Inner centromere protein

INCENP homologs in yeast

The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Its human homologue has been implicated in the tumorigenesis of diverse forms of cancer. Sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole-associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein (INCENP homolog) in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation (Kim, 1999).

Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1-Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome-spindle interactions or in the movement of kinetochores along microtubules (Kang, 2001).

How sister kinetochores attach to microtubules from opposite spindle poles during mitosis (bi-orientation) remains poorly understood. In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same spindle pole. IPL1 function was investigated in cells that cannot replicate their chromosomes but nevertheless duplicate their spindle pole bodies (SPBs). Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants. This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite spindle poles creates tension in the surrounding chromatin (Tanaka, 2002).

Fission yeast Bir1p/Cut17p/Pbh1p, the homolog of human Survivin, is a conserved chromosomal passenger protein that is required for cell division and cytokinesis. To study how Bir1p promotes accurate segregation of chromosomes, a temperature-sensitive allele, bir1-46, was generated and analyzed, and genetic screens were carried out to find genes that interact with bir1+. Psf2p, a component of the GINS complex required for DNA replication initiation, was identified as a high-copy-number suppressor of the bir1-46 growth defect. Loss of Psf2p function by depletion or deletion or by use of a temperature-sensitive allele, psf2-209, resulted in chromosome missegregation that was associated with mislocalization of Bir1p. The human homolog of Psf2p, PSF2, was required for proper chromosome segregation. In addition, it was observed that high-copy-number expression of Pic1p, the fission yeast homolog of INCENP (inner centromere protein), suppressed bir1-46. Pic1p exhibited a localization pattern typical of chromosomal passenger proteins. Deletion of pic1+ caused chromosome missegregation phenotypes similar to those of bir1-46. These data suggest that Bir1p and Pic1p act as part of a conserved chromosomal passenger complex and that Psf2p/GINS indirectly affects the localization and function of this complex in chromosome segregation, perhaps through an S-phase role in centromere replication (Huang, 2005).

C. elegans INCENP homologs

In animal cells, cytokinesis begins shortly after the sister chromatids move to the spindle poles. The inner centromere protein (Incenp) has been implicated in both chromosome segregation and cytokinesis, but it is not known exactly how it mediates these two distinct processes. Two Caenorhabditis elegans proteins, ICP-1 and ICP-2, with significant homology in their carboxyl termini to the corresponding region of vertebrate Incenp, have been identified. Embryos depleted of ICP-1 by RNA-mediated interference have defects in both chromosome segregation and cytokinesis. Depletion of the Aurora-like kinase AIR-2 results in a similar phenotype. The carboxy-terminal region of Incenp is also homologous to that in Sli15p, a budding yeast protein that functions with the yeast Aurora kinase Ipl1p. ICP-1 binds C. elegans AIR-2 in vitro, and the corresponding mammalian orthologs Incenp and AIRK2 can be co-immunoprecipitated from cell extracts. A significant fraction of embryos depleted of ICP-1 and AIR-2 completed one cell division over the course of several cell cycles. ICP-1 promotes the stable localization of ZEN-4 (also known as CeMKLP1), a kinesin-like protein required for central spindle assembly. It is concluded that ICP-1 and AIR-2 are part of a complex that is essential for chromosome segregation and for efficient completion of cytokinesis. It is proposed that this complex acts by promoting dissolution of sister chromatid cohesion and the assembly of the central spindle (Kaitna, 2000).

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, live and fixed assays were used in the one-cell stage C. elegans embryo. The consequences were compared of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical 'kinetochore null' phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A-containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation (Oegema, 2001).

INCENP sub-cellular distribution

A novel set of polypeptide antigens has been described that shows a dramatic change in structural localization during mitosis. Through metaphase these antigens define a new chromosomal substructure that is located between the sister chromatids. Because the antigens are concentrated in the pericentromeric region, they have been provisionally termed the INCENPs (inner centromere proteins). The INCENPs (two polypeptides of 155 and 135 kD) were identified with a monoclonal antibody that was raised against the bulk proteins of the mitotic chromosome scaffold fraction. These two polypeptides are the most tightly bound chromosomal proteins known. When scaffolds are prepared, 100% of the detectable INCENPs remain scaffold associated. The fate of the INCENPs at anaphase was therefore a surprise. As the sister chromatids separate, the INCENPs dissociate fully from them, remaining behind at the metaphase plate as the chromatids migrate to the spindle poles. During anaphase the INCENPs are found on coarse fibers in the central spindle, and also in close apposition to the cell membrane in the region of the forming contractile ring. During telophase, the INCENPs gradually become focused onto the forming midbody, together with which they are ultimately discarded. Several possible in vivo roles for the INCENPs are suggested by these data: regulation of sister chromatid pairing, stabilization of the plane of cleavage, and separation of spindle poles at anaphase (Cooke, 1987).

INCENP domain structure

It has been proposed that mitotic chromosomes transport certain cytoskeletal proteins to the metaphase plate so that these proteins are able to subsequently participate in the assembly of the anaphase spindle and the cleavage furrow. To understand how such proteins accomplish their dual chromosomal/cytoskeletal role, a molecular and functional analysis of the inner centromere proteins (INCENPs), founder members of the class of 'chromosome passenger proteins', has been initiated. cDNA clones encoding the open reading frames of the two chicken INCENPs were recovered. The predicted proteins, class I INCENP (96,357 D) and class II INCENP (100,931 D) are novel, and differ from each other by the inclusion of a 38-codon insert within the class II coding region. Transient expression of the chicken INCENPs in mammalian cells confirms that the signals and structures required for the transfer of these proteins from chromosomes to cytoskeleton are evolutionarily conserved. Furthermore, these studies reveal that INCENP association with the cytoskeleton is complex. The amino-terminal 42-amino acid residues are required for transfer of the INCENPs from the chromosomes to the mitotic spindle at anaphase, but not for binding of INCENPs to cytoplasmic microtubules. In contrast, an internal 200 amino acid coiled-coil domain is required for association with microtubules, but dispensable for spindle association. These experiments suggest that proteins required for assembly of specialized cytoskeletal structures during mitosis from anaphase onwards might be sequestered in the nucleus throughout interphase to keep them from disrupting the interphase cytoskeleton, and to ensure their correct positioning during mitosis (Mackay, 1993).

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. An autonomous centromere- and midbody-targeting module has been identified in the amino-terminal 68 amino acids of INCENP. Within this module, two evolutionarily conserved amino acid sequence motifs were identified: 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, a yeast two-hybrid screen was performed for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1(Hsalpha). 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 (Ainsztein, 1998).

INCENP is a tightly bound chromosomal protein that transfers to the spindle midzone at the metaphase/anaphase transition. An INCENP truncation mutant (INCENP382-839) associates with microtubules but does not bind to chromosomes, and coats the entire spindle throughout mitosis. Furthermore, an INCENP truncation mutant (INCENP43-839) previously shown not to transfer to the spindle at anaphase (Mackay, 1993), is shown in this study to bind chromosomes, but is unable to target to the centromere. Thus, association with the chromosomes, and specifically with centromeres, appears to be essential for INCENP targeting to the correct spindle subdomain at anaphase. An INCENP truncation mutant (INCENP1-405) that targets to centromeres but lacks the microtubule association region acquires strong dominant-negative characteristics. INCENP1-405 interferes with both prometaphase chromosome alignment and the completion of cytokinesis. INCENP1-405 apparently exerts its effect by displacing the endogenous protein from centromeres. These experiments provide evidence of an unexpected link between this chromosomal protein and cytokinesis, and suggest that one function of INCENP may be to integrate the chromosomal and cytoskeletal events of mitosis (Mackay, 1998).

The major result of this study is the finding that targeting of the chromosomal passenger protein INCENP and its various deletion constructs to chromosomes, and specifically to centromeres, is essential for both the localization and function of these proteins during mitosis. Furthermore, a truncated INCENP molecule (INCENP1-405) that targets to centromeres and remains trapped on the chromosomes behaves as a dominant-negative mutant that interferes with both metaphase chromosome alignment and cytokinesis in cultured cells. Thus, although chromosome associated for the majority of the cell cycle, INCENP can be considered a cytoskeletal protein whose centromeric localization appears to be required for the successful execution of prometaphase congression and cytokinesis (Mackay, 1998).

The chromosomal passenger complex (CPC) is a key regulator of chromosome segregation and cytokinesis. CPC functions are connected to its localization. The complex first localizes to centromeres and later associates with the central spindle and midbody. Survivin, Borealin, and INCENP are the three components of the CPC that regulate the activity and localization of its enzymatic component, the kinase Aurora B. This study determined the 1.4 Å resolution crystal structure of the regulatory core of the CPC, revealing that Borealin and INCENP associate with the helical domain of Survivin to form a tight three-helical bundle. siRNA rescue experiments with structure-based mutants were used to explore the requirements for CPC localization. Ihe intertwined structural interactions of the core components lead to functional interdependence. Association of the core 'passenger' proteins creates a single structural unit, whose composite molecular surface presents conserved residues essential for central spindle and midbody localization (Jeyaprakash, 2007).

The crystal structure of the regulatory core of the CPC presented in this study together with the structure of Aurora B in complex with unphosphorylated INCENP suggests a composite model for the functional holoCPC. The N-terminal domains of Borealin (15-76) and INCENP (1-46) together with Survivin form the core of the CPC. The CPC core (in the presence of full length INCENP) is sufficient to target to the central spindle and midbody, but centromere targeting requires also the C-terminal domain of Borealin. The data reported in this study suggest that there is no significant direct interaction of Aurora B with Survivin or Borealin, in line with the view that Aurora B is incorporated into the CPC via binding to the IN-box of INCENP. INCENP regulates the localization of Aurora B by interacting with Borealin and Survivin via its N-terminal domain. The role of the coiled-coil domain of INCENP that is predicted between the N and C terminus is not yet clear but might simply reside in connecting the two functional modules of the protein. The experiments reported in this study unambiguously show that the core of the CPC is formed by the molecular interaction between the CPC components in a 1:1:1 ratio. In light of the recent report on the activation of Aurora B by the chromosomal enrichment of CPC, it is possible that in the presence of appropriate interacting proteins at the centromeres and central spindle, the CPC components might assemble into a larger complex of oligomeric nature (Jeyaprakash, 2007 and references therein).

Previous attempts at dissecting the role of individual passenger proteins in regulating the Aurora B kinase suggested an extraordinary interdependence between the subunits. The structural basis for this interdependence becomes evident in light of the crystal structure of the core CPC. A complex network of intermolecular interactions observed within the passenger proteins stabilizes the core of the CPC and holds the subunits together. Although the function and regulation of Aurora B involves three different proteins, these operate as a single structural unit. Specific disruption of any single passenger protein results in the impairment of the structural unit and in the failure of CPC targeting. This unit forms a composite molecular surface that is required to localize the CPC to the central spindle and midbody. In future studies it will be interesting to explore the relationship between this composite surface and potential interaction partners of the CPC at the central spindle as well as the effect of phosphorylation and microtubules on the localization of Aurora B and INCENP (Jeyaprakash, 2007).

INCENP interaction with Aurora B kinase

Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins, which include INCENP, the Aurora-related serine-threonine protein kinase AIRK2 and the unidentified human autoantigen TD-60, 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, whereas interference with AIRK2 function primarily affects cytokinesis. INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and 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 is termed the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalize exactly in human cells, and INCENP is required to target AIRK2 correctly to centromeres and the central spindle (Adams, 2000).

The inner centromere protein (INCENP) is required for correct chromosome segregation and cytokinesis. The human INCENP gene has been idenified by library screening and reverse transcription-polymerase chain reaction (RT-PCR) and localized to chromosomal region 11q12. HsINCENP is a single-copy gene that consists of 17 exons and covers 25 kb of genomic DNA. The gene is expressed at highest levels in the colon, testis and prostate, consistent with its likely role in cell proliferation. HsINCENP encodes a highly basic protein of 915 amino acids that localizes to metaphase chromosomes and to the mitotic spindle and equatorial cortex at anaphase. It has been shown that INCENP is stockpiled in a complex with the Aurora-B/XAIRK2 kinase in Xenopus eggs. Consistent with such an interaction, the two proteins colocalize on human metaphase chromosomes. Levels of Aurora-B are increased in several human cancers, and HsINCENP protein levels are also significantly increased in several colorectal cancer cell lines (Adams, 2001c).

How the events of mitosis are coordinated is not well understood. Intriguing mitotic regulators include the chromosomal passenger proteins. Loss of either of the passengers inner centromere protein (INCENP) or the Aurora B kinase results in chromosome segregation defects and failures in cytokinesis. Furthermore, INCENP and Aurora B have identical localization patterns during mitosis and directly bind each other in vitro. These results led to the hypothesis that INCENP is a direct substrate of Aurora B. This study shows that the C. elegans Aurora B kinase AIR-2 specifically phosphorylates the C. elegans INCENP ICP-1 at two adjacent serines within the carboxyl terminus. Furthermore, the full length and a carboxyl-terminal fragment of ICP-1 stimulated AIR-2 kinase activity. This increase in AIR-2 activity required that AIR-2 phosphorylate ICP-1 because mutation of both serines in the AIR-2 phosphorylation site of ICP-1 abolished the potentiation of AIR-2 kinase activity by ICP-1. Thus, ICP-1 is directly phosphorylated by AIR-2 and functions in a positive feedback loop that regulates AIR-2 kinase activity. Since the Aurora B phosphorylation site within INCENP and the functions of INCENP and Aurora B have been conserved among eukaryotes, the feedback loop that this study has identified is also likely to be evolutionarily conserved (Bishop, 2002).

Aurora family serine/threonine kinases control mitotic progression, and their deregulation is implicated in tumorigenesis. Aurora A and Aurora B, the best-characterized members of mammalian Aurora kinases, are 60% identical but bind to unrelated activating subunits. The structure of the complex of Aurora A with the TPX2 activator has been reported previously. This study reports the crystal structure of Aurora B in complex with the IN-box segment of the inner centromere protein (INCENP) activator and with the small molecule inhibitor Hesperadin. The Aurora B:INCENP complex is remarkably different from the Aurora A:TPX2 complex. INCENP forms a crown around the small lobe of Aurora B and induces the active conformation of the T loop allosterically. The structure represents an intermediate state of activation of Aurora B in which the Aurora B C-terminal segment stabilizes an open conformation of the catalytic cleft, and a critical ion pair in the kinase active site is impaired. Phosphorylation of two serines in the carboxyl terminus of INCENP generates the fully active kinase (Sessa, 2005).

INCENP imparts two distinct changes on Aurora B: (1) INCENP molds the phosphorylated activation loop of Aurora B into its active conformation via an allosteric mechanism; (2) INCENP forces a rotation of the αC helix and a concomitant opening of the catalytic cleft that disrupts the ion pair between Lys122Au-B and Glu141Au-B. The phosphorylation of the C-terminal TSS motif of INCENP might reverse these effects, generating a fully active kinase. These studies identify structurally distinct states of Aurora B. It will now be important to test if each of these states represents a discrete drug target (Sessa, 2005).

Aurora family serine/threonine kinases control mitotic progression, and their deregulation is implicated in tumorigenesis. Aurora A and Aurora B, the best-characterized members of mammalian Aurora kinases, are approximately 60% identical but bind to unrelated activating subunits. The structure of the complex of Aurora A with the TPX2 activator has been reported previously. This study reports the crystal structure of Aurora B in complex with the IN-box segment of the inner centromere protein (INCENP) activator and with the small molecule inhibitor Hesperadin. The Aurora B:INCENP complex is remarkably different from the Aurora A:TPX2 complex. INCENP forms a crown around the small lobe of Aurora B and induces the active conformation of the T loop allosterically. The structure represents an intermediate state of activation of Aurora B in which the Aurora B C-terminal segment stabilizes an open conformation of the catalytic cleft, and a critical ion pair in the kinase active site is impaired. Phosphorylation of two serines in the carboxyl terminus of INCENP generates the fully active kinase (Sessa, 2005).

Chromatin-induced spindle assembly depends on regulation of microtubule-depolymerizing proteins by the chromosomal passenger complex (CPC), consisting of Incenp, Survivin, Dasra (Borealin), and the kinase Aurora B, but the mechanism and significance of the spatial regulation of Aurora B activity remain unclear. This study shows that the Aurora B pathway is suppressed in the cytoplasm of Xenopus egg extract by phosphatases, but that it becomes activated by chromatin via a Ran-independent mechanism. While spindle microtubule assembly normally requires Dasra-dependent chromatin binding of the CPC, this function of Dasra can be bypassed by clustering Aurora B-Incenp by using anti-Incenp antibodies, which stimulate autoactivation among bound complexes. However, such chromatin-independent Aurora B pathway activation promotes centrosomal microtubule assembly and produces aberrant achromosomal spindle-like structures. It is proposed that chromosomal enrichment of the CPC results in local kinase autoactivation, a mechanism that contributes to the spatial regulation of spindle assembly and possibly to other mitotic processes (Kelly, 2007).

How does chromatin activate Aurora B-dependent phosphorylation? Four lines of evidence support a model in which Aurora B is activated by increasing the local concentration of CPC molecules on chromatin: (1) Chromatin can bind to multiple molecules of the CPC and induce Aurora B pathway activation; (2) Antibody alone can activate Aurora B kinase activity, and this activity is dependent on having multiple binding sites; (3) The responses of the small microtubule-destabilizing protein Op18 hyperphosphorylation induced by sperm nuclei and antibodies are similar and Ran independent; (4) Op18 hyperphosphorylation induced by antibody clustering is insensitive to the geometry of attachment (Kelly, 2007).

Full activation of Aurora B requires Aurora B-mediated phosphorylation of the C-terminal TSS motif of Incenp, and structural analysis suggests that this phosphorylation must occur in trans. Thus, the simplest model is that the Incenp TSS motif is actively dephosphorylated in the cytoplasm, but chromatin increases the local concentration of the CPC, resulting in initiation of a positive feedback loop among bound CPC holocomplexes. It is worth noting other possible mechanisms: clustering may also activate Aurora B independent of phosphorylation, as is the case for kinases such as Raf and EGFR, or chromatin or its associated molecules might directly induce a non-clustering-mediated structural change in Aurora B (Kelly, 2007).

It is also possible that chromatin exerts its effect on the Aurora B pathway by inhibiting protein phosphatase activities. However, the data indicate that chromatin directly stimulates the kinase activity of Aurora B, since Dasra proteins (which are required for loading of the CPC onto chromatin) are needed for spindle assembly. Importantly, more than 90% of Dasra A is associated with Incenp and Aurora B in the cytoplasm of Xenopus egg extracts. In addition, it has been reported that recombinant human Dasra B/Borealin does not affect the in vitro kinase activity of Aurora B. Thus, it is unlikely that Dasra proteins stimulate the enzymatic activity of Aurora B simply by virtue of their interactions (Kelly, 2007).

The spatial distribution of phosphorylated substrates around chromatin can be finely regulated by the level of phosphatase activity, and substrate diffusibility and stability, whereas the amplitude of the gradient is most sensitive to kinase activity. For example, the freely diffusible Op18-tubulin interaction is abrogated in the vicinity of chromosomes (4-8 microm) by a gradient of Op18 phosphorylation, the extent of which is mainly determined by phosphatase activity/concentration and the Op18 diffusion rate. Alternatively, if the substrate is immobilized on chromosomes, kinase activity dictates the behavior of the phospho-substrate. MCAK, a protein that is bound to centromeric chromatin, is more efficiently phosphorylated at Ser196 by Aurora B on centromeres of unaligned chromosomes than on aligned chromosomes. This raises the question of whether a change in chromatin status between sister kinetochores can effectively regulate Aurora B activity by modulating its local concentration. In summary, these results illustrating that Aurora B is activated by increased local concentration have important implications for the several roles of this complex throughout mitosis (Kelly, 2007).

INCENP, Survivin and Borealin

Survivin is a mammalian protein that carries a motif typical of the inhibitor of apoptosis (IAP) proteins, first identified in baculoviruses. Although baculoviral IAP proteins regulate cell death, the yeast Survivin homolog Bir1 is involved in cell division. To determine the function of Survivin in mammals, the pattern of localization of Survivin protein during the cell cycle was analyzed, and its gene was deleted by homologous recombination in mice. In human cells, Survivin appears first on centromeres bound to a novel para-polar axis during prophase/metaphase, relocates to the spindle midzone during anaphase/telophase, and disappears at the end of telophase. In the mouse, Survivin is required for mitosis during development. Null embryos show disrupted microtubule formation, become polyploid, and fail to survive beyond 4.5 days post coitum. This phenotype, and the cell-cycle localization of Survivin, resemble closely those of INCENP. Because the yeast homolog of INCENP, Sli15, regulates the Aurora kinase homolog Ipl1p, and the yeast Survivin homolog Bir1 binds to Ndc10p, a substrate of Ipl1p, yeast Survivin, INCENP and Aurora homologs function in concert during cell division. Therefore, in vertebrates, Survivin and INCENP have related roles in mitosis, coordinating events such as microtubule organization, cleavage-furrow formation and cytokinesis. Like their yeast homologs Bir1 and Sli15, they may also act together with the Aurora kinase (Uren, 2000).

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. (1) Interference with the function of Survivin/BIR1, INCENP, or Aurora-B kinase leads to similar defects in mitosis and cytokinesis. (2) INCENP and Aurora-B exist in a complex in Xenopus eggs and in mammalian cultured cells. (3) Interference with Survivin or INCENP function causes Aurora-B kinase to be mislocalized in mitosis in both C. elegans and vertebrates. Evidence is provided 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 both 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. The data provide the first biochemical evidence that Survivin can interact directly with members of the chromosomal passenger complex (Wheatley, 2001b).

The function of the Aurora B kinase at centromeres and the central spindle is crucial for chromosome segregation and cytokinesis, respectively. This study investigates regulation of human Aurora B by its complex partners, inner centromere protein (INCENP) and survivin. Overexpression of a catalytically inactive, dominant-negative mutant of Aurora B impairs the localization of the entire Aurora B/INCENP/survivin complex to centromeres and the central spindle and severely disturbs mitotic progression. Similar results were also observed after depletion, by RNA interference, of either Aurora B, INCENP, or survivin. These data suggest that Aurora B kinase activity and the formation of the Aurora B/INCENP/survivin complex both contribute to its proper localization. Using recombinant proteins, it was found that Aurora B kinase activity is stimulated by INCENP and that the C-terminal region of INCENP is sufficient for activation. Under identical assay conditions, survivin does not detectably influence kinase activity. Human INCENP is a substrate of Aurora B and mass spectrometry identified three consecutive residues (threonine 893, serine 894, and serine 895) containing at least two phosphorylation sites. A nonphosphorylatable mutant (TSS893-895AAA) is a poor activator of Aurora B, demonstrating that INCENP phosphorylation is important for kinase activation (Honda, 2003).

The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Borealin, a novel member of the complex, is described in this study. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin. 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 (Gassmann, 2004).

The chromosomal passenger complex (CPC), consisting of the serine/threonine kinase Aurora B, the inner centromere protein INCENP, Survivin, and Borealin/DasraB, has essential functions at the centromere in ensuring correct chromosome alignment and segregation. Despite observations that small interfering RNA-mediated knockdown of any one member of the CPC abolishes localization of the other subunits, it remains unclear how the complex is targeted to the centromere. A ternary subcomplex of the CPC comprising Survivin, Borealin, and the N-terminal 58 amino acids of INCENP has been identified in vitro and in vivo. This subcomplex is essential and sufficient for targeting to the centromere. Notably, Aurora B kinase, the enzymatic core of the CPC, is not required for centromere localization of the subcomplex. CPC targeting to the centromere does not depend on CENP-A and hMis12, two core components for kinetochore/centromere assembly, and evidence is provided that the CPC may be directed to centromeric DNA directly via the Borealin subunit. These findings thus establish a functional module within the CPC that assembles on the N terminus of INCENP and controls centromere recruitment (Klein, 2006).

Survivin is a component of the chromosomal passenger complex (CPC) that plays a role in maintenance of an active spindle checkpoint and in cytokinesis. To study whether these different functions can be attributed to distinct domains within the Survivin protein, Survivin-depleted cells were complemented with a variety of point- and deletion-mutants of Survivin. An intact baculovirus IAP repeat (BIR) domain is required for proper spindle checkpoint functioning, but dispensable for cytokinesis. In line with this, mutants lacking an intact BIR domain localize normally to the central spindle, but their localization to inner centromeres is severely perturbed. Consequently, these mutants fail to recruit Aurora B, Borealin/Dasra B, and BubR1 to centromeres and kinetochores, but they have retained the ability to recruit Aurora B and Borealin/Dasra B to the midzone and midbody. Thus, the C terminus of Survivin is sufficient for central spindle localization and execution of cytokinesis, but the additional presence of a functional BIR domain is essential for centromere targeting and spindle checkpoint function. Importantly, the data show that the function of the CPC at the centromere can be separated from its function at the central spindle and that execution of cytokinesis does not require prior concentration of the CPC at centromeres (Lens, 2006).

The chromosomal passenger complex (CPC) coordinates chromosomal and cytoskeletal events of mitosis. The enzymatic core of this complex (Aurora-B) is guided through the mitotic cell by its companion chromosomal passenger proteins, inner centromere protein (INCENP), Survivin and Borealin/Dasra-B, thereby allowing it to act at the right place at the right time. This study addressed the individual contributions of INCENP, Survivin and Borealin to the proper functioning of this complex. INCENP has an important role in stabilizing the complex, and Borealin acts to promote binding of Survivin to INCENP. Importantly, when Survivin is directly fused to INCENP, this hybrid can restore CPC function at the centromeres and midbody, even in the absence of Borealin and the centromere-targeting domain of INCENP. Thus, Survivin is an important mediator of centromere and midbody docking of Aurora-B during mitosis (Vader, 2006).

INCENP binds to beta-tubulin

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. 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 (Wheatley, 2001a).

CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell

Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. A role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homolog, has been examined. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. These data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis (Zeitlin, 2001).

Other INCENP interactions

EVI5 has been shown to be a novel centrosomal protein in interphase cells. 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, immunoprecipitation and GST pull-down approaches were used 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 (Faitar, 2006).

Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as cyclin-dependent kinase 1 (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but the mechanisms of their coordination remain unknown. Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization and kinase activity of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. It is proposed that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics (Goto, 2006).

Centromeric α-satellite RNA is essential for the nucleolar localization of CENPC1 and INCENP

The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. This study demonstrates that centromeric α-satellite RNA and proteins CENPC1 (see Drosophila Cenp-C) and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I-dependent manner. The nucleolar targeting of CENPC1 and INCENP requires α-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following α-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, the nucleolar localization sequences on CENPC1 and INCENP have been identified. Evidence that CENPC1 is an RNA-associating protein that binds α-satellite RNA in an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and 'RNA replenishment' experiments, α-satellite RNA is show to be a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. These data suggest that centromere satellite RNA directly facilitates the accumulation and assembly of centromere-specific nucleoprotein components at the nucleolus and mitotic centromere, and that the sequestration of these components in the interphase nucleolus provides a regulatory mechanism for their timely release into the nucleoplasm for kinetochore assembly at the onset of mitosis (Wong, 2007).

The centromere is a specialized structure on chromosomes for microtubule attachment to ensure the equal partitioning of chromosomes during cell division. This structure comprises two defined domains: the central core for the assembly of the kinetochore and the flanking pericentric heterochromatin for centromere cohesion. In Schizosaccharomyces pombe, the outer centromeric repeat sequences give rise to small interfering RNAs (siRNA) that participate in chromatin repression. The depletion of Dicer (a nuclease required for the processing of siRNAs) in a chicken cell line leads to the disruption of heterochromatin assembly and cohesion. However, Dicer depletion has no observable effect on the binding of the core kinetochore proteins CENPA and CENPC1, indicating that while the evolutionarily conserved RNA interference (RNAi) machinery is crucial for the establishment of the pericentric heterochromatin, it may not be essential for the core kinetochore region. A recent study in maize has further described the association of single-stranded centromeric transposable element and repeat RNA with the core kinetochore complex that is distinct from those at pericentric heterochromatin; however, the functional significance of the observed centromere RNA transcripts is unclear. Furthermore, little is known about the subnuclear distribution of centromere RNA, and the pathway and significance, if any, of such RNA in kinetochore formation and function (Wong, 2007).

The nucleolus is a specialized organelle of the interphase nucleus where ribosomal DNA is transcribed, pre-rRNA is processed, and ribosome subunits are assembled. In addition, this organelle is known to serve a variety of other essential cellular and cell cycle control functions by allowing the timely sequestration of specific trans-acting factors and the biogenesis of many cellular ribonucleoprotein particles (RNPs) including transfer tRNAs, small nuclear snRNAs, different mRNAs, and telomerase RNA. Specifically, numerous studies have directly demonstrated the accumulation of different centromere-associated proteins at the nucleolus, including borealin (See Drosophila Borealin-related), PARP1, and PARP2 (Wong, 2007 and references therein).

This study shows the enrichment of centromeric α-satellite RNA and centromere proteins CENPC1 and INCENP in the interphase nucleolus. Evidence is presented that the centromere satellite RNA is required for the assembly of centromere-associated nucleoprotein components at the nucleolus and kinetochore (Wong, 2007).

This study demonstrates the nucleolar accumulation of the constitutive centromere protein CENPC1, the centromere-associated chromosomal passenger protein INCENP, and centromeric α-satellite RNA. The nucleolus localization sequence (NoLS) motifs for both of these proteins coincide with the nucleus localization sequence (NLS) motifs, and are found at the N-terminal portions of the proteins. These NoLS motifs share significant homology with those of other nucleolus-associated proteins. Some of these proteins, including borealin, PARP1 and PARP2, and INCENP, also bind directly to the centromere (Wong, 2007).

The presence of centromeric α-satellite RNA is closely associated with the nucleolar localization of CENPC1 and INCENP, since RNase treatment induces a complete disruption of this localization. More specifically, in the RNA 'rescue' study, it was shown that the incubation of RNase-treated cells with α-satellite RNA results in the in situ targeting of myc-tagged CENPC1 and INCENP recombinant proteins to the nucleolus. Experiments involving ActD treatment further demonstrate that the nucleolar accumulation of α-satellite RNA, CENPC1, and INCENP is dependent on active RNA polymerase I transcription. Previous studies have similarly shown that the nucleolar localization of centromere proteins borealin and PARP1 and PARP2 is dependent on active nucleolar transcriptional activity. Recent studies in S. pombe have shown that RNA polymerase II and IV (a unique RNA polymerase in plants) are essential for the generation of siRNAs. The transcription machinery that modulates the transcription of centromere repeats or siRNA synthesis in vertebrates is still unknown. However, it is unlikely that RNA polymerase I is directly driving the transcription of the nucleolar α-satellite RNA, as indicated by nuclear run-on transcription data. Furthermore, the depletion of α-satellite RNA-FISH signals is unlikely to be due to the down-regulation of some specific proteins, since the conditions used for ActD treatment did not affect the expression levels of proteins in general, as exemplified by those of CENPC1 and INCENP. In this context, the mechanism of action for the RNA polymerase I remains undefined, although it may be directly involved in providing a structural network or complex that retains the various centromere components in the nucleolus (Wong, 2007).

The nucleolus serves the important function of ribosome synthesis. Increasing numbers of studies now show that it is also a multitasking organelle that engages in other important cellular functions. An example of the pluri-functionality of the nucleolus is the cell cycle-dependent nucleolar localization of the telomere components hTERT (reverse transcriptase catalytic subunit of telomerase), hTR (telomerase RNA), and telomeric DNA-binding protein TERF2, where these components have been shown to be released from the nucleolus at late S and G2/M phase at a time that coincides with telomere elongation. The data suggest that the nucleolus may similarly sequester centromeric components such as centromere RNA and proteins for timely delivery to the chromosomes for kinetochore assembly at mitosis. The centromere proteins, in particular the chromosomal passenger proteins such as borealin, PARP1 and PARP2, and INCENP, display dynamic changes in distribution patterns during various stages of the cell cycle. Although these proteins only bind the centromere following mitotic onset, they are expressed much earlier in mid-S to G2 phase and have been shown to accumulate in the nucleolus. During this period, the nucleolus may serve a repository role for the temporal storage of centromere RNA and proteins, and control the timely release of these components into the nucleoplasm for kinetochore assembly at M phase. It is also possible that the assembly of functional kinetochore nucleoprotein complexes may take place in the nucleolus (Wong, 2007).

Treatment of metaphase cells with single-stranded RNA-specific RNases (but not with a double-stranded RNA-specific RNase) results in the significant but incomplete delocalization of CENPC1, and the complete delocalization of two centromere-associated chromosomal passenger proteins INCENP and survivin, from the metaphase centromere. Similar treatments have no significant effect on the centromeric localization of CENPA, CENPB, and CENPE. These results indicate that the assembly of INCENP and survivin, and to an extent CENPC1, but not CENPA, CENPB, and CENPE, at the metaphase centromere is dependent on the presence of single-stranded RNA (Wong, 2007).

Several lines of evidence suggest that at least a component of this single-stranded RNA is directly transcribed from the centromeric α-satellite DNA, and that this α-satellite RNA transcript associates directly with CENPC1. (1) Recombinant GST-fusion protein truncation constructs and site-specific mutagenesis were used to show that CENPC1 binds single-stranded α-satellite RNA probes derived from chromosomes 2, 4, 9, or 13/21, and ranging in size from 167-342 nucleotides (nt). This RNA-binding activity resides within the central region (amino acids 426-551) as well as the C-terminal portion of the protein. (2) By performing RT-PCR on RNA isolated following RNA-ChIP using an anti-CENPC1 antibody, in vivo binding of single-stranded α-satellite RNA with the CENPC1-associated kinetochore protein complex has been demonstrated. The RT-PCR results indicate that the kinetochore-associated α-satellite RNA has a size range corresponding to multiples of the 171-nt α-satellite repeating unit. These studies show the presence of single-stranded α-satellite RNA of 171 nt and larger and belonging to the different chromosome-specific α-satellite subfamilies. However, the possibility cannot be excluded that shorter RNA or α-satellite RNA of other sequences may be present, as the present detection may bias against these RNAs (Wong, 2007).

(3) It has been shown that the in situ replenishment of single-stranded α-satellite RNA significantly restores the relocalization of myc-tagged CENPC1 and INCENP to the centromeres of some of the chromosomes following RNase treatment. The incomplete rescue of the recruitment of the myc-tagged recombinant proteins to the kinetochore could be due to the inappropriate folding or modification of the recombinant proteins or replenishing RNA used. Further investigations are needed to discern these possibilities and define the characteristics and possible heterogeneity associated with these RNA transcripts (Wong, 2007).

(4) It has been shown that wild-type GFP-CENPC1, but not the mutant construct with mutated residues within the NoLs domain, is able to partially restore the functionality of the CENPC1-depleted kinetochore in the CENPC1 RNAi-knockdown cells and rescue the cells from the inhibition of cell growth. The failure of the over-expression of the mutated GFP-CENPC1 protein in improving the rate of cell growth suggests that the NoLS domain is essential for the proper function of CENPC1 (Wong, 2007).

Together, this analysis indicates that, in addition to the important role played by the short, double-stranded siRNA at the pericentric heterochromatin, a distinct class of longer, single-stranded centromeric α-satellite RNA is an important structural component of the human centromere (Wong, 2007).

Central to this model is the finding that centromeric α-satellite RNA is essential for the enrichment of centromere proteins CENPC1, INCENP, and/or survivin at the nucleolus and mitotic centromere. It is proposed that the nucleolar sequestration of centromere-specific nucleoprotein components provides a regulatory mechanism for the timely release of these components into the nucleoplasm for kinetochore assembly at the onset of mitosis. The finding that CENPC1 binds centromere satellite RNA, whereas previous in vitro studies have shown that CENPC1 is also DNA-binding, together suggest that this protein has a dual RNA- and DNA-binding function. It is therefore possible that one pool of CENPC1 may play a constitutive centromere DNA-binding role that persists throughout the cell cycle, while a second pool of this protein may act directly as, or be part of, a chaperone mechanism to relocate centromeric α-satellite RNA and centromere proteins, including INCENP, from the nucleolus onto the mitotic centromere. The RNA dependence of the nucleolar localization of both CENPC1 and INCENP suggests that these proteins may associate with the centromeric α-satellite RNA and be assembled into a nucleoprotein complex in the nucleolus. This nucleolar chaperone complex may further include other centromere-associated proteins such as borealin, PARP1, and PARP2, since these components have also been shown to accumulate in the nucleolus. In further support of the proposed dual role of CENPC1, significantly higher quantitative immunofluorescence signals have consistently been observed for CENPC1 (but not other constitutive centromere proteins examined, including CENPA and CENPB) on the metaphase centromere compared with those of the interphase centromere. Of interest, a recent study (Kwon, 2007) has shown that CENPC1 is targeted to interphase or metaphase centromeres through interactions with different sets of centromere proteins, providing further evidence that CENPC1 is localized to the centromere via two independent pathways during interphase or the mitosis stage. The present study has shown that treatment with single-stranded RNA-specific RNases results in a significant, but not complete, delocalization of CENPC1 from the metaphase centromere, consistent with the idea that only the RNA-, but not the DNA-, associated pool of CENPC1 (and components of its complex, such as INCENP) is sensitive to the RNase treatment. The data demonstrate that survivin does not localize to the nucleolus, although its localization to the metaphase centromere is also RNA-dependent. This can be explained by the incorporation of survivin into the metaphase chromosomes following the breakdown of the nucleolar membrane, via INCENP and/or borealin; previous work (Klein, 2006) has shown that survivin forms a functional complex with these two proteins for targeting to the centromere (Wong, 2007).

Cdk1 negatively regulates midzone localization of the mitotic kinesin Mklp2 and the chromosomal passenger complex

The survival of eukaryotes depends on the accurate coordination of mitosis with cytokinesis. Key for the coordination of both processes is the chromosomal passenger complex (CPC) comprising Aurora-B, INCENP, survivin, and borealin. The translocation of the CPC from centromeres to the spindle midzone, a structure composed of antiparallel microtubules, at anaphase onset is critical for the completion of cytokinesis. In mammalian cells, the mitotic kinesin Mklp2 is essential for recruitment of the CPC to the spindle midzone. However, the mechanism regulating the binding of Mklp2 to microtubules has remained unknown. This study demonstrates that Mklp2 and the CPC mutually depend on each other for midzone localization; i.e., Mklp2 is mislocalized in INCENP-RNAi cells and vice versa. Remarkably, INCENP is required for localization of Mklp2 to the ends of stable microtubules in cells with low Cdk1 activity. In vitro assays revealed that the association between the CPC and Mklp2 is negatively regulated by Cdk1. Collectively, these data suggest that anaphase onset triggers the association between the CPC and Mklp2 and that this association targets the CPC-Mklp2 complex to the ends of stable microtubules in the spindle midzone (Hümmer, 2009).

Mutation of INCENP

INCENP is a tightly bound chromosomal protein that transfers to the spindle midzone at the metaphase/anaphase transition. An INCENP truncation mutant (INCENP382-839) associates with microtubules but does not bind to chromosomes, and coats the entire spindle throughout mitosis. Furthermore, an INCENP truncation mutant (INCENP43-839) shown not to transfer to the spindle at anaphase, binds chromosomes, but is unable to target to the centromere. Thus, association with the chromosomes, and specifically with centromeres, appears to be essential for INCENP targeting to the correct spindle subdomain at anaphase. An INCENP truncation mutant (INCENP1-405) that targets to centromeres but lacks the microtubule association region acquires strong dominant-negative characteristics. INCENP1-405 interferes with both prometaphase chromosome alignment and the completion of cytokinesis. INCENP1-405 apparently exerts its effect by displacing the endogenous protein from centromeres. These experiments provide evidence of an unexpected link between this chromosomal protein and cytokinesis, and suggest that one function of INCENP may be to integrate the chromosomal and cytoskeletal events of mitosis (Mackay, 1998).

INCENP is a chromosomal passenger protein that relocates from the centromere to the spindle midzone during the metaphase-anaphase transition, ultimately being discarded in the cell midbody at the completion of cytokinesis. Using homologous recombination, Incenp gene-targeted heterozygous mice were generated that are phenotypically indistinguishable from their wild-type littermates. Intercrossing the hetero-zygotes results in no live-born homozygous Incenp -disrupted progeny, indicating an early lethality. Day 3.5 affected pre-implantation embryos contain large, morphologically abnormal cells that fail to fully develop a blastocoel cavity or thrive in utero and in culture. Chromatin and tubulin immunocytochemical stainings of these and day 2.5 affected embryos reveal a high mitotic index, no discernible metaphase or anaphase stages, complete absence of midbodies, micronuclei formation, morphologically irregular macronuclei with large chromosome complements, multipolar mitotic configurations, binucleated cells, internuclear bridges and abnormal spindle bundling. The phenotype is consistent with a defect in the modulation of microtubule dynamics, severely affecting chromosome segregation and resulting in poorly resolved chromatin masses, aberrant karyokinesis and internuclear bridge formation. These latter occurrences could pose a physical barrier blocking cytokinesis (Cutts, 1999).

ISWI remodeling complexes in Xenopus are regulated by INCENP-aurora B

Components of mitotic chromosomes assembled in Xenopus laevis egg extracts have been characterized and collectively referred to as Xenopus chromosome-associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, XCAP-F has been isolated; it is the Xenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains the Xenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, it has been found that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B (MacCallum, 2002).

Signaling downstream of Aurora B/Survivin

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway was studied. The results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3 (see Drosophila Bub3), and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other (Vigneron, 2004).

Inner centromere protein : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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