Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

Functional analysis of a Bcl-xL phosphorylation mutant series has revealed that cells expressing Bcl-xL(Ser49Ala) mutant are less stable at G2 checkpoint after DNA damage and enter cytokinesis more slowly after microtubule poisoning, than cells expressing wild-type Bcl-xL. These effects of Bcl-xL(Ser49Ala) mutant seem to be separable from Bcl-xL function in apoptosis. Bcl-xL(Ser49) phosphorylation is cell cycle-dependent. In synchronized cells, phospho-Bcl-xL(Ser49) appears during the S phase and G2, whereas it disappears rapidly in early mitosis during prometaphase, metaphase and early anaphase, and re-appears during telophase and cytokinesis. During DNA damage-induced G2 arrest, an important pool of phospho-Bcl-xL(Ser49) accumulates in centrosomes which act as essential decision centers for progression from G2 to mitosis. During telophase/cytokinesis, phospho-Bcl-xL(Ser49) is found with dynein motor protein. In a series of in vitro kinase assays, specific small interfering RNA and pharmacological inhibition experiments, polo kinase 3 (PLK3) was implicated in Bcl-xL(Ser49) phosphorylation. These data indicate that, during G2 checkpoint, phospho-Bcl-xL(Ser49) is another downstream target of PLK3, acting to stabilize G2 arrest. Bcl-xL phosphorylation at Ser49 also correlates with essential PLK3 activity and function, enabling cytokinesis and mitotic exit.     

The PTEN-Akt pathway impacts the integrity and composition of mitotic centrosomes

Loss of the tumor suppressor PTEN is observed in many human cancers that display increased chromosome instability and aneuploidy. The subcellular fractions of PTEN are associated with different functions that regulate cell growth, invasion and chromosome stability. In this study, we show a novel role for PTEN in regulating mitotic centrosomes. PTEN localization at mitotic centrosomes peaks between prophase and metaphase, paralleling the centrosomal localization of PLK-1 and γ-tubulin and coinciding with the time frame of centrosome maturation. In primary keratinocytes, knockdown of PTEN increased whole-cell levels of γ-tubulin and PLK-1 in an Akt-dependent manner and had little effect on recruitment of either protein to mitotic centrosomes. Conversely, knockdown of PTEN reduced centrosomal levels of pericentrin in an Akt-independent manner. Inhibition of Akt activation with MK2206 reduced the whole-cell and centrosome levels of PLK-1 and γ-tubulin and also prevented the recruitment of PTEN to mitotic centrosomes. This reduction in centrosome-associated proteins upon inhibition of Akt activity may contribute to the increase in defects in centrosome number and separation observed in metaphase cells. Concomitant PTEN knockdown and Akt inhibition reduced the frequency of metaphase cells with centrosome defects when compared with MK2206 treatment alone, indicating that both PTEN and pAkt are required to properly regulate centrosome composition during mitosis. The findings presented in this study demonstrate a novel role for PTEN and Akt in controlling centrosome composition and integrity during mitosis and provide insight into how PTEN functions as a multifaceted tumor suppressor.     

RED, a spindle pole-associated protein, is required for kinetochore localization of MAD1, mitotic progression, and activation of the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) is essential for ensuring the proper attachment of kinetochores to the spindle and, thus, the precise separation of paired sister chromatids during mitosis. The SAC proteins are recruited to the unattached kinetochores for activation of the SAC in prometaphase. However, it has been less studied whether activation of the SAC also requires the proteins that do not localize to the kinetochores. Here, we show that the nuclear protein RED, also called IK, a down-regulator of human leukocyte antigen (HLA) II, interacts with the human SAC protein MAD1. Two RED-interacting regions identified in MAD1 are from amino acid residues 301-340 and 439-480, designated as MAD1(301-340) and MAD1(439-480), respectively. Our observations reveal that RED is a spindle pole-associated protein that colocalizes with MAD1 at the spindle poles in metaphase and anaphase. Depletion of RED can cause a shorter mitotic timing, a failure in the kinetochore localization of MAD1 in prometaphase, and a defect in the SAC. Furthermore, the RED-interacting peptides MAD1(301-340) and MAD1(439-480), fused to enhanced green fluorescence protein, can colocalize with RED at the spindle poles in prometaphase, and their expression can abrogate the SAC. Taken together, we conclude that RED is required for kinetochore localization of MAD1, mitotic progression, and activation of the SAC.     

Translocation of XRCC1 and DNA ligase IIIalpha from centrosomes to chromosomes in response to DNA damage in mitotic human cells

DNA single-strand breaks (SSBs) are the most frequent lesions caused by oxidative DNA damage. They disrupt DNA replication, give rise to double-strand breaks and lead to cell death and genomic instability. It has been shown that the XRCC1 protein plays a key role in SSBs repair. We have recently shown in living human cells that XRCC1 accumulates at SSBs in a fully poly(ADP-ribose) (PAR) synthesis-dependent manner and that the accumulation of XRCC1 at SSBs is essential for further repair processes. Here, we show that XRCC1 and its partner protein, DNA ligase IIIα, localize at the centrosomes and their vicinity in metaphase cells and disappear during anaphase. Although the function of these proteins in centrosomes during metaphase is unknown, this centrosomal localization is PAR-dependent, because neither of the proteins is observed in the centrosomes in the presence of PAR polymerase inhibitors. On treatment of metaphase cells with H₂O₂, XRCC1 and DNA ligase IIIα translocate immediately from the centrosomes to mitotic chromosomes. These results show for the first time that the repair of SSBs is present in the early mitotic chromosomes and that there is a dynamic response of XRCC1 and DNA ligase IIIα to SSBs, in which these proteins are recruited from the centrosomes, where metaphase-dependent activation of PAR polymerase occurs, to mitotic chromosomes, by SSBs-dependent activation of PAR polymerase.     

The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1

BACKGROUND: The mitotic kinases, Cdk1, Aurora A/B, and Polo-like kinase 1 (Plk1) have been characterized extensively to further understanding of mitotic mechanisms and as potential targets for cancer therapy. Cdk1 and Aurora kinase studies have been facilitated by small-molecule inhibitors, but few if any potent Plk1 inhibitors have been identified. RESULTS: We describe the cellular effects of a novel compound, BI 2536, a potent and selective inhibitor of Plk1. The fact that BI 2536 blocks Plk1 activity fully and instantaneously enabled us to study controversial and unknown functions of Plk1. Cells treated with BI 2536 are delayed in prophase but eventually import Cdk1-cyclin B into the nucleus, enter prometaphase, and degrade cyclin A, although BI 2536 prevents degradation of the APC/C inhibitor Emi1. BI 2536-treated cells lack prophase microtubule asters and thus polymerize mitotic microtubules only after nuclear-envelope breakdown and form monopolar spindles that do not stably attach to kinetochores. Mad2 accumulates at kinetochores, and cells arrest with an activated spindle-assembly checkpoint. BI 2536 prevents Plk1's enrichment at kinetochores and centrosomes, and when added to metaphase cells, it induces detachment of microtubules from kinetochores and leads to spindle collapse. CONCLUSIONS: Our results suggest that Plk1's accumulation at centrosomes and kinetochores depends on its own activity and that this activity is required for maintaining centrosome and kinetochore function. Our data also show that Plk1 is not required for prophase entry, but delays transition to prometaphase, and that Emi1 destruction in prometaphase is not essential for APC/C-mediated cyclin A degradation.     

Thrombopoietin-induced Polyploidization of Bone Marrow Megakaryocytes Is Due to a Unique Regulatory Mechanism in Late Mitosis

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process.     

Cohesion and centromere activity are required for phosphorylation of histone H3 in maize

Haspin‐mediated phosphorylation of histone H3 at threonine 3 (H3T3ph) promotes proper deposition of Aurora B at the inner centromere to ensure faithful chromosome segregation in metazoans. However, the function of H3T3ph remains relatively unexplored in plants. Here, we show that in maize (Zea mays L.) mitotic cells, H3T3ph is concentrated at pericentromeric and centromeric regions. Additional weak H3T3ph signals occur between cohered sister chromatids at prometaphase. Immunostaining on dicentric chromosomes reveals that an inactive centromere cannot maintain H3T3ph at metaphase, indicating that a functional centromere is required for H3T3 phosphorylation. H3T3ph locates at a newly formed centromeric region that lacks detectable CentC sequences and strongly reduced CRM and ZmBs repeat sequences at metaphase II. These results suggest that centromeric localization of H3T3ph is not dependent on centromeric sequences. In maize meiocytes, H3T3 phosphorylation occurs at the late diakinesis and extends to the entire chromosome at metaphase I, but is exclusively limited to the centromere at metaphase II. The H3T3ph signals are absent in the afd1 (absence of first division) and sgo1 (shugoshin) mutants during meiosis II when the sister chromatids exhibit random distribution. Further, we show that H3T3ph is mainly located at the pericentromere during meiotic prophase II but is restricted to the inner centromere at metaphase II. We propose that this relocation of H3T3ph depends on tension at the centromere and is required to promote bi‐orientation of sister chromatids.     

Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, we develop an improved time-lapse video microscopy technique that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, green fluorescent protein (GFP)-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Our mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: first promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, we show that Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and that regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both Caenorhabditis elegans and mammals. We conclude that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release.     

Checking Out the Centrosome

Centrosomes consist of a pair of barrel-shaped microtubule assemblies called centrioles, surrounded by a pericentriolar matrix. The only well-characterized function of centrosomes is to organize both interphase microtubule arrays responsible for cell polarity and the mitotic spindle, which mediates the strictly bipolar separation of chromosomes. In addition to these established functions it has been speculated that centrosomes might be involved in several different cell cycle regulatory events like entry into mitosis, cytokinesis, G1/S transition and monitoring of DNA damage. These assumptions are mainly based on a rapidly growing list of centrosome-associated regulatory proteins such as p53, Brca1, Chk1, Chk2, TopBP1, Aurora-A, Plk1, cyclin B1, and Cdk1. However, only very few direct links between their localization to the centrosome and specific cellular functions have been unraveled until recently. This review will focus on recent advances in the understanding of the role of centrosomes as integrators of positive and negative pathways for mitotic entry.     

Determinants of Rad21 localization at the centrosome in human cells

Cohesin proteins help maintain the physical associations between sister chromatids that arise in S-phase and are removed in anaphase. Recent studies found that cohesins also localize to the centrosomes, the organelles that organize the mitotic bipolar spindle. We find that the cohesin protein Rad21 localizes to centrosomes in a manner that is dependent upon known regulators of sister chromatid cohesion as well as regulators of centrosome function. These data suggest that Rad21 functions at the centrosome and that the regulators of Rad21 coordinate the centrosome and chromosomal functions of cohesin.     

UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores

The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase.     

Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase

BACKGROUND: The linkage between duplicated chromosomes (sister chromatids) is established during S phase by the action of cohesin, a multisubunit complex conserved from yeast to humans. Most cohesin dissociates from chromosome arms when the cell enters mitotic prophase, leading to the formation of metaphase chromosomes with two cytologically discernible chromatids. This process is known as sister-chromatid resolution. Although two mitotic kinases have been implicated in this process, it remains unknown exactly how the cohesin-mediated linkage is destabilized at a mechanistic level. RESULTS: The wings apart-like (Wapl) protein was originally identified as a gene product that potentially regulates heterochromatin organization in Drosophila melanogaster. We show that the human ortholog of Wapl is a cohesin-binding protein that facilitates cohesin's timely release from chromosome arms during prophase. Depletion of Wapl from HeLa cells causes transient accumulation of prometaphase-like cells with chromosomes that display poorly resolved sister chromatids with a high level of cohesin. Reduction of cohesin relieves the Wapl-depletion phenotype, and depletion of Wapl rescues premature sister separation observed in Sgo1-depleted or Esco2-depleted cells. Conversely, overexpression of Wapl causes premature separation of sister chromatids. Wapl physically associates with cohesin in HeLa-cell nuclear extracts. Remarkably, in vitro reconstitution experiments demonstrate that Wapl forms a stoichiometric, ternary complex with two regulatory subunits of cohesin, implicating its noncatalytic function in inactivating cohesin's ability to interact with chromatin. CONCLUSIONS: Wapl is a new regulator of sister chromatid resolution and promotes release of cohesin from chromosomes by directly interacting with its regulatory subunits.     

Genes occupy a fixed and symmetrical position on sister chromatids

A high-resolution fluorescence methodology for nonisotopic in situ hybridization was applied to determine the positions occupied by several single-copy genes, DNA sequences, and integrated viral genomes on sister chromatids. The lateral and longitudinal mapping of the probes was performed on prometaphase and metaphase chromosomes. A fixed lateral position, exterior or median in relation to the longitudinal axis of the chromatids, was observed for a given probe, with a symmetrical position of the double fluorescent spots. This position appears to be independent of chromosome condensation stage from prometaphase to metaphase. These observations suggest an opposite helical-handedness conformation of DNA on both chromatids with a mirror symmetry. They support the model of chromosome packaging recently proposed by Boy de la Tour and Laemmli. Moreover, our results indicate that the last stages of chromosome condensation occur by packing down the coils without further coiling.     

Regulated assembly of the mitotic spindle: a perspective from two ends

Chromosome segregration and cell division requires the regulated assembly of the mitotic spindle apparatus. This mitotic spindle is composed of condensed chromosomes attached to a dynamic array of microtubules. The microtubule array is nucleated by centrosomes and organized by associated structural and motor proteins. Mechanical linkages between sister chromatids and microtubules are critical for spindle assembly and chromosome segregation. Defects in either chromosome or centrosome segregation can lead to aneuploidy and are correlated with cancer progression. In this review, we discuss current models of how centrosomes and chromosomes organize the spindle for their equal distribution to each daughter cell.     

Proteome analysis of the human mitotic spindle

The accurate distribution of sister chromatids during cell division is crucial for the generation of two cells with the same complement of genetic information. A highly dynamic microtubule-based structure, the mitotic spindle, carries out the physical separation of the chromosomes to opposite poles of the cells and, moreover, determines the cell division cleavage plane. In animal cells, the spindle comprises microtubules that radiate from the microtubule organizing centers, the centrosomes, and interact with kinetochores on the chromosomes. Malfunctioning of the spindle can lead to chromosome missegregation and hence result in aneuploidy, a hallmark of most human cancers. Despite major progress in deciphering the temporal and spatial regulation of the mitotic spindle, its composition and function are not fully understood. A more complete inventory of spindle components would therefore constitute an important advance. Here we describe the purification of human mitotic spindles and their analysis by MS/MS. We identified 151 proteins previously known to associate with the spindle apparatus, centrosomes, and/or kinetochores and 644 other proteins, including 154 uncharacterized components that did not show obvious homologies to known proteins and did not contain motifs indicative of a particular localization. Of these uncharacterized proteins, 17 were tagged and localized in transfected mitotic cells, resulting in the identification of six genuine spindle components (KIAA0008, CdcA8, KIAA1187, FLJ12649, FLJ90806, and C20Orf129). This study illustrates the strength of a proteomic approach for the analysis of isolated human spindles and identifies several novel spindle components for future functional studies.     

Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells

Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation.     

Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing

Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore-microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore-microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore-microtubule attachments and subsequently silencing the spindle checkpoint.     

SUMOylation of the C-terminal domain of DNA topoisomerase IIα regulates the centromeric localization of Claspin

DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity.     

TopBP1 deficiency impairs V(D)J recombination during lymphocyte development

TopBP1 was initially identified as a topoisomerase II‐β‐binding protein and it plays roles in DNA replication and repair. We found that TopBP1 is expressed at high levels in lymphoid tissues and is essential for early lymphocyte development. Specific abrogation of TopBP1 expression resulted in transitional blocks during early lymphocyte development. These defects were, in major part, due to aberrant V(D)J rearrangements in pro‐B cells, double‐negative and double‐positive thymocytes. We also show that TopBP1 was located at sites of V(D)J rearrangement. In TopBP1‐deficient cells, γ‐H2AX foci were found to be increased. In addition, greater amount of γ‐H2AX product was precipitated from the regions where TopBP1 was localized than from controls, indicating that TopBP1 deficiency results in inefficient DNA double‐strand break repair. The developmental defects were rescued by introducing functional TCR αβ transgenes. Our data demonstrate a novel role for TopBP1 as a crucial factor in V(D)J rearrangement during the development of B, T and iNKT cells.