INCENP Centromere and Spindle Targeting: Identification of Essential Conserved Motifs and Involvement of Heterochromatin Protein HP1

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13–amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11–amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1^Hsα. Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.     

Role of chromosomal passenger complex in chromosome segregation and cytokinesis

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 Ipl1-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.     

INCENP is required for proper targeting of Survivin to the centromeres and the anaphase spindle during mitosis

Three lines of investigation have suggested that interactions between Survivin and the chromosomal passenger proteins INCENP and Aurora-B kinase may be important for mitotic progression. First, interference with the function of Survivin/BIR1, INCENP, or Aurora-B kinase leads to similar defects in mitosis and cytokinesis [1-7] (see [8] for review). Second, INCENP and Aurora-B exist in a complex in Xenopus eggs [9] and in mammalian cultured cells [7]. Third, interference with Survivin or INCENP function causes Aurora-B kinase to be mislocalized in mitosis in both C. elegans and vertebrates [5, 7, 9]. Here, we provide evidence that Survivin, Aurora-B, and INCENP interact physically and functionally. Direct visualization of Survivin-GFP in mitotic cells reveals that it localizes identically to INCENP and Aurora-B. Survivin binds directly to both Aurora-B and INCENP in yeast two-hybrid and in vitro pull-down assays. The in vitro interaction between Survivin and Aurora-B is extraordinarily stable in that it resists 3 M NaCl. Finally, Survivin and INCENP interact functionally in vivo; in cells in which INCENP localization is disrupted, Survivin adheres to the chromosomes and no longer concentrates at the centromeres or transfers to the anaphase spindle midzone. Our data provide the first biochemical evidence that Survivin can interact directly with members of the chromosomal passenger complex.     

INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow

Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins [1], which include the inner centromere protein (INCENP [2]), the Aurora-related serine-threonine protein kinase AIRK2 [3,4] and the unidentified human autoantigen TD-60 [5], have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis [6,7], whereas interference with AIRK2 function primarily affects cytokinesis [3,8]. Here, we report that INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and that INCENP and AIRK2 kinase bind one another in vitro. This association was found to be evolutionarily conserved. Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalized exactly in human cells, and INCENP was required to target AIRK2 correctly to centromeres and the central spindle.     

Borealin: a novel chromosomal passenger required for stability of the bipolar mitotic spindle

The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B-INCENP subcomplex.     

INCENP and Aurora B promote meiotic sister chromatid cohesion through localization of the Shugoshin MEI-S332 in Drosophila

The chromosomal passenger complex protein INCENP is required in mitosis for chromosome condensation, spindle attachment and function, and cytokinesis. Here, we show that INCENP has an essential function in the specialized behavior of centromeres in meiosis. Mutations affecting Drosophila incenp profoundly affect chromosome segregation in both meiosis I and II, due, at least in part, to premature sister chromatid separation in meiosis I. INCENP binds to the cohesion protector protein MEI-S332, which is also an excellent in vitro substrate for Aurora B kinase. A MEI-S332 mutant that is only poorly phosphorylated by Aurora B is defective in localization to centromeres. These results implicate the chromosomal passenger complex in directly regulating MEI-S332 localization and, therefore, the control of sister chromatid cohesion in meiosis.     

Fission Yeast Bub1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis

The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1 ⁺ is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Δbub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1 ⁺ function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase–anaphase transition.     

Colocalization of TD-60 and INCENP throughout G2 and mitosis: evidence for their possible interaction in signalling cytokinesis

TD-60 and INCENP are two members of the chromosome passenger protein family, and each has been suggested to play a role in the control of cytokinesis. Here we demonstrate by confocal immunofluorescence microscopy that TD-60 and INCENP distribute identically throughout the cell cycle. Both appear coordinately in G2-phase nuclei and become concentrated at centromeres during prophase. TD-60 and INCENP both then leave the chromosome together during anaphase and redistribute to the spindle midzone, as do other chromosome passenger proteins, and traverse the entire equatorial diameter from cortex to cortex. By image overlay and pixel count analysis we show that TD-60 and INCENP are distinct among known chromosome passenger proteins in extending beyond the spindle to the cortex. Further, we show that the cytokinesis-associated protein kinase AIM-1 also shares this distribution property. We suggest that this redistribution is required to signal cytokinesis. TD-60 and INCENP also show identical localization in cells that exit mitosis in the presence of dihydrocytochalasin B (DCB), an inhibitor of actin assembly. Such cells can resume cleavage upon removal of DCB and in a recovery subpopulation that cleaves only on one side, these proteins both colocalize to the cortex only where a cleavage furrow forms. Given the coincident distribution of TD-60 and INCENP during both interphase and mitosis, we suggest that these proteins may cooperate, perhaps within a protein complex, in signalling cytokinesis. Such a mechanism, using chromosome passenger proteins, may ensure that cytokinesis occurs only between the separated chromatids, and only after they have segregated. Received: 12 August 1998; in revised form: 1 September 1998 / Accepted: 2 September 1998     

Chromosomal passengers and the (aurora) ABCs of mitosis

Chromosomal passengers are proteins that move from centromeres to the spindle midzone during mitosis. Recent experiments show that the passengers inner centromere protein (INCENP) and aurora-B kinase are in a macromolecular complex that might also contain a third passenger, survivin. The chromosomal passenger complex functions throughout mitosis in chromosome condensation and segregation, and at the end of mitosis, in the completion of cytokinesis.     

Cleavage Furrows Formed between Centrosomes Lacking an Intervening Spindle and Chromosomes Contain Microtubule Bundles, INCENP, and CHO1 but Not CENP-E

PtK₁ cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundles as well as INCENP and CHO1. Together these data suggest that the mechanism defining the site of furrow formation during mitosis in vertebrates does not depend on the presence of underlying microtubule bundles and chromosomes or on the stable association of INCENP or CHO1. The data also suggest that the completion of cytokinesis requires the presence of microtubule bundles and specific proteins (e.g., INCENP, CHO1, etc.) that do not include CENP-E.     

Identification of a Mid-anaphase Checkpoint in Budding Yeast

Activation of a facultative, dicentric chromosome provides a unique opportunity to introduce a double strand DNA break into a chromosome at mitosis. Time lapse video enhanced-differential interference contrast analysis of the cellular response upon dicentric activation reveals that the majority of cells initiates anaphase B, characterized by pole–pole separation, and pauses in mid-anaphase for 30–120 min with spindles spanning the neck of the bud before completing spindle elongation and cytokinesis. The length of the spindle at the delay point (3–4 μm) is not dependent on the physical distance between the two centromeres, indicating that the arrest represents surveillance of a dicentric induced aberration. No mid-anaphase delay is observed in the absence of the RAD9 checkpoint gene, which prevents cell cycle progression in the presence of damaged DNA. These observations reveal RAD9- dependent events well past the G2/M boundary and have considerable implications in understanding how chromosome integrity and the position and state of the mitotic spindle are monitored before cytokinesis.     

Essential roles of Drosophila inner centromere protein (INCENP) and aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation

We have performed a biochemical and double-stranded RNA-mediated interference (RNAi) analysis of the role of two chromosomal passenger proteins, inner centromere protein (INCENP) and aurora B kinase, in cultured cells of Drosophila melanogaster. INCENP and aurora B function is tightly interlinked. The two proteins bind to each other in vitro, and DmINCENP is required for DmAurora B to localize properly in mitosis and function as a histone H3 kinase. DmAurora B is required for DmINCENP accumulation at centromeres and transfer to the spindle at anaphase. RNAi for either protein dramatically inhibited the ability of cells to achieve a normal metaphase chromosome alignment. Cells were not blocked in mitosis, however, and entered an aberrant anaphase characterized by defects in sister kinetochore disjunction and the presence of large amounts of amorphous lagging chromatin. Anaphase A chromosome movement appeared to be normal, however cytokinesis often failed. DmINCENP and DmAurora B are not required for the correct localization of the kinesin-like protein Pavarotti (ZEN-4/CHO1/MKLP1) to the midbody at telophase. These experiments reveal that INCENP is required for aurora B kinase function and confirm that the chromosomal passengers have essential roles in mitosis.     

INCENP binds directly to tubulin and requires dynamic microtubules to target to the cleavage furrow

Inner centromere protein (INCENP) is a chromosomal passenger protein with an essential role in mitosis. At the metaphase/anaphase transition, some INCENP transfers from the centromeres to the central spindle; the remainder then transfers to the equatorial cortex prior to cleavage furrow formation. The molecular associations dictating INCENP behavior during mitosis are currently unknown. Here we show that targeting INCENP to the cleavage plane requires dynamic microtubules, but not F-actin. When microtubules are eliminated, INCENP is dispersed across the entire cell cortex. Yeast two-hybrid and in vitro binding data demonstrate that INCENP binds directly to beta-tubulin via a conserved domain encompassing residues 48-85. Furthermore, INCENP binds to microtubules polymerized from purified tubulin in vitro and appears to bundle microtubules when expressed in the interphase cytoplasm. These data indicate that INCENP is a microtubule-binding protein that targets to the equatorial cortex through interactions requiring microtubules.     

The kinase activity of aurora B is required for kinetochore-microtubule interactions during mitosis

As a component of the "chromosomal passenger protein complex," the aurora B kinase is associated with centromeres during prometaphase and with midzone microtubules during anaphase and is required for both mitosis and cytokinesis. Ablation of aurora B causes defects in both prometaphase chromosomal congression and the spindle checkpoint; however, the mechanisms underlying these defects are unclear. To address this question, we have examined chromosomal movement, spindle organization, and microtubule motor distribution in NRK cells transfected with a kinase-inactive, dominant-negative mutant of aurora B, aurora B(K-R). In cells overexpressing aurora B(K-R) fused with GFP, centromeres moved in a synchronized and predominantly unidirectional manner, as opposed to the independent, bidirectional movement in control cells expressing a similar level of wild-type aurora B-GFP. In addition, most kinetochores became physically separated from spindle microtubules, which appeared as a striking bundle between the spindle poles. These defects were associated with a microtubule-dependent depletion of motor proteins dynein and CENP-E from kinetochores. Our observations suggest that aurora B regulates the association of motor proteins with kinetochores during prometaphase. Interactions of kinetochore motors with microtubules may in turn regulate the organization of microtubules, the movement of prometaphase chromosomes, and the release of the spindle checkpoint.     

Centromere localization of INCENP-Aurora B is sufficient to support spindle checkpoint function

During mitosis, the chromosomal passenger complex (CPC) comprising the Aurora B kinase, INCENP, survivin and borealin is essential for correcting non-bipolar chromosome attachments and for cytokinesis. In addition, the CPC might fullfil a role in the mitotic spindle assembly checkpoint (SAC), but this activity might be related to its role in correcting non-bipolar chromosome attachments. Here, we demonstrate that treatment of mitotic cells with the antibiotic actinomycin D causes a displacement of an intact and active CPC from centromeres onto chromosome arms, which results in chromosome misalignment, cytokinesis failure and SAC override, but still preserves histone H3 phosphorylation on chromosome arms. This surprising and unique scenario allows the reconstitution of endogenous Aurora B at centromeres/inner kinetochores by expressing a Cenp-B-INCENP fusion protein. We find that although the selective recruitment of endogenous Aurora B to centromeres/inner kinetochores is not sufficient to restore chromosome alignment and cytokinesis, it can restore Cenp-A phosphorylation at kinetochores, BubR1 recruitment to kinetochores and SAC activity after spindle disruption. These results indicate that INCENP-Aurora B localized at centromeres/inner kinetochores is sufficient to mediate SAC activity upon spindle disruption.     

Cell division control by the Chromosomal Passenger Complex

The Chromosomal Passenger Complex (CPC) consisting of Aurora B kinase, INCENP, Survivin and Borealin, is essential for genomic stability by controlling multiple processes during both nuclear and cytoplasmic division. In mitosis it ensures accurate segregation of the duplicated chromosomes by regulating the mitotic checkpoint, destabilizing incorrectly attached spindle microtubules and by promoting the axial shortening of chromosomal arms in anaphase. During cytokinesis the CPC most likely prevents chromosome damage by imposing an abscission delay when a chromosome bridge connects the two daughter cells. Moreover, by controlling proper cytoplasmic division, the CPC averts tetraploidization. This review describes recent insights on how the CPC is capable of conducting its various functions in the dividing cell to ensure chromosomal stability.     

EVI5 protein associates with the INCENP-aurora B kinase-survivin chromosomal passenger complex and is involved in the completion of cytokinesis

EVI5 has been shown to be a novel centrosomal protein in interphase cells. In this report, we demonstrate using immunofluorescence microscopy that EVI5 has a dynamic distribution during mitosis, being associated with the mitotic spindle through anaphase and remaining within the midzone and midbody until completion of cytokinesis. Knockdown of EVI5 using siRNA results in a multinucleate phenotype, which is consistent with an essential role for this protein in the completion of cytokinesis. The EVI5 protein also undergoes posttranslational modifications during the cell cycle, which involve phosphorylation in early mitosis and proteolytic cleavage during late mitosis and cytokinesis. Since the subcellular distribution of the EVI5 protein was similar to that characteristic of chromosomal passenger proteins during the terminal stages of cytokinesis, we used immunoprecipitation and GST pull-down approaches to demonstrate that EVI5 is associated with the aurora B kinase protein complex (INCENP, aurora B kinase and survivin). Together, these data provide evidence that EVI5 is an essential component of the protein machinery facilitating the final stages of cell septation at the end of mitosis.     

Activation of the MKK/ERK Pathway during Somatic Cell Mitosis: Direct Interactions of Active ERK with Kinetochores and Regulation of the Mitotic 3F3/2 Phosphoantigen

The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal–regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. We have examined the regulation of ERK and MKK in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK₁ cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. J. Cell Biol. 122:1311–1321). The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment.     

ISWI is a RanGTP-dependent MAP required for chromosome segregation

Production of RanGTP around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)–containing factors. Here, we show that the NLS protein ISWI, a known chromatin-remodeling ATPase, is a RanGTP-dependent microtubule (MT)-associated protein. Recombinant ISWI induces MT nucleation, stabilization, and bundling in vitro. In Xenopus culture cells and egg extract, ISWI localizes within the nucleus in interphase and on spindles during mitosis. Depletion of ISWI in egg extracts does not affect spindle assembly, but in anaphase spindle MTs disappear and chromosomes do not segregate. We show directly that ISWI is required for the RanGTP-dependent stabilization of MTs during anaphase independently of its effect on chromosomes. ISWI depletion in Drosophila S2 cells induces defects in spindle MTs and chromosome segregation in anaphase, and the cells eventually stop growing. Our results demonstrate that distinctly from its role in spindle assembly, RanGTP maintains spindle MTs in anaphase through the local activation of ISWI and that this is essential for proper chromosome segregation.     

Cytochalasin D blocks chromosomal attachment to the spindle in the green algaOedogonium

Summary Mitosis in living cells ofOedogonium observed by time-lapse, was blocked by cytochalasin D (CD; 25–100 g/ml). Normal prometaphase to anaphase takes 10–15 min; blockage of entry into anaphase by CD was reversible up to 2–2.5 h in CD and washout was followed within 10–20 min by normal anaphase and cytokinesis. After 3–6 h in CD, unseparated chromatids segregated randomly into two groups as the spindle slowly elongated considerably, becoming distorted and twisted. During this pseudoanaphase, chromatids sometimes split irregularly and this was stimulated by late washout of CD. CD affected chromosomal attachment to the spindle. If applied at prophase and prometaphase, spindle fibres entered the nucleus; chromosomes moved vigorously and irregularly. A few achieved metaphase only briefly. Treatment at metaphase caused chromosomes to irregularly release and after random movement, all slowly gathered at either pole. Upon removal of CD, chromosomes rapidly achieved metaphase and anaphase A and B soon followed. If CD took effect during anaphase, chromatids detaching from the spindle oscillated rapidly along it; anaphase and cytokinesis (phycoplast formation) were delayed as the cell attempted to correct for abnormal chromosomal behaviour. Thus, CD prevents normal kinetochore attachment to the spindle and actin may be the target for this response.Abbreviations A-LP anaphase-like prometaphase - CD cytochalasin D - MT microtubule