Chromosomal passenger proteins associate with chromosomes early in mitosis and transfer to the spindle at ana/telophase. Recent results show that aurora B/AIM-1 (aurora and IpI1-like midbody-associated protein kinase), which is responsible for mitotic histone H3 phosphorylation, INCENP (Inner Centromere protein) and Survivin/BIR are in a macromolecular complex as novel chromosomal passenger proteins. Aurora B/AIM-1 can bind to Survivin and the C-terminal region of INCENP, respectively, and colocalizes with both proteins to the centromeres, midzone and midbody. Disruption of either aurora B/AIM-1 or INCENP function leads to sever defects in chromosome segregation and cytokinesis. Moreover, the formation of the central spindle through anaphase to cytokinesis is also disrupted severely.
These data suggest that chromosomal passenger complex is required for proper chromosome segregation by phosphorylating histone H3, and cytokinesis by ensuring the correct assembly of the midzone and midbody microtubule. Chromosomal passenger protein complex may couple chromosome segregation with cytokinesis.

Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation, Errors in this process result in aneuploidy which can lead to cell death or oncogenesis, In this paper we describe a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G(2)/M phase, The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis, Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division, Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death, These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells.

EXPOSURE to ultraviolet light arrests the function of mammalian fibroblasts in the G1 phase of the cell cycle, as well as the S and G2 phases. Although p21, an inhibitor of cyclin-dependent kinase (Cdk) that is induced by DNA damage mag partly account for the arrest in G1 (ref. 1), the mechanism is little understood. Here we show that tyrosine phosphorylation of Cdk4 is required for this arrest. In rat fibroblast, Cdk4 is tyrosine-phosphorylated during G1 progression, and its dephosphorylation is required for S phase. When cells are ultraviolet-irradiated, their arrest in G1 is accompanied by an increase in phosphorylation level. Conversely, cells expressing unphosphorylatable Cdk4(F17) fail to arrest in G1, and suffer significantly elevated chromosomal aberrations and cell death.

Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle. (C) 2015 Elsevier Inc. All rights reserved.

CDK5RAP2 is a centrosomal protein that regulates the recruitment of a gamma-tubulin ring complex (gamma-TuRC) onto centrosomes and microtubules (MTs) dynamics as a member of MT plus-end-tracking proteins (+TIPs). In our previous report, we found mammalian Cep169 as a CDK5RAP2 binding partner, and Cep169 accumulates at the distal ends of MTs and centrosomes, and coincides with CDK5RAP2. Depletion of Cep169 induces MT depolymerization, indicating that Cep169 targets MT tips and regulates stability and dynamics of MTs. However, how Cep169 contributes to the stabilization of MT remains unclear. Here we show that Cep169 is able to stabilize MTs and induces formation of long MT bundles with intense acetylation of MTs with CDK5RAP2, when expressed at higher levels in U2OS cells. In addition, we demonstrated that Cep169 forms homodimers through its N-terminal domain and directly interacts with MTs through its C-terminal domain. Interestingly, Cep169 mutants, which lack each domains, completely abolished the activity, respectively. Therefore, Cep169 bundles MTs and induces solid structure of MTs by crosslinking each adjacent MTs as a homodimer. (C) 2015 Elsevier Inc. All rights reserved.

Chromosome segregation machinery is controlled by mechanochemical regulation. Tension in a mitotic spindle, which is balanced by molecular motors and polymerization-depolymerization dynamics of microtubules, is thought to be essential for determining the timing of chromosome segregation after the establishment of the kinetochore-microtubule attachments. It is not known, however, whether and how applied mechanical forces modulate the tension balance and chemically affect the molecular processes involved in chromosome segregation. Here we found that a mechanical impulse externally applied to mitotic HeLa cells alters the balance of forces within the mitotic spindle. We identified two distinct mitotic responses to the applied mechanical force that either facilitate or delay anaphase onset, depending on the direction of force and the extent of cell compression. An external mechanical impulse that physically increases tension within the mitotic spindle accelerates anaphase onset, and this is attributed to the facilitation of physical cleavage of sister chromatid cohesion. On the other hand, a decrease in tension activates the spindle assembly checkpoint, which impedes the degradation of mitotic proteins and delays the timing of chromosome segregation. Thus, the external mechanical force acts as a crucial regulator for metaphase progression, modulating the internal force balance and thereby triggering specific mechanochemical cellular reactions.

The asymmetric and highly stereoselective synthesis of compound 1, which corresponds exactly to the DEF-ring moiety of (-)-FR182877, and the biological activities of its derivatives are described. All derivatives of 1 showed no activity in the tubulin polymerization assay, but one derivative was shown to have the ability to induce mitotic arrest by interfering with microtubule dynamics, and the cellular effects are similar to those of paclitaxel.

Centrosome duplication is tightly coupled with the cell cycle and neither too many nor too few centrosomes are induced in a normal cell. To study how centrosome assembly is regulated, we analyzed the abnormal process of multiple centrosome replications in Chinese hamster ovary (CHO) cells induced by hydroxyurea (HU), which is known to uncouple the centrosome cycle from the cell cycle. Green fluorescent protein (GFP)-tagged centrin2 expressed in CHO cells labels both centrioles and the pericentriolar material (PCM). Counting fluorescent spots of GFP-centrin in synchronized cells showed that in G(1)/S-arrested cells, centrioles are initially duplicated in a template manner. Further treatment with HU overrides the suppression of excess centriole/centrosome replication in a cell where the full complement of centrioles/centrosomes already exists. Time- lapse fluorescence microscopy revealed that small centrin- containing foci emerged in the cytoplasm during HU treatment. These foci are surrounded by a PCM cloud and their number continuously increases as cells are exposed to HU for longer periods of time. Both the centrosome and cytoplasmic foci are highly mobile, continuously changing their position in a manner dependent on microtubules/microtubule dynamics. The centrosome number increases as small foci grow in size and resolve into recognizable centrosomes. As this occurs in a random fashion, the cells arrested longer with HU induced highly heterogeneous numbers of centrosomes.

Vpr, the viral protein R of human immunodeficiency virus type 1, induces G(2) cell cycle arrest and apoptosis in mammalian cells via ATR (for "ataxia-telangiectasia-mediated and Rad3-related") checkpoint activation. The expression of Vpr induces the formation of the gamma-histone 2A variant X (H2AX) and breast cancer susceptibility protein 1 (BRCA1) nuclear foci, and a C-terminal domain is required for Vpr-induced ATR activation and its nuclear localization. However, the cellular target of Vpr, as well as the mechanism of G2 checkpoint activation, was unknown. Here we report that Vpr induces checkpoint activation and G2 arrest by binding to the CUS1 domain of SAP145 and interfering with the functions of the SAP145 and SAP49 proteins, two subunits of the multimeric splicing factor 3b (SF3b). Vpr interacts with and colocalizes with SAP145 through its C-terminal domain in a speckled distribution. The depletion of either SAP145 or SAP49 leads to checkpoint-mediated G2 cell cycle arrest through the induction of nuclear foci containing gamma-112AX and BRCA1. In addition, the expression of Vpr excludes SAP49 from the nuclear speckles and inhibits the formation of the SAP145-SAP49 complex. To conclude, these results point out the unexpected roles of the SAP145-SAP49 splicing factors in cell cycle progression and suggest that cellular expression of Vpr induces checkpoint activation and G(2) arrest by interfering with the function of SAP145-SAP49 complex in host cells.

Cep135 is a 135-kDa, coiled-coil centrosome protein important for microtubile organization in mammalian cells [Ohta et al., 2002: J. Cell Biol. 156: 87-99]. To identify Cep135-interacting molecules, we screened yeast two-hybrid libraries. One clone encoded dynamitin, a p50 dynactin subunit, which localized at the centrosome and has been shown to be involved in anchoring microtubules to centrosomes. The central domain of p50 binds to the C-terminal sequence of Cep135; this was further confirmed by immunoprecipitation and immunostaining of CHO cells co-expressing the binding domains for Cep135 and p50. Exogenous p50 lacking the Cep135-binding domain failed to locate at the centrosome, suggesting that Cep135 is required for initial targeting of the centrosome. Altered levels of Cep135 and p50 by RNAi and protein overexpression caused the release of endogenous partner molecules from centrosomes. This also resulted in dislocation of other centrosomal molecules, such as gamma-tubulin and pericentrin, ultimately leading to disorganization of microtubute patterns. These results suggest that Cep 135 and p50 play an important role in assembly and maintenance of functional microtubule-organizing centers. Cell Motil. Cytoskeleton 58:53-66, 2004. (C) 2004 Wiley-Liss, Inc.

During mitosis, the mitotic spindle, a bipolar structure composed of microtubules (MTs) and associated motor proteins(1,2), segregates sister chromatids to daughter cells. Initially some MTs emanating from one centrosome attach to the kinetochore at the centromere of one of the duplicated chromosomes. This attachment allows rapid poleward movement of the bound chromosome. Subsequent attachment of the sister kinetochore to MTs growing from the other centrosome results in the bi-orientation of the chromosome, in which interactions between kinetochores and the plus ends of MTs are formed and stabilized(2). These processes ensure alignment of chromosomes during metaphase and their correct segregation during anaphase. Although many proteins constituting the kinetochore have been identified and extensively studied, the signalling responsible for MT capture and stabilization is unclear(1,2). Small GTPases of the Rho family regulate cell morphogenesis by organizing the actin cytoskeleton and regulating MT alignment and stabilization(3). We now show that one member of this family, Cdc42, and its effector, mDia3, regulate MT attachment to kinetochores.

Aurora-B is an evolutionally conserved protein kinase that regulates several mitotic events including cytokinesis. We previously demonstrated the possible existence of a protein kinase that phosphorylates at least Ser-72 on vimentin, the most widely expressed intermediate filament protein, in the cleavage furrow-specific manner. Here we showed that vimentin-Ser-72 phosphorylation occurred specifically at the border of the Aurora-B-localized area from anaphase to telophase. Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation. In vitro analyses revealed that Aurora-B phosphorylates vimentin at similar to2 mol phosphate/mol of substrate for 30 min and that this phosphorylation dramatically inhibits vimentin filament formation. We further identified eight Aurora-B phosphorylation sites, including Ser-72 on vimentin, and then constructed the mutant vimentin in which these identified sites are changed into Ala. Cells expressing this mutant formed an unusually long bridge-like intermediate filament structure between unseparated daughter cells. We then identified important phosphorylation sites for the bridge phenotype. Our findings indicate that Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation and controls vimentin filament segregation in cytokinetic process.

Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its, subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.

The mammalian polo-like kinase (Plk) plays a critical role in M-phase progression. Plk is phosphorylated and activated by an upstream kinase(s), which has not yet been identified in mammalian cells. Phosphopeptide mapping and phosphoamino acid analyses of Plk labeled in vivo and phosphorylated in vitro by Xenopus polo-like kinase kinase-1 (xPlkk1) or by lymphocyte-oriented kinase, its most closely related mammalian enzyme, indicate that Thr-210 is a major phosphorylation site in activated Plk from mitotic HeLa cells. Although the amino acid sequence surrounding Ser-137 is similar to that at Thr-210 and is conserved in Plk family members, Ser-137 is not detectably phosphorylated in mitotic mammalian cells or by xPlkk1 in vitro. Nevertheless, the substitution of either Thr-210 or Ser-137 with Asp (T210D or S137D) elevates the kinase activity of Plk. The kinase activity of the double mutant S137D/T210D is not significantly different from that of T210D or S137D, demonstrating that substitution of both residues does not have an additive effect on Plk activity. Expression of the S137D mutant construct arrested HeLa cells in early S-phase with slightly separated centrosomes, whereas cells expressing wild type and T210D were arrested or delayed in M-phase. These data indicate that the Ser-137 may have an unexpected and novel role in the function of Plk.

Phosphorylation of histone H3 at Ser-10 is required for maintenance of proper chromosome dynamics during mitosis. AIM-1, a mammalian Ipl1/aurora kinase involved in H3 phosphorylation, is transcriptionally overexpressed in many tumor cell lines. Increased expression of the AIM-1 gene has been observed in human colorectal tumors of advanced grade and stage. Here we report that forced exogenous overexpression of AIM-1 in Chinese hamster embryo cells causes increased mitotic Ser-10 phosphorylation with concomitant induction of lagging chromosomes during mitosis. Lagging chromosomes could also be induced by transfection with mutated histone H3 (S10E), which is thought to maintain Ser-10 in the phosphorylated state. In the present study, chromosome number instability and increased tumor invasiveness were noted in constitutively AIM-1-overexpressing cells in vivo. Increased mitotic Ser-10 phosphorylation was also observed in various colorectal tumor cells with high AIM-1 expression levels. These data suggest that increased H3 histone phosphorylation as a result of AIM-1 overexpression is a major precipitating factor of chromosome instability and, thus, may play a role in carcinogenesis.

CHO1 is a kinesin-like protein of the mitotic kinesin-like protein (MKLP)1 subfamily present in central spindles and midbodies in mammalian cells. It is different from other subfamily members in that it contains an extra similar to300 by in the COOH-terminal tail. Analysis of the chicken genomic sequence showed that heterogeneity is derived from alternative splicing, and exon 18 is expressed in only the CHO1 isoform. CHO1 and its truncated isoform MKLP1 are coexpressed in a single cell. Surprisingly, the sequence encoded by exon 18 possesses a capability to interact with F-actin, suggesting that CHO1 can associate with both microtubule and actin cytoskeletons. Microinjection of exon 18-specific antibodies did not result in any inhibitory effects on karyokinesis and early stages of cytokinesis. However, almost completely separated daughter cells became reunited to form a binulceate cell, suggesting that the exon 18 protein may not have a role in the formation and ingression of the contractile ring in the cortex. Rather, it might be involved directly or indirectly in the membrane events necessary for completion of the terminal phase of cytokinesis.

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ip1)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3-dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-l(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.

The rat AIM-1 gene encoding an Aurora- and Ipl1-like midbody-associated protein serine/threonine kinase has a mitotic regulator function playing a key role in the onset of cytokinesis during mitosis. This report presents a cDNA sequence and megakaryocytic differentiaion-dependent expression profile of the human AIM-1 gene. The nucleotide sequences of the human AIM-1 were identified from cDNAs of three cell lines, including cervical carcinoma HeLa cells, colorectal tumor SW480 cells, and normal human diploid skin fibroblast NHDF cells, and no mutation was found. The expression levels of AIM-1 transcript were markedly reduced during differentiation into megakaryocytic cell lineage in human leukemia cells induced by 12-o-tetradecanoylphorbol-13-acetate (TPA), suggesting that the downregulation of AIM-1 contributes to the differentiation by repeated duplication of DNA without cytokinesis (endomitosis). (C) 1998 Elsevier Science B.V. All rights reserved.

Aurora- and Ipl1-like midbody-associated protein (AIM-1) is a serine/threonine threonine kinase that is structurally related to Drosophila aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. A kinase-negative form of AIM-1 inhibits the formation of cleavage furrow without affecting nuclear division, indicating that the gene controls entry into cytokinesis during M phase in mammalian cells. A human gene that encodes the protein AIM-1 was overexpressed in colorectal and other tumor cell lines. The regulation of AIM-1 expression was cell cycle dependent in normal and tumor cells, and the maximum accumulation was observed at G(2)-M. Exogenous overexpression of wildtype AIM-1 produced multinuclearity in human cells, suggesting that the excess amount of AIM-1 had a dominant-negative effect on the overexpressing cells. In long-term culture of AIM-1-overexpressing cells, multiple nuclei in a cell were occasionally fused, and then an increased ploidy and aneuploidy were induced. Thus, the overexpression of AIM-1 in colorectal tumor cell lines is thought to have a causal relationship with multinuclearity and increased ploidy. Cytokinesis error caused by AIM-1 overexpression is a major factor in the predisposition of tumor cells to the perturbation of chromosomal integrity that is commonly observed in human neoplasia. Thus, defects of pathways essential for mitotic regulation are important during human cancer development.

Mitogen-activated protein (MAP) kinase kinase (MAPKK) is a recently characterized activator of MAP kinase (MAPK), and is considered to be regulated by a protooncogene product c-Raf-1. It is, however, unclear whether the signals originating from c-Raf-1 utilize this phosphorylation cascade to lead to oncogenesis. To clarify this point, we isolated rat MAPKK cDNAs, and identified two distinct cDNAs encoding MAPKK and a highly related kinase, both with molecular weights of approximately 45 kDa (MEK1 and MEK2). Genomic Southern blot analyses suggested that MAPKK may form a large gene family.