Kinetochore Localization Research Articles

A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B

By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint (SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the CPC without affecting the bulk of Aurora B activity in HeLa cells. Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 and SAC override at high nocodazole concentrations. 5-ITu also impaired kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed by concomitant inhibition of phosphatase activity. Forcing localization of Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but failed to rescue the SAC override. This result suggests that a target of 5-ITu, possibly Haspin itself, may further contribute to SAC signaling downstream of Aurora B.

Protein Tyrosine Phosphatase Shp2 (Ptpn11) Plays an Important Role in Maintenance of Chromosome Stability

Both activating and inactivating mutations in protein tyrosine phosphatase Ptpn11 (encoding Shp2) are associated with tumorigenesis. However, the underlying mechanisms remain unclear. Here, we show that Shp2 plays an important role in mitosis, dysregulation of which results in chromosome instability and cancer predisposition. Depletion of Shp2 compromised the mitotic checkpoint. Shp2-depleted cells exhibited a delay in mitotic entry and an earlier mitotic exit. Moreover, Shp2 deficiency caused defective kinetochore-microtubule attachment, chromosome misalignment, chromosomal congression defects, lagging chromosomes, and chromosome missegregation. Reintroduction of wild-type Shp2, but not a catalytically deficient mutant, restored the checkpoint function and chromosome alignment at metaphase in Shp2-deficient cells, establishing a requirement for the catalytic activity of Shp2 during mitosis. Further analyses revealed that Shp2 was required for the optimal activation of the mitotic kinases PLK1 and Aurora B and thereby the proper kinetochore localization and phosphorylation of BubR1, a core mitotic checkpoint protein that is also critical for chromosome alignment. Together, our findings show a previously unrecognized role for Shp2 in the maintenance of chromosome stability and suggest a new mechanism by which dysregulation of Shp2 signaling contributes to malignancy development.

Mitotic Regulator SKAP Forms a Link between Kinetochore Core Complex KMN and Dynamic Spindle Microtubules

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent study shows that mitotic motor CENP-E cooperates with SKAP to orchestrate an accurate chromosome movement in mitosis. However, it remains elusive how kinetochore core microtubule binding activity KMN (KNL1-MIS12-NDC80) regulates microtubule plus-end dynamics. Here, we identify a novel interaction between MIS13 and SKAP that orchestrates accurate interaction between kinetochore and dynamic spindle microtubules. SKAP physically interacts with MIS13 and specifies kinetochore localization of SKAP. Suppression of MIS13 by small interfering RNA abrogates the kinetochore localization of SKAP. Total internal reflection fluorescence microscopic assays demonstrate that SKAP exhibits an EB1-dependent, microtubule plus-end loading and tracking in vitro. Importantly, SKAP is essential for kinetochore oscillations and dynamics of microtubule plus-ends during live cell mitosis. Based on those findings, we reason that SKAP constitutes a dynamic link between spindle microtubule plus-ends and mitotic chromosomes to achieve faithful cell division.

Plk1 Phosphorylates Sgt1 at the Kinetochores To Promote Timely Kinetochore-Microtubule Attachment

Accurate chromosome segregation during cell division maintains genomic integrity and requires the proper establishment of kinetochore-microtubule attachment in mitosis. As a key regulator of mitosis, Polo-like kinase 1 (Plk1) is essential for this attachment process, but the molecular mechanism remains elusive. Here we identify Sgt1, a cochaperone for Hsp90, as a novel Plk1 substrate during mitosis. We show that Sgt1 dynamically localizes at the kinetochores, which lack microtubule attachments during prometaphase. Plk1 is required for the kinetochore localization of Sgt1 and phosphorylates serine 331 of Sgt1 at the kinetochores. This phosphorylation event enhances the association of the Hsp90-Sgt1 chaperone with the MIS12 complex to stabilize this complex at the kinetochores and thus coordinates the recruitment of the NDC80 complex to form efficient microtubule-binding sites. Disruption of Sgt1 phosphorylation reduces the MIS12 and NDC80 complexes at the kinetochores, impairs stable microtubule attachment, and eventually results in chromosome misalignment to delay the anaphase onset. Our results demonstrate a mechanism for Plk1 in promoting kinetochore-microtubule attachment to ensure chromosome stability.

95-P

Aim In the last fifteen years, published reports have described KIR gene-content frequency distributions in more than 120 populations worldwide. However, there have been limited studies examining these data in aggregate to detect overall patterns of variation at regional and global levels. Here, we present KIR gene-content data for 113 worldwide populations collected as part of the 15th and 16th International Histocompatibility and Immunogenetics Workshops. Methods The data were contributed by 31 laboratories during the five year course of this project. All populations were genotyped for KIR gene-content (presence/absence) for all loci. Data are examined for an overview of global patterns of carrier frequency distributions for individual loci, as well as variation in gene-content profiles (genotypes) and estimated haplotypes for the centromeric and telomeric regions and across the entire KIR complex. Forty populations were also genotyped for HLA-B and -C ligands, and the data analyzed for correlation between KIR and HLA ligand frequencies within populations. Results Nearly 100 unique genotypes are present in the populations under study. Nearly half of these genotypes are observed in only a single population, while several are present in nearly all populations. Most KIR gene-content variation can be attributed to a few common geneotypes; six gene-content haplotypes account for the majority of the total observed haplotypic variation in all world regions except Africa. Distinct patterns of variation are observed for the centromeric vs. telomeric KIR loci and haplotypes. In the populations genotyped for HLA ligands, a significant correlation between KIR2DL2 and HLA-C group frequencies is observed. Conclusions This work represents the most comprehensive summary of global KIR gene-content variation to date, and lays a foundation for our understanding of human population diversity in the KIR complex. In addition, it lends further support to the notion of co-evolution of KIR and HLA in human populations.

Centromeres of filamentous fungi

How centromeres are assembled and maintained remains one of the fundamental questions in cell biology. Over the past 20 years, the idea of centromeres as precise genetic loci has been replaced by the realization that it is predominantly the protein complement that defines centromere localization and function. Thus, placement and maintenance of centromeres are excellent examples of epigenetic phenomena in the strict sense. In contrast, the highly derived "point centromeres" of the budding yeast Saccharomyces cerevisiae and its close relatives are counter-examples for this general principle of centromere maintenance. While we have learned much in the past decade, it remains unclear if mechanisms for epigenetic centromere placement and maintenance are shared among various groups of organisms. For that reason, it seems prudent to examine species from many different phylogenetic groups with the aim to extract comparative information that will yield a more complete picture of cell division in all eukaryotes. This review addresses what has been learned by studying the centromeres of filamentous fungi, a large, heterogeneous group of organisms that includes important plant, animal and human pathogens, saprobes, and symbionts that fulfill essential roles in the biosphere, as well as a growing number of taxa that have become indispensable for industrial use.

Mps1 promotes rapid centromere accumulation of Aurora B

Aurora B localization to mitotic centromeres, which is required for proper chromosome alignment during mitosis, relies on Haspin-dependent histone H3 phosphorylation and on Bub1-dependent histone H2A phosphorylation--which interacts with Borealin through a Shugoshin (Sgo) intermediate. We demonstrate that Mps1 stimulates the latter recruitment axis. Mps1 activity enhances H2A-T120ph and is critical for Sgo1 recruitment to centromeres, thereby promoting Aurora B centromere recruitment in early mitosis. Importantly, chromosome biorientation defects caused by Mps1 inhibition are improved by restoring Aurora B centromere recruitment. As Mps1 kinetochore localization reciprocally depends on Aurora B, we propose that this Aurora B-Mps1 recruitment circuitry cooperates with the Aurora B-Haspin feedback loop to ensure rapid centromere accumulation of Aurora B at the onset of mitosis.

Putting CENP-A in its place

The centromere is the chromosomal region that directs kinetochore assembly during mitosis in order to facilitate the faithful segregation of sister chromatids. The location of the human centromere is epigenetically specified. The presence of nucleosomes that contain the histone H3 variant, CENP-A, are thought to be the epigenetic mark that indicates active centromeres. Maintenance of centromeric identity requires the deposition of new CENP-A nucleosomes with each cell cycle. During S-phase, existing CENP-A nucleosomes are divided among the daughter chromosomes, while new CENP-A nucleosomes are deposited during early G1. The specific assembly of CENP-A nucleosomes at centromeres requires the Mis18 complex, which recruits the CENP-A assembly factor, HJURP. We will review the unique features of centromeric chromatin as well as the mechanism of CENP-A nucleosome deposition. We will also highlight a few recent discoveries that begin to elucidate the factors that temporally and spatially control CENP-A deposition.

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment.

Keeping centromeric identity

MIS18α interacts with DNMT3A and DNMT3B to ensure CENPA centromeric localization.

Spindle checkpoint–independent inhibition of mitotic chromosome segregation byDrosophilaMps1

Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.

Genetic Positioning of Centromeres through Half-Tetrad Analysis in Gynogenetic Diploid Families of the Zhikong Scallop (Chlamys farreri)

Centromere mapping is a powerful tool for improving linkage maps, investigating crossover events, and understanding chiasma interference during meiosis. Ninety microsatellite markers selected across all linkage groups (LGs) from a previous Chlamys farreri genetic map were studied in three artificially induced meiogynogenetic families for centromere mapping by half-tetrad analysis. Inheritance analyses showed that all 90 microsatellite loci conformed to Mendelian inheritance in the control crosses, while 4.4% of the microsatellite loci showed segregation departures from an expected 1:1 ratio of two homozygote classes in meiogynogenetic progeny. The second division segregation frequency (y) of the microsatellites ranged from 0.033 to 0.778 with a mean of 0.332, confirming the occurrence of partial chiasma interference in this species. Heterogeneity of y is observed in one of 42 cases in which markers were typed in more than one family, suggesting variation in gene-centromere recombination among families. Centromere location was mostly in accordance with the C. farreri karyotype, but differences in marker order between linkage and centromere maps occurred. Overall, this study makes the genetic linkage map a more complete and informative tool for genomic studies and it will also facilitate future research of the structure and function of the scallop centromeres.

Plk1 regulates the kinesin-13 protein Kif2b to promote faithful chromosome segregation

Solid tumors are frequently aneuploid, and many display high rates of ongoing chromosome missegregation in a phenomenon called chromosomal instability (CIN). The most common cause of CIN is the persistence of aberrant kinetochore-microtubule (k-MT) attachments, which manifest as lagging chromosomes in anaphase. k-MT attachment errors form during prometaphase due to stochastic interactions between kinetochores and microtubules. The kinesin-13 protein Kif2b promotes the correction of k-MT attachment errors in prometaphase, but the mechanism restricting this activity to prometaphase remains unknown. Using mass spectrometry, we identified multiple phosphorylation sites on Kif2b, some of which are acutely sensitive to inhibition of Polo-like kinase 1 (Plk1). We show that Plk1 directly phosphorylates Kif2b at threonine 125 (T125) and serine 204 (S204), and that these two sites differentially regulate Kif2b function. Phosphorylation of S204 is required for the kinetochore localization and activity of Kif2b in prometaphase, and phosphorylation of T125 is required for Kif2b activity in the correction of k-MT attachment errors. These data demonstrate that Plk1 regulates both the localization and activity of Kif2b during mitosis to promote the correction of k-MT attachment errors to ensure mitotic fidelity.

The Cotton Centromere Contains a Ty3-gypsy-like LTR Retroelement

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species.

Phosphoregulation of Spc105 by Mps1 and PP1 Regulates Bub1 Localization to Kinetochores

Kinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that copurifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores, and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein.

Roles of Mis18α in Epigenetic Regulation of Centromeric Chromatin and CENP-A Loading

The Mis18 complex has been identified as a critical factor for the centromeric localization of a histone H3 variant, centromeric protein A (CENP-A), which is responsible for the specification of centromere identity in the chromosome. However, the functional role of Mis18 complex is largely unknown. Here, we generated Mis18α conditional knockout mice and found that Mis18α deficiency resulted in lethality at early embryonic stage with severe defects in chromosome segregation caused by mislocalization of CENP-A. Further, we demonstrate Mis18α's crucial role for epigenetic regulation of centromeric chromatin by reinforcing centromeric localization of DNMT3A/3B. Mis18α interacts with DNMT3A/3B, and this interaction is critical for maintaining DNA methylation and hence regulating epigenetic states of centromeric chromatin. Mis18α deficiency led to reduced DNA methylation, altered histone modifications, and uncontrolled noncoding transcripts in centromere region by decreased DNMT3A/3B enrichment. Together, our findings uncover the functional mechanism of Mis18α and its pivotal role in mammalian cell cycle.

The evolutionary life cycle of the resilient centromere

The centromere is a chromosomal structure that is essential for the accurate segregation of replicated eukaryotic chromosomes to daughter cells. In most centromeres, the underlying DNA is principally made up of repetitive DNA elements, such as tandemly repeated satellite DNA and retrotransposable elements. Paradoxically, for such an essential genomic region, the DNA is rapidly evolving both within and between species. In this review, we show that the centromere locus is a resilient structure that can undergo evolutionary cycles of birth, growth, maturity, death and resurrection. The birth phase is highlighted by examples in humans and other organisms where centromere DNA deletions or chromosome rearrangements can trigger the epigenetic assembly of neocentromeres onto genomic sites without typical features of centromere DNA. In addition, functional centromeres can be generated in the laboratory using various methodologies. Recent mapping of the foundation centromere mark, the histone H3 variant CENP-A, onto near-complete genomes has uncovered examples of new centromeres which have not accumulated centromere repeat DNA. During the growth period of the centromere, repeat DNA begins to appear at some, but not all, loci. The maturity stage is characterised by centromere repeat accumulation, expansions and contractions and the rapid evolution of the centromere DNA between chromosomes of the same species and between species. This stage provides inherent centromere stability, facilitated by repression of gene activity and meiotic recombination at and around the centromeres. Death to a centromere can result from genomic instability precipitating rearrangements, deletions, accumulation of mutations and the loss of essential centromere binding proteins. Surprisingly, ancestral centromeres can undergo resurrection either in the field or in the laboratory, via as yet poorly understood mechanisms. The underlying principle for the preservation of a centromeric evolutionary life cycle is to provide resilience and perpetuity for the all-important structure and function of the centromere.

Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting

The spindle checkpoint prevents aneuploidy by delaying anaphase onset until all sister chromatids achieve proper microtubule attachment. The kinetochore-bound checkpoint protein complex Mad1-Mad2 promotes the conformational activation of Mad2 and serves as a catalytic engine of checkpoint signaling. How Mad1 is targeted to kinetochores is not understood. Here, we report the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. Mad1 CTD forms a homodimer and, unexpectedly, has a fold similar to those of the kinetochore-binding domains of Spc25 and Csm1. Nonoverlapping Mad1 fragments retain detectable kinetochore targeting. Deletion of the CTD diminishes, does not abolish, Mad1 kinetochore localization. Mutagenesis studies further map the functional interface of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Our results indicate that CTD is a part of an extensive kinetochore-binding interface of Mad1, and rationalize graded kinetochore targeting of Mad1 during checkpoint signaling.

Heterochromatin protein 1 homologue Swi6 acts in concert with Ers1 to regulate RNAi-directed heterochromatin assembly

In fission yeast, the RNAi pathway is required for centromeric heterochromatin assembly. siRNAs derived from centromeric transcripts are incorporated into the RNA-induced transcriptional silencing (RITS) complex and direct it to nascent homologous transcripts. The RNA-induced transcriptional silencing-bound nascent transcripts further recruit the RNA-directed RNA polymerase complex (RDRC) to promote dsRNA synthesis and siRNA production. Heterochromatin coated with Swi6/Heterochromain Protein 1 is then formed following recruitment of chromatin modification machinery. Swi6 is also required for the upstream production of siRNA, although the mechanism for this has remained obscure. Here, we demonstrate that Swi6 recruits RDRC to heterochromatin through Ers1, an RNAi factor intermediate. An ers1(+) mutant allele (ers1-C62) was identified in a genetic screen for mutants that alleviate centromeric silencing, and this phenotype was suppressed by overexpression of either the Hrr1 RDRC subunit or Clr4 histone H3-K9 methyltransferase. Ers1 physically interacts with Hrr1, and loss of Ers1 impairs RDRC centromeric localization. Although Ers1 failed to bind Clr4, a direct interaction with Swi6 was detected, and centromeric localization of Swi6 was enhanced by Clr4 overexpression in ers1-C62 cells. Consistent with this, deletion of swi6(+) reduced centromeric localization of Ers1 and RDRC. Moreover, tethering of Ers1 or Hrr1 to centromeric heterochromatin partially bypassed Swi6 function. These findings demonstrate an alternative mechanism for RDRC recruitment and explain the essential role of Swi6/Heterochromain Protein 1 in RNAi-directed heterochromatin assembly.

Structure of Human Mad1 C‐terminal Domain Reveals Its Kinetochore‐Targeting Function

Accurate chromosome segregation during mitosis is critical for maintaining genomic stability. The spindle checkpoint is a cellular surveillance system that ensures the fidelity of chromosome segregation. The kinetochore‐bound checkpoint protein complex Mad1–Mad2 promotes the conformational activation of Mad2 and serves as a catalytic engine of checkpoint signaling. How Mad1 is targeted to kinetochores is not understood, however. To understand the functional importance of the C‐terminal region of Mad1, we solved the crystal structure of human Mad1 C‐terminal domain (CTD) and found that it formed a homodimer and had a fold similar to that of the known kinetochore‐targeting domain of Spc25 (a subunit of the Ndc80 complex). Indeed, Mad1‐ ∆CTD was defective in kinetochore targeting in human cells. The mutagenesis studies validate a role of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Our results thus reveal novel functions of Mad1‐CTD in kinetochore targeting. Deletion of CTD does not abolish Mad1 kinetochore localization, however. Non‐overlapping Mad1 fragments retain detectable kinetochore targeting. Therefore, our results indicate that CTD is a part of an extensive kinetochore‐binding interface of Mad1, and rationalize graded kinetochore targeting of Mad1 during checkpoint signaling.