CENP-A is required for accurate chromosome segregation and sustained kinetochore association of BubR1

CENP-A is an evolutionarily conserved, centromere-specific variant of histone H3 that is thought to play a central role in directing kinetochore assembly and in centromere function. Here, we have analyzed the consequences of disrupting the CENP-A gene in the chicken DT40 cell line. In CENP-A-depleted cells, kinetochore protein assembly is impaired, as indicated by mislocalization of the inner kinetochore proteins CENP-I, CENP-H, and CENP-C as well as the outer components Nuf2/Hec1, Mad2, and CENP-E. However, BubR1 and the inner centromere protein INCENP are efficiently recruited to kinetochores. Following CENP-A depletion, chromosomes are deficient in proper congression on the mitotic spindle and there is a transient delay in prometaphase. CENP-A-depleted cells further proceed through anaphase and cytokinesis with unequal chromosome segregation, suggesting that some kinetochore function remains following substantial depletion of CENP-A. We furthermore demonstrate that CENP-A-depleted cells exhibit a specific defect in maintaining kinetochore localization of the checkpoint protein BubR1 under conditions of checkpoint activation. Our data thus point to a specific role for CENP-A in assembly of kinetochores competent in the maintenance of mitotic checkpoint signaling.     

BubR1 is an effector of multiple mitotic kinases that specifies kinetochore: Microtubule attachments and checkpoint

BubR1 is a critical component of the mitotic checkpoint but has also been shown to play an essential role in establishing kinetochore:microtubule attachments. BubR1 is hyperphosphorylated in mitosis and recent studies in human and Xenopus have identified 9 phosphorylation sites. Plk1-dependent phosphorylations (T792, T1008 and S676) were reported to stimulate BubR1 kinase activity, promote kinetochore microtubule attachments, monitor kinetochore tension, as well as the recruitment of Mad2 checkpoint protein to kinetochores. Plk1-independent sites (S435, S543, S670 and S1043) were also identified and some of these were found to be sensitive to the loss of microtubule attachment but not tension. Functional studies showed that phosphorylation of S670 is critical for correcting aberrant attachments. Once end-on attachments are established, dephosphorylation of S670 appeared to be important for generating tension to signal anaphase onset. The collective data when combined with early EM studies that showed BubR1 is present at both the inner and outer kinetochore plates suggest that BubR1 maybe an effector of multiple kinases that specifies its roles in microtubule attachments and checkpoint functions.     

BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1

The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.     

Depletion of BubR1 promotes premature centrosomal localization of cyclin B1 and accelerates mitotic entry

The role of BubR1 has been established mainly in mitosis as an essential mitotic checkpoint protein although it is expressed throughout the cell cycle. To explore a possible role of BubR1 in regulating the G2 phase of cell cycle, we have employed siRNA–mediated hBubR1 knockdown in HeLa cells. Here, we demonstrate that reducing BubR1 levels during the G2 phase causes accelerated mitotic entry. As expected, BubR1 depletion leads to degradation of cyclin B1 in the G2 phase. Intriguingly, cyclin B1 is prematurely targeted to centrosomes appearing at early G2 phase in BubR1-depleted cells despite its low levels. This is in contrast to control cells where cyclin B1 appears at the centrosomes in early prophase based on cell cycle-specific localization of CENP-F. Furthermore, cyclin B/Cdk1 kinase activity in early G2 is aberrantly high in BubR1-depleted cells. Together, our results indicate that hBubR1 depletion triggers premature centrosomal localization of cyclin B1 probably leading to premature mitotic entry. This study is the first to suggest a role of hBubR1 in controlling centrosome targeting of cyclin B1 and timing of mitotic entry.     

Caspase-mediated specific cleavage of BubR1 is a determinant of mitotic progression

The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Delta579Delta610) was resistant to paclitaxel-induced degradation. Expression of BubR1Delta579Delta610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.     

Cdc5-dependent asymmetric localization of bfa1 fine-tunes timely mitotic exit

In budding yeast, the major regulator of the mitotic exit network (MEN) is Tem1, a GTPase, which is inhibited by the GTPase-activating protein (GAP), Bfa1/Bub2. Asymmetric Bfa1 localization to the bud-directed spindle pole body (SPB) during metaphase also controls mitotic exit, but the molecular mechanism and function of this localization are not well understood, particularly in unperturbed cells. We identified four novel Cdc5 target residues within the Bfa1 C-terminus: (452)S, (453)S, (454)S, and (559)S. A Bfa1 mutant in which all of these residues had been changed to alanine (Bfa1(4A)) persisted on both SPBs at anaphase and was hypo-phosphorylated, despite retaining its GAP activity for Tem1. A Bfa1 phospho-mimetic mutant in which all of these residues were switched to aspartate (Bfa1(4D)) always localized asymmetrically to the SPB. These observations demonstrate that asymmetric localization of Bfa1 is tightly linked to its Cdc5-dependent phosphorylation, but not to its GAP activity. Consistent with this, in kinase-defective cdc5-2 cells Bfa1 was not phosphorylated and localized to both SPBs, whereas Bfa1(4D) was asymmetrically localized. BFA1(4A) cells progressed through anaphase normally but displayed delayed mitotic exit in unperturbed cell cycles, while BFA1(4D) cells underwent mitotic exit with the same kinetics as wild-type cells. We suggest that Cdc5 induces the asymmetric distribution of Bfa1 to the bud-directed SPB independently of Bfa1 GAP activity at anaphase and that Bfa1 asymmetry fine-tunes the timing of MEN activation in unperturbed cell cycles.     

Drosophila basement membrane collagen col4a1 mutations cause severe myopathy

Recent data from clinical and mammalian genetic studies indicate that COL4A1 mutations manifest with basement membrane defects that result in muscle weakness, cramps, contractures, dystrophy and atrophy. In-depth studies of mutant COL4A1-associated muscle phenotype, however, are lacking and significant details of the muscle-specific pathomechanisms remain unknown. In this study, we have used a comprehensive set of Drosophila col4a1 and col4a2 mutants and a series of genetic and mutational analyses, gene, protein expression, and immunohistochemistry experiments in order to establish a Drosophila model and address some of these questions. The Drosophila genome contains two type IV collagen genes, col4a1 and col4a2. Mutant heterozygotes of either gene are viable and fertile, whereas homozygotes are lethal. In complementation analysis of all known mutants of the locus and a complementation matrix derived from these data we have identified the dominant lesions within the col4a1, but not within the col4a2 gene. Expression of a col4a1 transgene partially rescued the dominant and recessive mutant col4a1 alleles but not the col4a2 mutations that were all recessive. Partial complementation suggested that col4a1 gene mutations have strong antimorph effect likely due to the incorporation of the mutant protein into the triple helix. In col4a1 mutants, morphological changes of the oviduct muscle included severe myopathy with centronuclear myofibers leading to gradual development of female sterility. In larval body wall muscles ultrastructural changes included disturbance of A and I bands between persisting Z bands. In the most severely affected DTS-L3 mutant, we have identified four missense mutations within the coding region of the col4a1 gene two of which affected the Y within the Gly-X-Y unit and a 3' UTR point mutation. In conclusion, our Drosophila mutant series may serve as an effective model to uncover the mechanisms by which COL4A1 mutations result in compromised myofiber-basement membrane interactions and aberrant muscle function.     

Centromere/kinetochore localization of human centromere protein A (CENP-A) exogenously expressed as a fusion to green fluorescent protein

Three human centromere proteins, CENP-A, CENP-B and CENP-C, are a set of autoantigens specifically recognized by anticentromere antibodies often produced by patients with scleroderma. Microscopic observation has indicated that CENP-A and CENP-C localize to the inner plate of metaphase kinetochore, while CENP-B localizes to the centromere heterochromatin beneath the kinetochore. The antigenic structure, called "prekinetochore", is also present in interphase nuclei, but little is known about its molecular organization and the relative position of these antigens. Here, to visualize prekinetochore in living cells, we first obtained a stable human cell line, MDA-AF8-A2, in which human CENP-A is exogenously expressed as a fusion to a green fluorescent protein of Aequorea victoria. Simultaneous staining with anti-CENP-B and anti-CENP-C antibodies showed that the recombinant CENP-A colocalized with the endogenous CENP-C and constituted small discrete dots attaching to larger amorphous mass of CENP-B heterochromatin. When the cell growth was arrested in G1/ S phase with hydroxyurea, CENP-B heterochromatin was sometimes highly extended, while the relative location between GFP-fused CENP-A and the endogenous CENP-C was not affected. These results indicated that the fluorescent CENP-A faithfully localizes to the centromere/kinetochore throughout the cell cycle. We then obtained several mammalian cell lines where the same GFP-fused human CENP-A construct was stably expressed and their centromere/kinetochore is fluorescent throughout the cell cycle. These cell lines will further be used for visualizing the prekinetochore locus in interphase nuclei as well as analyzing kinetochore dynamics in the living cells.     

Identification and purification of a soluble region of BubR1: a critical component of the mitotic checkpoint complex

The mitotic checkpoint complex (MCC) ensures the fidelity of chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle. In essence, this MCC-induced delay is achieved via the inhibition of the anaphase-promoting complex (APC). Among the components of the MCC, BubR1 plays two major roles in the functions of the mitotic checkpoint. First, BubR1 is able to inhibit APC activity, either by itself or as a component of the MCC, by sequestering a APC coactivator, known as Cdc20. Second, BubR1 activates mitotic checkpoint signaling cascades by binding to the centromere-associated protein E, a microtubule motor protein. Obtaining highly soluble BubR1 is a prerequisite for the study of its structure. BubR1 is a multi-domain protein, which includes a KEN box motif, a mad3-like region, a Bub3 binding domain, and a kinase domain. We obtained a soluble BubR1 construct using a three-step expression strategy. First, we obtained two constructs from BLAST sequence homology searches, both of which were expressed abundantly in the inclusion bodies. We then adjusted the lengths of the two constructs by secondary structure prediction, thereby generating partially soluble constructs. Third, we optimized the solubility of the two constructs by either chopping or adding a few residues at the C-terminus. Finally, we obtained a highly soluble BubR1 construct via the Escherichia coli expression system, which allowed for a yield of 10.8 mg/L culture. This report may provide insight into the design of highly soluble constructs of insoluble multi-domain proteins.     

Pharicin A,a novel natural ent-kaurene diterpenoid,induces mitotic arrest and mitotic catastrophe of cancer cells by interfering with BubR1 function

In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in leukemia and solid tumor-derived cells identified by their morphology, DNA content and mitotic marker analyses. Pharicin A-induced mitotic arrest is associated with unaligned chromosomes, aberrant BubR1 localization and deregulated spindle checkpoint activation. Pharicin A directly binds to BubR1 in vitro, which is correlated with premature sister chromatid separation in vivo. Pharicin A also induces mitotic arrest in paclitaxel-resistant Jurkat and U2OS cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing mitotic progression and initiating mitotic catastrophe, which merits further preclinical and clinical investigations for cancer drug development.Key words: pharicin A, mitotic arrest, leukemia, tumor cells, spindle checkpoint     

CENP-F is a novel microtubule-binding protein that is essential for kinetochore attachments and affects the duration of the mitotic checkpoint delay

Centromeric protein F (CENP-F) is a 367-kDa human kinetochore protein that was identified a decade ago, but its function was only recently revealed by studies that used small interfering RNA to deplete the protein from cells. All studies showed that CENP-F is important for chromosome alignment, but these studies differed as to whether CENP-F is important to the mitotic checkpoint. We report here that CENP-F is essential for cells to sustain a prolonged mitotic delay in response to unattached kinetochores. Cells depleted of CENP-F exit mitosis in the presence of defective kinetochore attachments resulting from treatment with nocodazole, or the depletion of kinetochore proteins CENP-E and hSgo1. Kinetochores depleted of CENP-F exhibited a reduction in the amounts of the mitotic checkpoint proteins Mad1, Mad2, hBUBR1, hBUB1, and hMps1. We postulate that CENP-F is not an essential component of the mitotic checkpoint but facilitates the duration of the mitotic delay. Separately, we show that CENP-F is a novel microtubule-binding protein that possesses two microtubule-binding domains at opposite ends of the molecule. The C-terminal microtubule-binding domain was found to stimulate microtubule polymerization in vitro. These activities provide a biochemical explanation for how CENP-F contributes to kinetochore attachments in vivo.Electronic Supplementary Material Supplementary material is available for this article at .     

Pharicin A,a novel natural ent-kaurene diterpenoid,induces mitotic arrest and mitotic catastrophe of cancer cells by interfering with BubR1 function

In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon xerophilus, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in leukemia and solid tumor-derived cells identified by their morphology, DNA content, and mitotic marker analyses. Pharicin A-induced mitotic arrest is associated with unaligned chromosomes, aberrant BubR1 localization, and deregulated spindle checkpoint activation. Pharicin A directly binds to BubR1 in vitro, which is correlated with premature sister chromatid separation in vivo. Pharicin A also induces mitotic arrest in paclitaxel-resistant Jurkat and U2OS cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing mitotic progression and initiating mitotic catastrophe, which merits further preclinical and clinical investigations for cancer drug development.     

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.     

Protein interaction domain mapping of human kinetochore protein Blinkin reveals a consensus motif for binding of spindle assembly checkpoint proteins Bub1 and BubR1

The kinetochore is a supramolecular structure essential for microtubule attachment and the mitotic checkpoint. Human blinkin/human Spc105 (hSpc105)/hKNL1 was identified originally as a mixed-lineage leukemia (MLL) fusion partner and later as a kinetochore component. Blinkin directly binds to several structural and regulatory proteins, but the precise binding sites have not been defined. Here, we report distinct and essential binding domains for Bub1 and BubR1 (here designated Bubs) at the N terminus of blinkin and for Zwint-1 and hMis14/hNsl1 at the C terminus. The minimal binding sites for Bub1 and BubR1 are separate but contain a consensus KI motif, KI(D/N)XXXF(L/I)XXLK. RNA interference (RNAi)-mediated replacement with mutant blinkin reveals that the Bubs-binding domain is functionally important for chromosome alignment and segregation. We also provide evidence that hMis14 mediates hNdc80 binding to blinkin at the kinetochore. The C-terminal fragment of blinkin locates at kinetochores in a dominant-negative fashion by displacing endogenous blinkin from kinetochores. This negative dominance is relieved by mutations of the hMis14 binding PPSS motif on the C terminus of blinkin or by fusion of the N sequence that binds to Bub1 and BubR1. Taken together, these results indicate that blinkin functions to connect Bub1 and BubR1 with the hMis12, Ndc80, and Zwint-1 complexes, and disruption of this connection may lead to tumorigenesis.     

Plk1-dependent phosphorylation of optineurin provides a negative feedback mechanism for mitotic progression

Plk1 activation is required for progression through mitotic entry to cytokinesis. Here we show that at mitotic entry, Plk1 phosphorylates Optineurin (Optn) at serine 177 and that this dissociates Optn from the Golgi-localized GTPase Rab8, inducing its translocation into the nucleus. Mass spectrometry analysis revealed that Optn is associated with a myosin phosphatase complex (MP), which antagonizes the mitotic function of Plk1. Our data also indicate that Optn functionally connects this complex to Plk1 by promoting phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). Accordingly, silencing Optn expression increases Plk1 activity and induces abscission failure and multinucleation, which were rescued upon expression of wild-type (WT) Optn, but not a phospho-deficient mutant (S177A) that cannot translocate into the nucleus during mitosis. Overall, these results highlight an important role of Optn in the spatial and temporal coordination of Plk1 activity.     

Cytogenetic localization of a putative regulatory gene affecting an NADP+-dependent enzyme in Drosophila melanogaster

The cytogenetic localization of a putative regulatory locus affecting an NADP+-dependent enzyme is described. This locus, Mex, is in region 8D10-12 to 9A1-2 on the X chromosome of Drosophila melanogaster. Hyperploidy for this region is associated with significant increases in NADP+-dependent malic enzyme specific activity and specific immunologically cross-reacting material. This is the first report of a specific locus, unlinked to the locus coding for the major polypeptide of the enzyme, which affects an NADP+-dependent enzyme in Drosophila melanogaster.     

In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila

The gene polo encodes a highly conserved serine/threonine protein kinase that has been implicated in several functions during cell division. Polo-like kinases are important positive regulators of cell cycle progression and have also been implicated in the exit from mitosis through the activation of the anaphase-promoting complex. Several data indicate that Plks are required for centrosome function, bipolar spindle organisation and cytokinesis. The intracellular localisation of Plks reflects their multiple roles in cell division, however, in vivo studies that describe the distribution of this protein during different stages of mitosis have never been performed. In the present work, we report the in vivo distribution of a GFP-POLO fusion protein expressed in stable transformants and analysed during the early embryonic development of Drosophila melanogaster. The GFP-POLO protein can be detected in unfertilised oocytes associated with the centromeric region of chromosomes of the polar body and followed until the formation of mitotic domains in later development. Detailed analysis of the dynamic localisation of GFP-POLO during syncytial mitotic cycles shows the timing of localisation to the centrosomes, centromeres and midbody. The results also indicate that GFP-POLO is present in astral microtubules early in mitosis, accumulates around the nuclear envelope until nuclear envelop breakdown and at metaphase associates to spindle microtubules. These in vivo studies show a highly dynamic association of POLO with multiple compartments of the mitotic apparatus. Furthermore, the wide distribution of the GFP-POLO protein to all compartments of the mitotic apparatus provides a valuable tool for future studies on cell cycle during development.     

The mitotic arrest in response to hypoxia and of polar bodies during early embryogenesis requires Drosophila Mps1

Mps1 kinase plays an evolutionary conserved role in the mitotic spindle checkpoint. This system precludes anaphase onset until all chromosomes have successfully attached to spindle microtubules via their kinetochores. Mps1 overexpression in budding yeast is sufficient to trigger a mitotic arrest, which is dependent on the other mitotic checkpoint components, Bub1, Bub3, Mad1, Mad2, and Mad3. Therefore, Mps1 might act at the top of the mitotic checkpoint cascade. Moreover, in contrast to the other mitotic checkpoint components, Mps1 is essential for spindle pole body duplication in budding yeast. Centrosome duplication in mammalian cells might also be controlled by Mps1 , but the fission yeast homolog is not required for spindle pole body duplication. Our phenotypic characterizations of Mps1 mutant embryos in Drosophila do not reveal an involvement in centrosome duplication, while the mitotic spindle checkpoint is defective in these mutants. In addition, our analyses reveal novel functions. We demonstrate that Mps1 is also required for the arrest of cell cycle progression in response to hypoxia. Finally, we show that Mps1 and the mitotic spindle checkpoint are responsible for the developmental cell cycle arrest of the three haploid products of female meiosis that are not used as the female pronucleus.