congression. n nOur analyses of centromere geometry in CENP-E-suppressed cells validate that CENP-E activity is essential for centromere stretch (Supplementary information, Table S1). Misaligned chromosomes and decreased centromere stretch in syntelin-treated cells suggest that inhibition of CENP-E motor activity results in abnormal interactions between the kinetochores and spindle microtubules. In syntelin-treated cells, cold-stable kinetochore-microtubule fibers were present on both aligned chromosomes and chromosomes near the pole (Supplementary information, Figure S3A). Interestingly, careful examination revealed that the kinetochores of lagging chromosome appeared to connect with spindle microtubules derived from the same pole (Supplementary information, Figure S3B; enlarged insets). n nTo study the precise kinetochore attachment in the absence of CENP-E motor activity, we carried out electron microscopic analysis on syntelin-treated HeLa cells. As shown in Figure 1D, spindle microtubules emanate from two opposing centrioles (asterisks) form a bipolar spindle with majority of chromosomes congressed near the spindle equator while a few chromosomes remain scattered around the poles (Figure 1D, boxed area). At a higher magnification, it was readily apparent that one chromosome near the pole displays a clear syntelic attachment in which the kinetochore connects to two sets of microtubules emanating from same centriole. Thus, CENP-E motor activity is essential for the accurate attachment of kinetochores to spindle microtubules (Supplementary information, Figure S3C). n nSmall molecules that modulate specific protein functions are valuable tools for dissecting complex processes in mammalian cell division. Having demonstrated the ability of syntelin to inhibit CENP-E motor function in cultured cells, we sought to test whether syntelin alters the chromosome dynamics during mitosis and whether its inhibitory effect is reversable. To this end, we adopted a protocol to synchronize cells at prometaphase and then added syntelin to probe for functional relevance of CENP-E in bipolar spindle formation and chromosome movements (illustrated in Figure 1E). We began real time imaging of prometaphase-synchronized HeLa cells expressing mCherry-tubulin and GFP-H2B soon after addition of syntelin to visualize chromosome congression and subsequent prometaphase-metaphase transition. In general, it takes an average of 41 ± 2 min (n = 8 cells) for HeLa cells to transit from prometaphase (mono-polar) to the anaphase onset of sister chromatid separation (Figure 1G; top panel). However, some chromosomes still failed to align at the equator in the syntelin-treated cells even after 120 min (Figure 1G; middle panel), consistent with the essential role of CENP-E in chromosome congression [4–6]. n nWe next examined if syntelin inhibition can be reversed using a protocol to synchronize cells at prometaphase followed by three washes to allow cells to recover from prometaphase chromosome misalignment arrest and to progress into metaphase and subsequent sister chromatid separation (Figure 1F). As predicted, syntelin removal was readily apparent as chromosomes in the released cell effectively approached metaphase alignment about 40 min after wash-out and subsequently progressed into anaphase onset (Figure 1G; bottom panel). The temporal order of metaphase-anaphase transition and telophase process in syntelin-released cells is indistinguishable from that of monastrol-released cells, demonstrating that mitotic arrest due to syntelin treatment is also rapidly reversible. Synthelin is thus a useful tool to dissect the molecular mechanisms underlying mitotic checkpoint regulation and correction of chromosome mal-orientation. n nSmall molecules that modulate specific protein functions are valuable experimental tools for dissecting complex processes in mammalian cells. By using a novel CENP-E motor syntelin inhibitor, we have been able to directly address, for the first time, the requirement of CENP-E in these processes. Because the phenotypes derived from CENP-E protein repression appear more extensive than those induced by syntelin, our data demonstrated the usefulness of small molecule inhibitors in dissecting the spatiotemporal dynamics of complex chromosome movements in mitosis. Indeed, kinetochore fibers form in the presence of syntelin, suggesting that CENP-E activity is not required for spindle microtubule capture, but rather accurately regulates these interactions to ensure correct bi-orientation and promote correct chromosome alignment. n nCENP-E has been implicated in spindle checkpoint satisfaction [4, 7] as well as chromosome alignment [5]. The temporal profiles of CENP-E distribution to the kinetochore in prometaphase cells and its dissociation from aligned kinetochores at metaphase are consistent with this notion. Indeed, inhibition of CENP-E motor activity retains active spindle checkpoint judged by the localization of Mad2 to the kinetochore of lagging chromosomes near the spindle poles (Supplementary information, Figure S4). This mitotic arrest is relieved and active checkpoint silenced upon removal of CENP-E inhibition by syntelin washout. Thus, in human cells at least, CENP-E activity appears to be directly required for spindle checkpoint silencing and correct kinetochore attachment. It would be of great interest to directly address and quantify the checkpoint kinase activity on syntelic attachment and examine what happens in live chromosome congression upon the removal of syntelin and correction of syntelic attachment. n nIn summary, our chemical biological study of mitotic kinesins led to identification of a novel CENP-E motor inhibitor, syntelin. Chromosomes in syntelin-treated cells frequently exhibit syntelic attachment in which both sister kinetochores attached to spindle microtubules from the same pole. Syntelin is thus a useful tool to study mitotic checkpoint regulation and correction of chromosome malorientation, and contributions of CENP-E to chromosome dynamics and plasticity.

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

Significance The spindle assembly checkpoint (SAC) works as a surveillance mechanism to ensure accurate segregation of genetic materials during cell division. Protein kinase monopolar spindle 1 (Mps1) plays a key role in SAC, but the mechanism of Mps1 action in chromosome segregation remains elusive. In this study, we identified a previously undefined structural determinant of Mps1 named “IRK” (internal region for kinetochore localization) and demonstrated its functional importance in accurate kinetochore–microtubule attachment. Mechanistically, a dynamic hierarchical interaction between Mps1 and the nuclear division cycle 80 complex (Ndc80C) orchestrates accurate mitosis, because persistent association of inactive Mps1 with Ndc80C via the IRK perturbs correct kinetochore–microtubule attachment. Our results provide a new mechanistic insight into the spatiotemporal dynamics of Mps1 activity at the kinetochore in mitosis. The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C–MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.

Whole-genome duplication increases tumor cell sensitivity to MPS1 inhibition

Several lines of evidence indicate that whole-genome duplication resulting in tetraploidy facilitates carcinogenesis by providing an intermediate and metastable state more prone to generate oncogenic aneuploidy. Here, we report a novel strategy to preferentially kill tetraploid cells based on the abrogation of the spindle assembly checkpoint (SAC) via the targeting of TTK protein kinase (better known as monopolar spindle 1, MPS1). The pharmacological inhibition as well as the knockdown of MPS1 kills more efficiently tetraploid cells than their diploid counterparts. By using time-lapse videomicroscopy, we show that tetraploid cells do not survive the aborted mitosis due to SAC abrogation upon MPS1 depletion. On the contrary diploid cells are able to survive up to at least two more cell cycles upon the same treatment. This effect might reflect the enhanced difficulty of cells with whole-genome doubling to tolerate a further increase in ploidy and/or an elevated level of chromosome instability in the absence of SAC functions. We further show that MPS1-inhibited tetraploid cells promote mitotic catastrophe executed by the intrinsic pathway of apoptosis, as indicated by the loss of mitochondrial potential, the release of the pro-apoptotic cytochrome c from mitochondria, and the activation of caspases. Altogether, our results suggest that MPS1 inhibition could be used as a therapeutic strategy for targeting tetraploid cancer cells.

Bub1 targeting to centromeres is sufficient for Sgo1 recruitment in the absence of kinetochores

Centromeric chromatin containing the histone H3 variant centromere protein A (CENP-A) directs kinetochore assembly through a hierarchical binding of CENPs, starting with CENP-C and CENP-T. Centromeres are also the chromosomal regions where cohesion, mediated by cohesin, is most prominently maintained in mitosis. While most cohesin dissociates from chromosome arms in prophase, Shugoshin 1 (Sgo1) prevents this process at centromeres. Centromeric localization of Sgo1 depends on histone H2A phosphorylation by the kinase Bub1, but whether additional interactions with kinetochore components are required for Sgo1 recruitment is unclear. Using the Xenopus egg cell-free system, we here show that both CENP-C and CENP-T can independently drive centromeric accumulation of Sgo1 through recruitment of Bub1 to the KNL1, MIS12, NDC80 (KMN) network. The spindle assembly checkpoint (SAC) kinase Mps1 is also required for this pathway even in the absence of checkpoint signaling. Sgo1 recruitment is abolished in chromosomes lacking kinetochore components other than CENP-A. However, forced targeting of Bub1 to centromeres is sufficient to restore Sgo1 localization under this condition.