InteractiveFly: GeneBrief

Cenp-C: Biological Overview | References

Faithful transmission of genetic information during mitotic divisions depends on bipolar attachment of sister kinetochores to the mitotic spindle and on complete resolution of sister-chromatid cohesion immediately before the metaphase-to-anaphase transition (see Organization of the animal kinetochore). Separase is thought to be responsible for sister-chromatid separation, but its regulation is not completely understood. Therefore, a screen was carried out for genetic loci that modify the aberrant phenotypes caused by overexpression of the regulatory separase complex subunits Pimples/securin and Three rows in Drosophila. An interacting gene was found to encode a constitutive centromere protein. Characterization of its centromere localization domain revealed the presence of a diverged CENPC motif. While direct evidence for an involvement of this Drosophila Cenp-C homolog in separase activation at centromeres could not be obtained, in vivo imaging clearly demonstrated that it is required for normal attachment of kinetochores to the spindle (Heeger, 2005).

Separase functions as a protease at the metaphase-to-anaphase transition of mitosis. At this crucial cell cycle transition, separase cleaves the α-kleisin subunit (Scc1/Mcd1/Rad21) of the cohesin complex and thereby promotes the final release of sister-chromatid cohesion. The careful control of separase activity during the cell division cycle involves regulatory subunits. Securin is a subunit that accumulates and associates with separase during interphase. It acts as an inhibitor of separase activity. Thus, the rapid degradation of securin at the metaphase-to-anaphase transition via the anaphase-promoting complex/cyclosome (APC/C) pathway of ubiquitin-dependent proteolysis results in separase activation. In Drosophila, securin is encoded by the pimples (pim) gene and the catalytic protease subunit by the Separase (Sse) gene. Drosophila Sse lacks the extensive N-terminal regulatory domain that is present in separases outside the dipterans because the corresponding gene region appears to have evolved into an independent gene, three rows (thr). Drosophila Thr binds to Sse and is required for sister-chromatid separation during mitosis (Heeger, 2005).

The precise role of Thr and the corresponding N-terminal domains in nondipteran separases is not understood. Moreover, Pim and other securins are not just separase inhibitors but also contribute in an unknown positive manner to sister-chromatid separation. In fission yeast, securin recruits separase to the mitotic spindle, and similar observations have been described in other organisms. Separase activation and transport on spindle microtubules might confine its action to the congressed chromosomes in metaphase plates and in particular to the pericentromeric region. This hypothetical scenario might explain why only a minute and preferentially pericentromeric pool of Scc1 appears to be cleaved by separase during mitosis of higher eukaryotic cells, while the large majority of Scc1 remains intact (Heeger, 2005).

To identify additional genes that might contribute to separase regulation and function, a screen was performed for chromosomal regions that act as genetic modifiers of the aberrant phenotypes resulting from overexpression of Pim or a dominant-negative Thr fragment during Drosophila eye development. Molecular characterization of an interacting locus revealed that it encodes a constitutive centromere protein. Mapping of its centromere localization domain in combination with sequence comparisons among Drosophilid orthologs allowed its identification as the most diverged Cenp-C homolog. Cenp-C was originally identified as a human autoantigen localized to centromeres and found to display limited sequence similarity to budding yeast Mif2, which was identified by mutations affecting the fidelity of chromosome transmission during mitosis. Homologs have also been described in nematodes (HCP-4) and plants. For simplicity, Cenp-C has been used as a designation for all these homologs. Interestingly, recent analyses have demonstrated that Cenp-C, as well as Cenp-A, a histone H3 variant present in centromeric nucleosomes, evolve rapidly and adaptively in many lineages, perhaps driven by the rapid evolution of centromeric satellite sequences, and in Drosophila, Cenp-C was supposed to be absent. Apart from providing further support for the striking sequence divergence of ubiquitous eukaryotic centromere components, these findings also raise the possibility that separase activity might be enhanced by such components (Heeger, 2005).

Identification of Drosophila Cenp-C closes a prominent gap in the arguments for homologous centromere organization. Centromeric DNA sequences have evolved extremely rapidly and appear to have driven the coevolution of centromeric proteins during eukaryote evolution. The resulting low sequence similarity between centromeric proteins has effectively concealed the existence of a common set of constitutive eukaryotic centromere proteins until very recently. The first features demonstrated to be shared among fungal, plant, and animal centromeres were centromere-specific histone H3 variants. In addition to Cenp-A homologs, only one further constitutive centromere component, Cenp-C, has so far been shown to be present in each of the three main eukaryotic branches. In combination with the recent identification of related Cenp-H-, Cenp-I-, and Mis12-like proteins in both vertebrates and yeast, these results provide strong support for the notion of a common set of constitutive centromere proteins. These proteins, which are centromeric throughout the cell cycle, appear to provide a foundation for kinetochore assembly and spindle attachment during mitosis by recruiting several distinct multisubunit complexes that also contain highly diverged proteins (Heeger, 2005).

The extensive sequence divergence characteristically observed among homologous eukaryotic centromere and kinetochore proteins is striking, especially in the light of their common fundamental cellular function. The average amino acid identity observed in a genome-wide comparison of D. melanogaster and D. pseudoobscura ortholog pairs is 77% and only 38% in case of the Cenp-C pair. Moreover, based on the ratio between radical charge mutations and conservative substitutions in D. melanogaster and D. pseudoobscura ortholog pairs, Cenp-C is one of 44 genes likely to have evolved under positive selection. Except for a few very restricted regions, comparison of D. melanogaster Cenp-C with the orthologs from D. erecta and D. yakuba, which are closer relatives than D. pseudoobscura and thus amenable to d_N/d_S analyses, did not reveal strong evidence for positive selection, in contrast to the recent findings in plant and mammalian lineages. However, these d_N/d_S analyses ignore insertions and deletions (indels), which have occurred considerably more often during Cenp-C evolution in Drosophilids than in the mammalian lineage. Most of the indels are observed within the minimally conserved central regions of Drosophilid Cenp-C. Similar variabilities resulting from recurrent exon duplications have been observed in the central region of the plant Cenp-C genes (Heeger, 2005).

The adaptively evolving regions of mammalian Cenp-C have been shown to bind to DNA in vitro, consistent with the proposed coevolution of centromeric DNA and protein sequences (Talbert, 2004). However, this DNA binding in vitro is not sequence-specific, suggesting that interactions with centromere-specific proteins contribute to centromere localization of Cenp-C. As in other organisms, Cid/Cenp-A is also required for centromere localization of Cenp-C in Drosophila. High-resolution light microscopy of mitotic chromosomes has indicated that human Cenp-C covers the poleward-oriented peripheral region of the Cenp-A-containing centromeric chromatin (Blower, 2002). Direct interactions between Cenp-A and Cenp-C have not yet been demonstrated in any organism. Attempts with yeast two-hybrid experiments were also unsuccessful (Heeger, 2005).

The CENPC motif has recently been identified as the only region conserved among the Cenp-C orthologs from fungi, plant, and animals (Talbert, 2004). In Drosophilids, even this short motif of ~24 amino acids is not fully conserved in its C-terminal part. These results suggest that this CENPC motif is crucial for centromere localizationA single-point mutation affecting one of the invariant positions in the CENPC motif interferes with centromere localization of the C-terminal domain of Cenp-C in a transfection assay. This mutation was identified as the only missense mutation interfering with centromere localization after extensive random mutagenesis. Further experiments will reveal whether and how the CENPC motif contacts Cid/Cenp-A nucleosomes. It is emphasized, however, that also in Drosophila Cenp-C, other regions than the CENPC motif clearly contribute to efficient centromere localization. Centromere localization of the CN subregion (1009-1205), for instance, is only detected in a transfection assay in live but not in fixed cells, while centromere localization of the larger C region (1009-1411) is resistant to fixation (Heeger, 2005).

The highest conservation among Drosophilid Cenp-C proteins is observed within the N-terminal third, which is neither required nor sufficient for normal centromere localization. Nevertheless, prolonged overexpression of this domain in proliferating eye and wing imaginal disc cells results in severe defects. The conserved N-terminal Cenp-C domains (R and DH) might bind to kinetochore proteins and titrate these away from the centromere when overexpressed. Biochemical and genetic characterizations in Saccharomyces cerevisiae and Caenorhabditis elegans have suggested that Cenp-C is not only associated with Cenp-A, but that it also recruits the next layer of kinetochore proteins, in particular the Mis12/Mtw1 and Ndc80 complexes, which remain to be identified in Drosophila (Heeger, 2005).

As in yeast (Meluh, 1995), chicken (Fukagawa, 1997; Fukagawa, 1999), and mice (Kalitsis, 1998), Cenp-C is also an essential gene in Drosophila. Antibody microinjection experiments in mammalian cells (Tomkiel, 1994); RNA interference in C. elegans (Moore, 2001; Oegema, 2001); and phenotypic analysis in yeast (Brown, 1993), chicken cells (Fukagawa, 2001), and mutant Drosophila embryos demonstrate that Cenp-C is required for normal chromosome segregation during mitosis. in vivo imaging in Cenp-C mutant embryos discloses these defects in detail. Previously, in vivo imaging has also been applied in C. elegans CENP-C(RNAi) embryos (Moore, 2001; Oegema, 2001). The formation of holocentric chromosomes and transient Cenp-C recruitment only during mitosis differentiates C. elegans from other metazoans like Drosophila and mammalian cells. Moreover, in contrast to the findings in C. elegans, chromosome congression into a central plane is still observed in the Drosophila Cenp-C mutants. Presumably, this chromosome congression reflects the function of residual maternally provided Cenp-C, which is still detectable at this stage of analysis. Moreover, time-lapse analyses demonstrate that chromosome congression is not entirely normal in the Cenp-C mutant embryos. Metaphase plate formation is delayed and often does not lead to the highly ordered arrangement of all chromosomes characteristically observed before anaphase onset in wild type. Occasional chromosomes fail to achieve bipolar attachment in the Cenp-C mutants. These chromosomes do not segregate normally during anaphase. Cenp-C is, therefore, clearly required for normal attachment of kinetochores to the mitotic spindle (Heeger, 2005).

Evidently, the insufficiently attached chromosomes in Cenp-C mutant embryos are unable to inhibit the onset of anaphase, even though the mitotic spindle checkpoint appears to be at least partially functional in Cenp-C mutants at the analyzed stage. However, assembly of mitotic spindle checkpoint proteins might fail, particularly on the kinetochores of those chromosomes that do not attach correctly to the mitotic spindle (Heeger, 2005).

In principle, the chromosome segregation defects observed in the Cenp-C mutants might not only reflect impaired interactions between kinetochores and spindle. Segregation of sister chromatids to the spindle poles also depends on complete resolution of sister-chromatid cohesion at the metaphase-to-anaphase transition. This final separation of sister chromatids is thought to be achieved by separase-mediated cleavage of the Scc1/Rad21 subunit of those cohesin complexes that perdure in the pericentromeric region until the metaphase-to-anaphase transition. Several observations are consistent with the idea that a localized full activation of separase might be assisted by centromeric proteins. Accordingly, mutations in Cenp-C might reduce separase activity and thereby explain the genetic interactions with the regulatory separase subunits Pim/securin and Thr that led to the identification of Drosophila Cenp-C. No coimmunoprecipitation of Cenp-C with separase complex proteins was observed and no effects of Cenp-C mutations on Pim and Thr levels was observed. This analysis has, therefore, not exposed clear evidence for separase activation at centromeres. In contrast, Cenp-C is clearly required for normal chromosome attachment to the mitotic spindle, and thus the observed genetic interactions most likely reflect a summation of negative effects on the efficiency of sister-chromatid separation (by separase) and segregation (by the spindle). However, it is emphasized that the results certainly do not rule out a local activation of separase within the centromeric region (Heeger, 2005).

Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes

Chromosome segregation during meiosis and mitosis depends on the assembly of functional kinetochores within centromeric regions. Centromeric DNA and kinetochore proteins show surprisingly little sequence conservation despite their fundamental biological role. However, identification in Drosophila of the most diverged orthologs identified so far, which encode components of a kinetochore protein network including the Ndc80 and Mis complexes, further emphasizes the notion of a shared eukaryotic kinetochore design. To determine its spatial organization, quantitative light microscopy was used to analyzed hundreds of native chromosomes from transgenic Drosophila strains coexpressing combinations of red and green fluorescent fusion proteins, fully capable of providing the essential wild-type functions. Thereby, Cenp-A/Cid, Cenp-C, Mis12 and the Ndc80 (CG9938) complex were mapped along the inter sister kinetochore axis with a resolution below 10 nm. The C terminus of Cenp-C was found to be near but well separated from the innermost component Cenp-A/Cid. The N terminus of Cenp-C is further out, clustered with Mis12 and the Spc25 end of the rod-like Ndc80 complex, which is known to bind to microtubules at its other more distal Ndc80/Nuf2 end (Schittenhelm, 2007).

Identification of Drosophila kinetochore proteins further exposes hidden similarities of kinetochore design in eukaryotes. In addition to the previously known, highly diverged Cenp-A/Cid and Cenp-C homologs, Drosophila expresses similarly diverged homologs of the Mis12 and Ndc80 complex network, which is also present in yeast, C. elegans, vertebrates, and presumably in plants as well. These ubiquitous CKC components have been localized along the intersister kinetochore axis with unprecedented spatial resolution. Early Drosophila embryos allow an efficient isolation of native mitotic chromosomes and thereby imaging with reduced background. Moreover, transgenic strains allow the expression of fluorescent fusion proteins, which were demonstrated to be fully functional by genetic complementation tests (Schittenhelm, 2007).

The position of fluorescent signal maxima has been determined within the kinetochore of native chromosomes released from embryos expressing fluorescent CKC fusion proteins. The CKC map is based on averaged data from hundreds of analyzed chromosomes. Therefore, its interpretation depends critically on the variability of kinetochore organization in individual chromosomes. For instance, in principle, a given component might be localized on the inner kinetochore side in 50% of the chromatids and on the outer side in the other half of the chromatids, resulting in a misleading central positioning in the CKC map. Theoretically, such variability should widen the distribution of the distances measured in individual chromosomes. However, kinetochore width is smaller than the spreading of the image of a point light source in the microscope, and several additional factors (like background, noise, pixelation) further limit the precision of the measurements. The effect of positional variability on distribution width of the measured values would therefore be very subtle. Moreover, none of the known CKC proteins has been firmly demonstrated to be a spatially invariable kinetochore component, precluding comparisons to an established standard distribution. However, the reproducible trilaminar structure of the kinetochore during prometaphase, that has been documented by EM, argues strongly against extensive organizational variability. It is emphasized that the difficulties in detecting subtle alterations in the distribution width of the measurements obtained for a given CKC component has important consequences even under the assumption that the spatial distribution of CKC components is essentially invariable in individual kinetochores. These difficulties prevent conclusions concerning the width occupied by a given CKC component within a kinetochore. For instance, Mis12 could either be confined to a single layer in the middle of the kinetochore or spread throughout the kinetochore, and both localization patterns would result in a central signal maximum. However, biochemical analyses of kinetochore proteins have so far revealed highly specific interactions, arguing strongly for a precise and restricted localization of CKC components. The following discussion is therefore based on the unproven but likely assumption that the kinetochore represents a precisely defined layered structure (Schittenhelm, 2007).

Based on previous analyses, Cenp-A, Cenp-C, and Mis12 are thought to be components of the inner plate of the characteristic trilaminar kinetochore structure apparent in the EM (Kline, 2006; Vos, 2006). The analyses indicate a significant separation between the inner most CKC component Cenp-A and all other CKC components analyzed here. Recently, Cenp-A nucleosomes purified from human cells were found to be intimately associated with the five proteins Cenp-M, Cenp-N, Cenp-T, Cenp-U, and Cenp-H in addition to Cenp-C (Foltz, 2006; Izuta, 2006; Okada, 2006). The apparent space between Cenp-A and Cenp-C might therefore be occupied by some of those proteins (Schittenhelm, 2007).

Many immunolocalization studies, including a recent study with Drosophila cells (Maiato, 2006), have failed to detect a comparable extensive spatial separation between Cenp-A/Cid and Cenp-C. However, immunolocalization with human chromosomes also revealed little overlap between Cenp-A and Cenp-C, with the latter extending over the top and bottom of a Cenp-A cylinder (Blower, 2002). Antigen accessibility problems, which were not excluded by Blower (2002), cannot affect the concurrent findings (Schittenhelm, 2007).

In this paper, Cenp-C is shown to be spread in a polar orientation across a central CKC region. The C-terminal domain of Cenp-C, which contains the most conserved region including the CENP-C motif (Talbert, 2004; Heeger, 2005), points toward the centromeric DNA. These C-terminal sequences are connected via minimally conserved spacer sequences to the N-terminal domain which is oriented toward the kinetochore spindle fibers. The N-terminal region of D. melanogaster Cenp-C contains some blocks which are highly conserved among Drosophilids (Heeger, 2005). These blocks might be involved in recruiting the next layer of kinetochore proteins which are suggested to include the Ndc80 and Mis12 complexes. Mis12 is close to the N-terminal Cenp-C region. Moreover, the Ndc80 complex component Spc25 (CG7242) appears to be even a bit closer but well separated by about 20 nm from the other Ndc80 component Nuf2 (CG8902). Apart from a polar Cenp-C orientation, these analyses therefore also indicate a polar orientation for the Ndc80 complex (Schittenhelm, 2007).

The tetrameric Ndc80 complex has a highly elongated, rod-like structure in vitro (Ciferri, 2005; Wei, 2005). The globular N-terminal domains of Ndc80 and Nuf2 are present on one end of the rod. The remainder of these two subunits forms an extended coiled coil which is further prolonged at its C-terminal end by binding to the N-terminal coiled coil region of the Spc24/Spc25 dimer. Closely associated C-terminal globular domains of Spc24 and Spc25 (Wei, 2006) form the other end of the rod. Scanning force microscopy and EM analyses have indicated that the coiled coil region separating the globular domains at the end of the Ndc80 complex has an extension of about 40 nm (Ciferri, 2005; Wei, 2005). This is twofold longer than the distance observed between fluorescent proteins at the N and C termini of Nuf2 and Spc25 in kinetochores of native Drosophila chromosomes. Many of the elongated Ndc80 complexes might not be perfectly oriented along the spindle axis, especially as the kinetochores in the preparations used in this study are not under tension. Such a nonuniform orientation could result in spatial distributions of the N and C termini of Nuf2 and Spc25, respectively, with signal maxima that are more closely spaced than their separation within an isolated complex. An analysis of the positions of CKC components in chromosomes that are bi-oriented within the spindle and under tension would clearly be of interest. However, the increased background levels present in living embryos have so far precluded such analyses (Schittenhelm, 2007).

The observed polar orientation of the Ndc80 complex within the kinetochore confirms the findings of a recent independent study (Deluca. 2006). Moreover, the observation that Ndc80 and Nuf2 kinetochore localization is no longer observed in the absence of Spc24 or Spc25 is consistent but does not prove an orientation of the complex with inner Spc24/Spc25 and outer Ndc80/Nuf2 globular domains, because absence of Spc24 or Spc25 for instance might simply result in an instability of other complex components, as often observed in the case of stable complexes (Schittenhelm, 2007 and references therein).

In budding yeast, the Ndc80 complex has been proposed to function as a connection between the inner components (CBF3 complex, Cenp-A/Cse4 nucleosome, Cenp-C/Mif2, Mis12/MIND complex) and the Dam/DASH complex which is required for bi-orientation and appears to form a ring around the single microtubule attaching to a yeast kinetochore. More recently, bacterial expression of the C. elegans KMN network composed of the Spc105/KNL-1, Mis12 and Ndc80 complexes has led to a convincing identification of two independent sites in this protein network that can bind directly to microtubules in vitro (Cheeseman, 2006). One of these microtubule binding sites is present within Spc105/KNL-1. The other is found within the globular N-terminal Ndc80 domain (Cheeseman, 2006) which is known to be within the outer kinetochore plates where kinetochore microtubules terminate (DeLuca, 2005). In vitro, the Ndc80 complex binds to microtubules at an angle (Cheeseman, 2006). A corresponding orientation of the Ndc80 complex within the kinetochore is fully consistent with the finding that the separation of the terminal globular domains of Spc25 and Nuf2 along the intersister kinetochore axis appears to be less than their separation along the axis of isolated complexes (Ciferri, 2005; Wei, 2005). Accordingly, the 'barbed end' of microtubules decorated with the Ndc80 complex would be predicted to correspond to the plus end (Schittenhelm, 2007 and references therein).

In conclusion, in addition to the identification of Drosophila Ndc80 and Mis12 complex components, this work provides a highly resolved structural framework integrating the most widely studied ubiquitous CKC components and a precise method for a future incorporation of additional proteins (Schittenhelm, 2007).

Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation

Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, factors required for centromere propagation were isolated using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila. The proteins CAL1 and CENP-C were identified as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. The mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 were identified as regulators of centromere propagation. CYCA was shown to be centromere localized, and CYCA and RCA1/Emi1 were shown to couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. These findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division (Erhardt, 2008).

This is the first example of a genome-wide RNAi screen for mislocalization of an endogenous chromosomal protein and provides the distinct advantage that the primary screen output is a direct readout of the phenotype of interest. This approach identified novel and known factors that control the assembly of centromeric chromatin and link centromere assembly and propagation to the cell cycle (Erhardt, 2008).

Although centromere assembly has been described as a hierarchical process directed by CENP-A, the data show that CID, CENP-C, and CAL1 are interdependent for centromere propagation, which is consistent with experiments in vertebrate cells showing interdependence between the CENP-H-CENP-I complex and CENP-A. However, studies in C. elegans and vertebrates have not detected a role for CENP-C in CENP-A chromatin assembly, suggesting that CENP-C plays a more prominent role in regulating centromere propagation in flies. Collectively, these results suggest that CENPs that depend on CENP-A for their localization may 'feed back' to control CENP-A assembly. Histone variants are assembled into chromatin both by histone chaperones (e.g., the histone H3.3-specific chaperone HIRA [histone regulatory A] that provides specificity to the CHD1 chromatin-remodeling ATPase) and by histone variant-specific ATPases (e.g., Swr1 that can use the general chaperone Nap1 or the specific chaperone Chz1 to assemble H2A.Z). CENP-C or CAL1 might facilitate centromere-specific CID localization by providing centromere specificity to a chromatin-remodeling ATPase in a manner analogous to HIRA or might direct the localization of chromatin assembly factors to the centromere. It will be interesting to determine what factors associate with CAL1 and CENP-C as a route to elucidating the mechanisms of centromere assembly and propagation (Erhardt, 2008).

The loading of CENP-A in human somatic cells and in Drosophila embryos occurs after anaphase initiation when APC^FZR/CDH1 activity is high. Ubiquitin-mediated proteolysis facilitates formation of a single centromere by degrading noncentromeric CENP-A, and subunits of the APC are localized to kinetochores. The results demonstrate that normal regulation of APC^FZR/CDH1 activity is required for centromere propagation, providing a link between centromere assembly and cell cycle regulation (Erhardt, 2008).

Two alternative models are proposed for the role of APC^FZR/CDH1 in centromere function. The first model is that CYCA is the relevant substrate of APC^FZR/CDH1 and that the kinase activity of the CYCA-Cdk1 complex is required for the localization of CID, CENP-C, and CAL1 to the centromere. CYCA is normally degraded as cells proceed through mitosis, suggesting that CYCA-Cdk1 would likely act during G2 or early M to phosphorylate a substrate involved in centromere assembly. The CID and CENP-C localization defect caused by CYCA depletion was rescued by the simultaneous depletion of FZR/CDH1 even though the levels of CYCA protein remained low in the double depletion. The rescue of the CID and CENP-C localization defect in cells with low CYCA protein suggests that maintaining high levels of CYCA-Cdk1 activity is not required for centromere propagation, but it cannot be ruled out that the residual CYCA protein in these cells is sufficient to rescue the centromeric phenotype when APC activity is compromised by FZR/CDH1 depletion (Erhardt, 2008).

The second model that is consistent with these observations is that one or more APC^FZR/CDH1 substrates (“X”) regulate the interdependent localization of CID, CENP-C, and CAL1 to the centromere. RCA1 and CYCA inhibit the APC in G2 to allow mitotic cyclin accumulation. An APC^FZR/CDH1 substrate could repress centromere assembly until anaphase/G1, when proteolysis would remove the repression in a manner analogous to replication licensing. If an APC^FZR/CDH1 substrate acted solely as a negative regulator of centromere assembly, FZR/CDH1 depletion should prevent CID assembly at centromeres, and premature APC^FZR/CDH1 activation by CYCA or RCA1 depletion might cause an increase of CID at centromeres as a result of premature assembly. It was observed that neither CDH1 nor CDC20 depletion alone impacted CID, CAL1, or CENP-C assembly at centromeres or the overall levels of these proteins but that premature APC activation resulted in failed centromere assembly (Erhardt, 2008).

A simple interpretation of the results is that CYCA-Cdk1 or another APC^FZR/CDH1 substrate acts during G2/metaphase before APC^FZR/CDH1 activation to make centromeres competent for assembly during anaphase and/or G1. Premature removal of the APC^FZR/CDH1 substrate would cause failure to relicense the centromeres for assembly in the next G1 phase. When compared with the process of replication licensing, in which the positive regulator CDC6 and the negative regulators geminin and CYCA are all substrates of APC^FZR/CDH1, the model of a single APC^FZR/CDH1 substrate that controls centromere licensing or propagation may be oversimplified. This study observed that defective centromere localization of CID and CENP-C after CYCA or RCA1 depletion was not rescued by CDC20 depletion, but a role for APC^FZY/CDC20 in centromere propagation cannot be ruled out because premature APC^FZR/CDH1 activation could mask a subsequent role for FZY/CDC20, which is activated at the metaphase/anaphase transition (Erhardt, 2008).

It is not yet known whether the localization of CYCA at centromeres is important for the regulation of centromere assembly. In Drosophila, it has been demonstrated that the subcellular localization of CYCA is not important for proper progression through the cell cycle; however, these experiments did not directly address whether mislocalization of CYCA prevented the association of CYCA with centromeres. It will be interesting to determine whether CID, CENP-C, and CAL1 localization require centromere-localized CYCA-Cdk1 activity or whether any of these proteins are a direct target of CYCA-Cdk1 (Erhardt, 2008).

The results suggest that CID or CAL1 levels are indirectly controlled by APC activity. Interestingly, the human M18BP1 has recently been proposed to act as a 'licensing factor' for centromere assembly. Although no clear homologues of M18BP1/KNL2 have been identified in Drosophila, both CAL1 in flies and M18BP1/KNL2 in other species are interdependent with CENP-A for centromere localization. Strikingly, levels of CAL1 and M18BP1/KNL2 are reduced on metaphase centromeres and increase coincident with CENP-A loading in late anaphase/telophase. Further analysis is required to determine whether CAL1 and M18BP1/KNL2 function analogously in centromere assembly. It will be important to determine whether fly homologues of other Mis18 complex components are associated with CAL1 and important for centromere assembly. Identifying the APC substrates involved in centromere assembly will be necessary to distinguish between these models and to determine how these proteins epigenetically regulate centromere assembly and couple this essential process to the cell cycle (Erhardt, 2008).

Detrimental incorporation of excess Cenp-A/Cid and Cenp-C into Drosophila centromeres is prevented by limiting amounts of the bridging factor Cal1

Propagation of centromere identity during cell cycle progression in higher eukaryotes depends critically on the faithful incorporation of a centromere-specific histone H3 variant encoded by CENPA in humans and cid in Drosophila. Cenp-A/Cid is required for the recruitment of Cenp-C, another conserved centromere protein. With yeast three-hybrid experiments, this study demonstrates that the essential Drosophila centromere protein Cal1 can link Cenp-A/Cid and Cenp-C. Cenp-A/Cid and Cenp-C interact with the N- and C-terminal domains of Cal1, respectively. These Cal1 domains are sufficient for centromere localization and function, but only when linked together. Using quantitative in vivo imaging to determine protein copy numbers at centromeres and kinetochores, it was demonstrates that centromeric Cal1 levels are far lower than those of Cenp-A/Cid, Cenp-C and other conserved kinetochore components, which scale well with the number of kinetochore microtubules when comparing Drosophila with budding yeast. Rather than providing a stoichiometric link within the mitotic kinetochore, Cal1 limits centromeric deposition of Cenp-A/Cid and Cenp-C during exit from mitosis. The low amount of endogenous Cal1 prevents centromere expansion and mitotic kinetochore failure when Cenp-A/Cid and Cenp-C are present in excess (Schittenhelm, 2010).

The centromeric regions of chromosomes direct formation of kinetochores, which allow chromosome attachment to spindle microtubules. Centromeres and kinetochores are therefore of paramount importance for faithful propagation of genetic information. However, centromeric DNA sequences are not conserved. Most eukaryotes (including Drosophila melanogaster and humans) have regional centromeres with up to several megabases of repetitive DNA. Importantly, these repetitive sequences are neither necessary nor sufficient for centromere function, indicating that there is an epigenetic centromere specification (Schittenhelm, 2010).

A centromere-specific histone H3 variant (CenH3) is thought to be crucial for epigenetic centromere marking. CenH3 proteins are present in all eukaryotes (e.g. CENP-A in humans and Cid in Drosophila). They replace histone H3 in canonical nucleosomes or possibly variant complexes. Depletion of CenH3 results in a failure to localize most or all other centromere and mitosis-specific kinetochore proteins. Strong overexpression of Drosophila Cenp-A/Cid results in incorporation at ectopic chromosomal sites, which in part also assemble ectopic kinetochores during mitosis (Schittenhelm, 2010).

Ectopic kinetochores result in chromosome segregation errors and genetic instability. Ectopic CenH3 incorporation therefore must be prevented. Although still fragmentary, the understanding of the molecular mechanisms that regulate CenH3 incorporation is progressing rapidly. In proliferating cells, an additional complement of CenH3 needs to be incorporated during each cell cycle. In syncytial Drosophila embryos, this occurs during exit from mitosis. Similar findings were made in human cells, where Cenp-A deposition occurs during late telophase and early G1 phase. The number of factors shown to be required for normal CenH3 deposition is increasing rapidly, which suggests that there is an intricate control mechanism. Various and often dedicated chaperones, chromatin modifying and remodelling factors, as well as other centromere components are involved (Schittenhelm, 2010).

In Drosophila, Cenp-C is incorporated into centromeres concomitantly with Cenp-A/Cid. High-resolution mapping with native Drosophila chromosomes has indicated that these two proteins do not have an identical localization within the kinetochore. Although these localization studies cannot exclude an association between subfractions of Cenp-A and Cenp-C, direct molecular interactions between these centromere proteins have not yet been reported. Recently, however, Cal1 has been identified in Drosophila and shown to be required for normal centromeric localization of Cenp-A/Cid and Cenp-C. Moreover, these three Drosophila centromere proteins can be co-immunoprecipitated from soluble chromatin preparations. Cal1 might therefore provide a physical link between Cenp-A/Cid and Cenp-C (Schittenhelm, 2010).

This study reports that Cal1 has distinct binding sites for Cenp-A/Cid and Cenp-C. It can link these proteins together according to yeast three-hybrid experiments. However, the level of centromeric Cal1 is far lower than that of Cenp-A/Cid and Cenp-C. Cal1 therefore cannot function as a stoichiometric linker connecting each monomer or dimer of Cenp-C to Cenp-A within the centromere. But the low levels of Cal1 effectively protect cells against mitotic defects resulting from increased centromeric incorporation of excess Cenp-A/Cid and Cenp-C (Schittenhelm, 2010).

cal1 is an essential gene that is expressed specifically in mitotically proliferating cells. To provide its function, the protein product needs its N-terminal domain, which interacts with Cenp-A/Cid, as well as its C-terminal domain, which interacts with Cenp-C. By contrast, the most rapidly diverging middle region of Cal1 seems to be of lesser importance because expression of the N-C version, which lacks the M domain, is sufficient to prevent the characteristic defects in cal1 mutant embryos. The obvious functionality of the N-C version also emphasizes the importance of the centromeric localization of Cal1. The complete Cal1 protein is observed not only at the centromere, but also in the nucleolus. The M region is both sufficient and required for nucleolar localization. However, because this M domain is not required for cal1 mutant rescue, the significance of the nucleolar Cal1 localization remains unclear (Schittenhelm, 2010).

Rescue of cal1 mutants is not observed when the N- and C-terminal domains of Cal1 are expressed without a covalent linkage. The ability to recruit Cenp-A/Cid and Cenp-C into a complex, as clearly evidenced by yeast three-hybrid experiments, is therefore likely to be crucial for Cal1 function. Co-immunoprecipitation of Cal1, Cenp-A/Cid and Cenp-C has previously indicated that these components can associate in vivo. However, quantification of protein levels, which is largely dependent on the accuracy of EGFP signal quantifications, demonstrates that Cenp-C is not exclusively anchored to centromeric chromatin via persistent and stoichiometric Cal1-mediated links to Cenp-A/Cid. Centromeric Cal1 levels are more than 40-times lower than those of Cenp-A/Cid and Cenp-C (Schittenhelm, 2010).

The centromeric amount of Cal1 is also far lower than that of the other kinetochore components that have been quantified (Spc105, Spc25, Nuf2). Interestingly, per kinetochore, the copy numbers of these components appear to be scaling well with the number of kinetochore microtubules (kMTs) when comparing the current results from Drosophila with those described for budding and fission yeast. Spc25 and Nuf2 are constituents of the heterotetrameric Ndc80 complex, which binds directly to kinetochore microtubules (kMTs) (see Models of kinetochore assembly). Eight copies of the Ndc80 complex are thought to bind a single kMT to the budding yeast kinetochore. In Drosophila, where the number of kMTs per kinetochore appears to be around 11, about seven copies appear to be present per kMT according to the quantification. This quantification of kinetochore proteins fits very well with the notion that the kinetochores of higher eukaryotes might be composed of several copies of a module that is present in one copy in budding yeast. By contrast, the centromere proteins Cenp-A and Cenp-C are scaling less well with the number of kMTs. The increased complexity of lateral co-ordination within animal kinetochores and of epigenetic specification of centromere identity might explain the higher relative amount of centromere proteins apparent in Drosophila. Despite this relative increase, centromeric Cenp-A/Cid allows packaging of only about 5% of the centromeric DNA in Drosophila under the assumption that Cenp-A/Cid nucleosomes wrap about 200 bp of a 200 kb centromere (Schittenhelm, 2010).

Although these quantifications exclude the notion that Cal1 functions as a stable stoichiometric linker of Cenp-A/Cid and Cenp-C in mitotic kinetochores, the overexpression experiments provide further support for a role as a centromere protein-loading factor. Moreover, these experiments reveal additional layers of regulation that prevent excess incorporation of centromere proteins within the centromeric region. They also indicate that such excess incorporation is highly detrimental to kinetochore function. Previous work in Drosophila has demonstrated that strong overexpression of Cenp-A/Cid (about 70-fold) can lead to ectopic kinetochore formation. However, almost all Cenp-A/Cid that is incorporated ectopically within the chromosome arm regions is degraded rapidly, which is also observed in yeast. This study shows that the limiting amounts of Cal1 provide additional, highly efficient protection against excessive chromosomal incorporation of Cenp-A/Cid. After bypassing this protection by Cal1 overexpression, even low levels of Cenp-A/Cid overexpression (about 2.5-fold) result in increased incorporation into centromeres (about 1.6-fold). When, in addition to Cal1 and Cenp-A/Cid, Cenp-C is also mildly co-overexpressed (about 3.5-fold), the levels of centromeric Cenp-A/Cid are further increased (about 2-fold) along with those of Cal1 and Cenp-C. Importantly, co-overexpression of these centromere proteins resulted not only in increased centromeric levels, but also in severe mitotic defects (Schittenhelm, 2010).

Although other interpretations are not excluded, these findings strongly suggest that the mitotic defects observed after overexpression of Cal1 and Cenp-A/Cid, and even more strongly when Cenp-C was also overexpressed, reflect the consequence of the increase in the centromeric levels of these proteins. The increase in centromeric levels of centromere proteins was accompanied by a significant increase in kinetochore proteins (Spc105 and the Mis12 and Ndc80 complex) but only to a very limited extent and only when all three centromere proteins were co-expressed. The increased amounts of centromeric Cenp-A/Cid observed after co-expression of Cal1 and Cenp-A/Cid, which were not accompanied by a statistically significant increase in kinetochore protein levels, might therefore be sufficient to disturb the spatial organization of the kinetochore, leading to inefficient chromosome congression, spindle checkpoint hyperactivation and chromosome segregation defects in anaphase (Schittenhelm, 2010).

Experiments in stg mutant embryos, demonstrate that co-overexpression of centromeric proteins during interphase is not sufficient to cause excess centromeric incorporation, consistent with the previously demonstrated dependence of centromeric deposition of Cenp-A/Cid and Cenp-C on exit from mitosis. Indeed, forcing progression through mitosis (by hs-stg induction) was observed to be sufficient to cause centromeric deposition of the overexpressed proteins. Moreover, the fact that the excess centromere proteins that were not yet incorporated into the centromere did not disturb the hs-stg induced mitosis, further supports the suggestion that the mitotic defects observed after co-expression of centromeric proteins depend on excessive incorporation into the centromere (Schittenhelm, 2010).

The severe mitotic defects observed after co-overexpression of Cal1, Cenp-A/Cid and Cenp-C emphasize the importance of careful control of centromere protein deposition. Several levels of control are effective. The interdependence of Cal1, Cenp-A/Cid and Cenp-C functions in conjunction with cell cycle control to prevent detrimental excessive centromeric incorporation. The cell cycle regulators cyclin A, Rca1/Emi1 and Fzr/Cdh1 have recently been implicated in the control of deposition of Cenp-A/Cid and Cenp-C at the centromere. How these and possibly additional cell cycle regulators control centromere protein deposition has yet to be clarified (Schittenhelm, 2010).

A possible scenario for centromere protein deposition in Drosophila might include a release of nucleolar Cal1 at the onset of mitosis, followed by conversion into a form that associates with non-centromeric soluble Cenp-A/Cid during exit from mitosis. After binding of soluble Cenp-A/Cid to the N-terminal domain of Cal1, its C-terminal domain might become exposed so that it can bind to centromeric Cenp-C and promote Cenp-A/Cid transfer onto the neighboring centromeric chromatin and thereby indirectly also additional Cenp-C deposition (Schittenhelm, 2010).

The mechanisms and the extent of control of centromeric Cenp-A deposition appear to have evolved. In fission yeast, overexpression of Cenp-A/Cid alone is sufficient to obtain excess centromeric Cenp-A/Cnp1, and this excess does not result in increased kinetochore protein levels. Spreading of Cenp-A within centromeric chromatin has also been clearly demonstrated in human cells after mild overexpression of Cenp-A. Mitotic defects were not detected in this case, perhaps because of the very limited increase in centromeric Cenp-A. Cal1 homologs from non-Drosophilid genomes have not yet been identified so far. Conversely, with the exception of Cenp-C, homologs of the 15 components of the vertebrate centromere chromatin-associated network (CCAN), which is related to the yeast Ctf19 and Sim4 complexes, have not been revealed in Drosophilid genomes, neither by thorough bioinformatic analyses nor by genome-wide RNAi screens. The CCAN seems also to be absent in C. elegans. It is conceivable therefore that Cal1 is a functional analog of the CCAN, which has also been implicated in Cenp-A loading. However, because the evolutionary sequence conservation of centromere and kinetochore components is generally very low, it remains a possibility that Cal1 homologs also exist and function in centromere loading of human Cenp-A and Cenp-C (Schittenhelm, 2010).

Assembly of Drosophila centromeric chromatin proteins during mitosis

Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. This study analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and Chromosome alignment defect 1 (CAL1), which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation (Mellone, 2011).

CID, CAL1 and CENP-C display different turnover and assembly dynamics, despite the fact that these essential centromeric components interact physically, and are interdependent for centromere localization. Epitope tagged SNAP-CID, SNAP-CAL1 and SNAP-CENP-C were expressed from the identical Copia promoter; thus it is unlikely that these distinctions are due to different rates of new protein synthesis. Using a pulse-chase strategy, it was shown that CID levels are reduced by ~50% after one cell cycle, which could result from semi-conservative distribution of pre-existing CID nucleosomes, or random redistribution of parental CID-H4 tetramers, to replicated sister chromatids. While CID and CENP-C display stable association with centromeres and 50:50 distribution after each cell cycle, 66% of TMR-CAL1 is replaced by new protein. Thus, CAL1 is either less stably bound, or its replenishment involves partial removal of pre-existing protein. Alternatively, CAL1 could undergo an even higher turnover and the quantification could be an underestimation; CAL1 could be entirely recruited de novo and the measured centromeric TMR-CAL1 could reflect recruitment from an initial soluble pool at the time of labeling (Mellone, 2011).

An additional difference is that while SNAP-CID and CAL1 are detectable at centromeres 1 h after quenching the SNAP epitopes, 10 h of chase time are necessary for CENP-C to be visible by fluorescent substrate tetramethylrhodamine (TMR) labeling. This suggests that at each cell cycle the recruitment of CID and CAL1 relies for the most part on newly-synthesized protein, while CENP-C recruitment also involves a pre-existing non-centromeric or soluble pool. Indeed, the cellular fractionation analysis demonstrated the presence of low levels of CENP-C in chromatin-free extracts, supporting the possibility that there is a soluble pool of CENP-C available to replenish the centromere-associated CENP-C diluted during the cell cycle (Mellone, 2011).

CENP-C is targeted to centromeres during multiple cell cycle stages, consistent with previous findings in human cells. In contrast, newly-synthesized CAL1 and CID are recruited to centromeres during discrete stages of mitosis. Using quench-chase-pulse time-courses in both asynchronous and arrested cultures, it was demonstrated that the contribution of interphase to CID loading is minimal, since the percent of interphase cells displaying newly-synthesized SNAP-CID and the signal intensity of TMR-CID differ dramatically from those measured for mitotic cells. These observations distinguish Drosophila from human HeLa cells, where CENP-A is recruited during G1, from fission yeast, where CENP-A assembles at centromeres in both S and G2 phases, as well as from plants and Dictyostelium (G2/prophase) (Mellone, 2011).

Both new CID and CAL1 are assembled at centromeres in mitosis, but each protein is recruited during discrete stages: prophase for CAL1 and metaphase for CID. It is possible that CID and CAL1 loading are initiated simultaneously in prophase, but CAL1 levels accumulate faster than CID at centromeres. Regardless, the observed temporal distinction suggests that CAL1 acts upstream of CID recruitment (see Model for centromere assembly in Drosophila cells.). Incorporation of nascent CAL1 at centromeres during prophase could be mediated by binding to pre-existing centromeric CID and CENP-C. This could in turn promote new incorporation of nascent CID during metaphase, either by gap-filling or exchange of space-holder histone H3 (Mellone, 2011).

Interestingly, a similar temporal distinction has been described for the human centromere proteins hMis18α, β and M18BP, which localize to centromeres in anaphase, before new CENP-A assembly in late telophase/G1. The lack of any physical interaction between hMis18α, β, M18BP and CENP-A, and the observation that hMis18α can localize to centromeres even if CENP-A is depleted, has led to the proposal that this complex may 'prime' centromeres to receive new CENP-A (Fujita, 2007) from the HJURP chaperone, whose centromeric targeting coincides temporally with deposition of new CENP-A. Homologs for hMis18 complex components and HJURP (or the budding and fission yeast Scm3 homologs) have not been identified in the Drosophila genome (Mellone, 2011).

Collectively these data support a model in which CAL1 performs functions attributable to both HJURP and hMis18, despite the lack of sequence homology. hMis18 proteins are recruited to centromeres before CENP-A, and CAL1 loading precedes CID assembly. However, the hMis18 complex does not interact with CENP-A, whereas CAL1 and CID are associated in chromatin-free extracts, identifying the first Drosophila protein that binds CID in its pre-nucleosomal form. HJURP also interacts with pre-nucleosomal CENP-A, and both HJURP and CAL1 strongly colocalize with nucleoli. Thus, CAL1 could 'prime' the centromere in prophase, and also mediate CID recruitment directly in metaphase (Mellone, 2011 and references therein).

It has been showm that gross-levels of centromeric GFP-CID and GFP-CENP-C did not visibly change through the cell cycle in time-lapse analysis, consistent with the 50:50 segregation observed in this study during one division. In contrast, GFP-CAL1 levels were significantly reduced in metaphase, increased again in telophase, and remained stable through interphase. The transient reduction in GFP-CAL1 levels at metaphase is intriguing, given that it coincides with new CID assembly. The observation that newly assembled TMR-CAL1 intensities were constant from prophase to cytokinesis suggests that most of the GFP-CAL1 reduction at metaphase and increase at telophase involves pre-existing protein. One model to account for these observations is that free CAL1 (not bound to CID) is recruited to centromeres in prophase where it performs a yet undefined ‘priming’ function; then, the subset of CAL1 bound to pre-nucleosomal CID escorts it to centromeres in metaphase while 'old' CAL1 is displaced (Model for centromere assembly in Drosophila cells.). The interdependency of CAL1, CID and CENP-C in centromere localization could be explained by the requirement of pre-existing CID and CENP-C for CAL1 assembly in prophase (Mellone, 2011).

The loading of CID and CAL1 in specific, early stages of mitosis also raises questions about the nature of the signal(s) that initiate assembly of centromeric chromatin. Centromere replenishment signaling by kinetochore-microtubule interactions is inconsistent with the demonstration that CID loading in metaphase is not affected by colchicine treatment, and therefore does not require spindles (as also observed in human cells), SAC inactivation, chromosome segregation, or inter-kinetochore tension. However, it has been shown that premature activation of the Anaphase Promoting Complex, by Cyclin A or RCA1 depletion, interferes with CID localization to centromeres, demonstrating that centromeric chromatin assembly is linked to key regulators of mitotic progression. Interestingly, Cyclin A localizes to centromeres and is degraded in metaphase; this study has demonstrated that metaphase loading depends on proteasome activity, which could include degradation of key mitotic regulators. MG132 treatment prior to BTP block prevented CID loading while transfecting cells with a non-degradable form of CYCA abrogated new CID recruitment in a subset of cells, and TMR-CID levels were significantly reduced in most cells. One possibility to explain the stronger impact of proteasome inhibition is that proteasome targets in addition to CYCA need to be degraded for efficient CID deposition. Alternatively, the presence of centromeric endogenous CYCA, which is probably degraded normally in the presence of excess ND-CYCA, might trigger a sufficient signal to initiate CID incorporation in some cells. Interestingly, Cyclin A is degraded in the presence of microtubule drugs and escapes inhibition of the APC by the SAC, which would explain why new CID recruitment takes place efficiently in the presence of colchicine. Proteasome and ubiquitin-ligase activities have been implicated in controlling proper CENP-A centromeric incorporation by degradation of euchromatic CENP-A in budding yeast and Drosophila. Understanding the relationship between the CENP-A degradation pathway and the implication of proteasome activity in the recruitment of nascent CENP-A will require further investigation (Mellone, 2011).

It is unclear at this point how degradation of CYCA contributes to CID assembly. One possibility is that high CDK-CYCA activity at the centromere inhibits CID recruitment, and that local inhibition of CDK activity through degradation of CYCA or other substrates triggers CID assembly. Understanding the role of degradation of Cyclin A and other APC and proteasome substrates in CID recruitment will be crucial to elucidating how centromere assembly is coupled to the cell cycle (Mellone, 2011).

The dynamics of centromere replenishment in Drosophila cultured cells differs from those observed in S. pombe and human HeLa cells. Early syncytial fly embryos display slightly later recruitment of new CID in anaphase, but this difference could be due to the unusually short nuclear cycles that lack G1 and G2 phases. Although CENP-A loading in HeLa cells is first observed in telophase, it is possible that the primary signal to initiate CENP-A loading (e.g. inhibiting local CDK-CYCA activity at the centromere) is conserved, and occurs during prophase or metaphase in both Drosophila and human cells (Mellone, 2011).

It is also puzzling that key proteins required in trans for CENP-A assembly, such as HJURP and CAL1, are not always conserved, in contrast to the universality of centromeric chromatin components such as CENP-A and CENP-C. It is possible that highly diverged proteins, such as CAL1, perform the same function(s) as human regulators such as HJURP and hMis18. Thus, although this data challenges the universality of centromere propagation dynamics in metazoans, it will be important to determine whether some mechanisms and signals required for CENP-A replenishment are conserved, despite different times of assembly in the cell cycle, and the lack of conservation for key regulatory proteins (Mellone, 2011).

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 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 (Yang, 1996; Politi, 2002), 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).

CENP-C is a structural platform for kinetochore assembly

Centromeres provide a region of chromatin upon which kinetochores are assembled in mitosis. Centromeric protein C (CENP-C) is a core component of this centromeric chromatin that, when depleted, prevents the proper formation of both centromeres and kinetochores. CENP-C localizes to centromeres throughout the cell cycle via its C-terminal part, whereas its N-terminal part appears necessary for recruitment of some but not all components of the Mis12 complex of the kinetochore. This study has found that all kinetochore proteins belonging to the KMN (KNL1/Spc105, the Mis12 complex, and the Ndc80 complex) network bind to the N-terminal part of Drosophila CENP-C. Moreover, the Mis12 complex component Nnf1 was shown to interact directly with CENP-C in vitro. To test whether CENP-C's N-terminal part was sufficient to recruit KMN proteins, it was targeted to the centrosome by fusing it to a domain of Plk4 kinase. The Mis12 and Ndc80 complexes and Spc105 protein were then all recruited to centrosomes at the expense of centromeres, leading to mitotic abnormalities typical of cells with defective kinetochores. Thus, the N-terminal part of Drosophila CENP-C is sufficient to recruit core kinetochore components and acts as the principal linkage between centromere and kinetochore during mitosis (Przewloka, 2011).

Centromeres are patches of chromatin defined by the presence of the specific histone H3 variant called centromeric protein A (CENP-A)/CenH3, also known as CID in Drosophila, Cse4 in S. cerevisiae, and Cnp1 in S. pombe. Shortly before each mitosis, kinetochores are assembled on centromeres in a process whose mechanism and regulation are largely unknown (Przewloka, 2011).

Recently published studies have pointed toward the centromeric protein CENP-C as a potential candidate for the regulatory role in kinetochore formation. CENP-C is localized at the centromeric chromatin throughout the cell cycle. It is embedded in the centromeric chromatin and, together with CENP-A, forms part of the core of the centromere. The carboxy-terminal part of CENP-C is known to be responsible for the binding to centromeres but by itself is insufficient to rescue the lack of CENP-C in vertebrate cells or Drosophila embryos. However, the exact function of CENP-C’s N-terminal part has not been defined. In vertebrates, CENP-C is also a component of the so-called constitutive centromere-associated network (CCAN) that comprises at least 14 proteins assembled in several subcomplexes (Przewloka, 2011).

Whereas CENP-C seems to be a component of the centromere in all organisms, the associated CCAN proteins appear to be absent from the centromeres in C. elegans and Drosophila. Because these organisms thus provide a fundamentally simplified centromere whose functions may be easier to dissect, the roles of CENP-C in kinetochore assembly were studied using D. melanogaster as a model (Przewloka, 2011).

In order to test the localization properties of the two functional parts of CENP-C, the open reading frame was arbitrarily divided, and both portions were expressed in Drosophila Dmel-2 culture cells. Stably transformed cell lines expressing a transgene encoding a fusion of EGFP to the C-terminal 623 amino acids of the 1411 amino acid CENP-C (which are referred to as EGFP::CENPC-C) were generated (Przewloka, 2011).

This transgene was under the control of the inducible metallothionein promoter to avoid potential dominant-negative phenotypes that might arise from constitutive expression. As expected, this CENPC-C fusion colocalized with the centromeric marker CENP-A/CID during the entire cell cycle. In contrast, a fusion of EGFP with the N-terminal 788 amino acids of CENP-C, EGFP::CENPC-N, localized to nuclei but not to centromeres when expressed from the metallothionein promoter. This suggests that this half of the protein must be transported actively into the nucleus, although a nuclear localization signal within this part was not identified previously. Moreover, its lack of centromeric localization suggests that this part of CENP-C is incapable of either dimerizing with full-length endogenous protein or interacting with other centromeric components (Przewloka, 2011).

This raised the question of what other proteins the N-terminal part of CENP-C might interact with. To address this, a stably transformed Drosophila cell line was built able to express a fusion of protein A::CENPC-N from the metallothionein promoter. After overnight induction of protein A::CENPC-N expression, the product of the transgene was affinity purified from cell extracts using IgG-coupled magnetic beads for analysis by mass spectrometry. No peptides from the centromeric proteins CENP-A/CID [18], CAL1 [19], or the C-terminal part of CENP-C were identified, in accordance with the observation that CENPC-N does not associate with centromeres. Surprisingly, however, it was possible to identify the entire set of kinetochore proteins belonging to theKMN (KNL1/Spc105, theMis12 complex, and the Ndc80 complex) network, including Spc105, Mis12, Nnf1, Spc25/Mitch, Nuf2, and Ndc80. In addition, proteins were identified belonging to the 14-3-3 family with high Mascot scores. The high levels of phosphorylation of CENP-C in cultured Drosophila cells and in Drosophila embryos would accord with being able to associate with 14-3-3 proteins that interact with phosphorylated targets.None of the abovementioned proteins copurified with protein A alone (Przewloka, 2011).

Because it was not possible to find any good candidate proteins that might provide a bridge between CENPC-N and KMN components, it was speculated that the interaction might be a direct one involving the innermost part of the kinetochore, the Mis12 complex. To test this possibility, the 35S-labeled Mis12 complex subunits Nnf1a and Nsl1 and also maltose-binding protein (MBP) as a negative control were synthesized in an in vitro transcription- translation system and whether they would interact with bacterially expressed CENP-C fragments was investigated. (Mis12 appeared to be either insoluble or very poorly expressed in this system, and hence its interaction could not be tested.) No binding of MBP, Nnf1a, or Nsl1 to GST::CENPC-C was detected. However, binding of Nnf1a to the N-terminal part of CENP-C was detected, whereas Nsl1 binding was poorly detectable. This suggests that this component of the Mis12 complex may be responsible for the association of the KMN network with CENP-C. It would accord with a recent study (Petrovic, 2010) that places Nnf1, of all the Mis12 complex components, as lying most proximal to the centromeric chromatin (Przewloka, 2011).

The finding that the N-terminal part of CENP-C can associate with all proteins of the KMN network led to a speculation about its potential role in kinetochore assembly and a test whether it would be sufficient to recruit the KMN network and other kinetochore proteins to ectopic sites. This part of CENP-C was targeted to centrosomes and whether the KMN network would be recruited to the same site was tested. To this end, a centrosome-targeting domain from Plk4 kinase was fused in frame with the EGFP::CENPC-N construct (this entire construct was termed ect-KTR₁, for ectopic kinetochore recruitment). As a control, the localization and recruitment capabilities were tested of the Plk4 centrosome localization domain (CLD) alone fused with EGFP. Expression of either construct in Dmel-2 cells from an inducible promoter resulted in the localization of EGFP at the centrosome, confirmed by costaining with the centrosomal markers dPLP/Cp309 (the Drosophila counterpart of pericentrin) and dSpd2/Cep192. The ect-KTR₁ fusion protein also localized to the nuclei of a substantial subset of cells during interphase, most likely reflecting the activity of its own strong nuclear localization signal. However, focus was placed on localization of these proteins in mitotic cells when kinetochores have normally assembled at the centromeres, thus allowing examination of the extent to which kinetochore proteins localized to either centromeres or centrosomes (Przewloka, 2011).

Because expression of either CENPC-N or ect-KTR caused frequent and severe mitotic phenotypes in which chromosomes became scattered so as often to lie in the vicinity of the poles, focus was placed on examining the localization of kinetochore proteins in cells where centrosomes and centromeres were well separated (Przewloka, 2011).

Cells were stained with several antibodies raised against kinetochore proteins. Expression of the CLD alone did not lead to any change of the localization of kinetochore markers. Kinetochore proteins belonging to the KMN network colocalized exclusively with CENP-A/CID in such cells. Expression of ect-KTR, on the other hand, led to the strong recruitment of all KMN network components tested to the centrosome and the frequent diminution of the levels of these proteins at the kinetochore. Thus, immunostaining clearly revealed Mis12, Nsl1, Nnf1a, and Spc105 to colocalize with ect-KTR and the centrosomal markers dPlp and Spd2. To determine the localization of the outer kinetochore plate Ndc80 complex, Dmel-2 cells were cotransfected with constructs expressing RFP-tagged ect-KTR and either EGFP-tagged Ndc80 or Nuf2. It was found in both cases that these components of the Ndc80 complex localized to centrosomes in ect-KTR-expressing cells. In contrast, neither of the centromeric markers CAL1 or CENP-A/CID colocalized with ect-KTR; they were found only at centromeres. Thus, the N-terminal part of CENP-C is not able to interact with centromeric proteins but is able to recruit the KMN network to an ectopic site in the cell (Przewloka, 2011).

It was also asked whether, in addition to the core KMN network proteins, other ancillary proteins known to associate temporarily with kinetochores might be relocated onto ectopically positioned CENPC-N. The localization was examined of the kinetochore-associated motor protein CENP-meta (the Drosophila counterpart of CENP-E) and the spindle assembly checkpoint proteins Mad2 and BubR1. It was found that, unlike the core KMN network proteins that were associated with centrosomes in all cells expressing ect-KTR, ancillary kinetochore proteins were only associated with centrosomes in a subset of ect-KTR-expressing cells. Thus, CENP-meta was predominantly centrosome associated in 78.8% of ect-KTR cells. Mad2 also showed similar localization to ectopic sites regardless of whether or not the checkpoint was activated by colchicine treatment. No strong signals of Mad2 were seen on scattered chromosomes in ect-KTR cells. This could be due to, for example, either depletion of a proper platform for binding of Mad2 on kinetochores such as the Ndc80 complex or the titration of Mad2 to centrosomes. This observation supports the idea that Mad2 binds to kinetochores, not centromeres. On the contrary, in the case of BubR1, its association with centrosomes in a subset of ect-KTR-expressing cells was much weaker than its association with centromeres in the same cells. This is in agreement with suggestions that BubR1 may have at least two independent binding sites: one on centromeres, which might be more exposed in cells expressing ect-KTR, and the other on kinetochores, which in cells expressing ect-KTR would be associated with centrosomes. In fact, this result supports the idea of BubR1 being recruited to centromeres rather than kinetochores. Because none of these ancillary kinetochore proteins associated with centrosomes in CLD-expressing cells, it is concluded that the ectopic localization of the N-terminal part of CENP-C was responsible for localizing the core KMN network to centrosomes, and that this then permitted recruitment of other kinetochore-associated proteins (Przewloka, 2011).

As indicated above, it was found frequent mitotic defects in cells expressing CENPC-N and ect-KTR, of which improperly congressed and scattered chromosomes were the most commonly observed aberrations. The scattering of chromosomes on elongated spindles was reminiscent of the phenotype observed after knockdown of components of the KMN network. It was observed that whereas spindles in CLD-expressing cells had a mean length of 6.83 that is typical of the parent Dmel-2 cell line, spindles in ect-KTR and CENPC-N cells were significantly elongated and had mean lengths of 8.7 and 8.04, respectively. To determine whether cells with elongated spindles were in anaphase, cells were costained with anti-CENP-A/CID and anti-cyclin B antibodies. This revealed that ect-KTR cells showing the mitotic phenotype had high levels of cyclin B and that the chromosomes had two centromeres and so were conjoined chromatids (Przewloka, 2011).

This strongly suggests that these cells were blocked in prometaphase with improperly congressed chromosomes and had not entered anaphase. Therefore, the chromosome scattering and the spindle elongation phenotype may be a consequence of the very long prometaphase block occurring as a result of the primary defect: severe congression problems (Przewloka, 2011).

It was hypothesized that the high incidence of mitotic defects present after ect-KTR or CENPC-N expression was caused by the titration out of one or more KMN network proteins by the ectopic construct. This interpretation was supported by the observation that immunolocalization signals of kinetochore proteins ectopically localized at centrosomes were usually stronger than the signals at centromeres in ect-KTR cells. To address this directly, fluorescence intensities of endogenous kinetochores were quantified after staining with anti-Mis12 and anti-Spc105 antibodies in relation to the intensities of the centromeric CENP-A/CID in CLD-, ect-KTR-, and CENPC-N-expressing cells. In ect-KTR- and CENPC-N-expressing cells, Mis12 levels at the kinetochore were reduced respectively to an average of 46% and 26.8% of the level in cells expressing the CLD construct alone. Similarly, levels of Spc105 were reduced to 29.2% and to 39.1% in ect-KTR and CENPC-N cells respectively. Thus, it is concluded that overexpression of ect-KTR or nontargeted CENPC-N indeed results in a depletion of kinetochore proteins from endogenous kinetochores, and that this in turn leads to mitotic defects (Przewloka, 2011).

Although the vast majority of CLD-expressing cells looked normal, a low level of background aberrations were noticed that may be attributable to the expression of the Plk4 fragment, which is able to drive production of extra centrosomes and which may affect the efficiency of spindle assembly. However, the finding of chromosomes scattered along elongated spindles was unique to CENPC-N- or ect- KTR-expressing cells and was not seen in CLD-expressing cells. This strong dominant-negative phenotype and the ectopic localization pattern of KMN network proteins in cells expressing ect-KTR lead to the unequivocal conclusion that the N-terminal domain of CENP-C is sufficient to recruit all of the major core kinetochore proteins, regardless of its localization in the cell (Przewloka, 2011).

Recent studies from a number of laboratories have provided a detailed description of the interaction network between subunits of the KMN supercomplex. This knowledge is key for reaching a full understanding of how kinetochores are formed early in mitosis and how they interact with microtubules of the mitotic spindle. Considerable progress has also been made in studies of centromere inheritance, composition, and function (Przewloka, 2011).

However, little is known yet about the interaction between these two complex structures. Previously, it was reported that kinetochore formation in Drosophila cells is absolutely dependent upon the presence of the centromeric protein CENP-C. The results presented in this study indicate that it is the N-terminal part of this protein that not only is responsible for binding to the KMN network but also is sufficient to recruit it to ectopic sites (Przewloka, 2011).

The consequential depletion of KMN proteins from the centromeres of mitotic chromosomes to centrosomes leads to strong mitotic defects. Together, these data support a model in which one face of CENP-C binds CENP-A-rich centromeric chromatin and the other binds components of the KMN network, providing a platform for kinetochore assembly at the onset of mitosis. That such a function of CENP-C may be conserved is strongly suggested from a contemporaneous study of its interacting partners in human cells (Screpanti, 2011). The length of the CENP-C molecule, the bifunctionality of its binding, and its potential for posttranslational modification together suggest that this protein might not only provide structural support for kinetochore assembly but also be a major hub for regulating centromere and kinetochore function. It will be important to learn more about such potential regulatory mechanisms, and also to determine whether CENP-C is the only component of the centromere to provide an interface between centromeres and kinetochores in either Drosophila or vertebrates (Przewloka, 2011).

CENP-C functions as a scaffold for effectors with essential kinetochore functions in mitosis and meiosis

The conserved kinetochore protein CENP-C plays a fundamental role in chromosome segregation, but its specific functions remain elusive. This study has gained insights into the role of CENP-C through identification of interacting effector proteins required for kinetochore function in fission yeast. Fta1/CENP-L is a primary effector that associates directly with Cnp3/CENP-C, and ectopic localization of Fta1 largely suppresses the mitotic kinetochore defects of cnp3Delta cells. Pcs1 functions downstream of Cnp3 to prevent merotelic attachment. In meiosis, Cnp3 further associates with and recruits Moa1, a meiosis-specific protein exclusively required for the mono-orientation of kinetochores. Genetic and biochemical analyses identified Cnp3 mutants that preserve intact mitotic kinetochore function but abolish the association with Moa1 and meiotic mono-orientation. Overall, therefore, these studies identify effectors of CENP-C in mitosis and meiosis and establish the concept that CENP-C serves as a scaffold for the specific recruitment of essential kinetochore proteins (Tanaka, 2011).

Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore

Kinetochores are proteinaceous scaffolds implicated in the formation of load-bearing attachments of chromosomes to microtubules during mitosis. Kinetochores contain distinct chromatin- and microtubule-binding interfaces, generally defined as the inner and outer kinetochore, respectively. The constitutive centromere-associated network (CCAN) and the Knl1-Mis12-Ndc80 complexes (KMN) network are the main multisubunit protein assemblies in the inner and outer kinetochore, respectively. The point of contact between the CCAN and the KMN network is unknown. Cenp-C is a conserved CCAN component whose central and C-terminal regions have been implicated in chromatin binding and dimerization. This study shows that a conserved motif in the N-terminal region of Cenp-C binds directly and with high affinity to the Mis12 complex. Expression in HeLa cells of the isolated N-terminal motif of Cenp-C prevents outer kinetochore assembly, causing chromosome missegregation. The KMN network is also responsible for kinetochore recruitment of the components of the spindle assembly checkpoint, and checkpoint impairment was observed in cells expressing the Cenp-C N-terminal segment. These studies unveil a crucial and likely universal link between the inner and outer kinetochore (Screpanti, 2011).

Search PubMed for articles about Drosophila Cenp-C

Blower, M. D., Sullivan, B. A. and Karpen, G. H (2002) Conserved organization of centromeric chromatin in flies and humans. Dev Cell 2: 319-330. Medline abstract: 11879637

Brown, M. T., Goetsch, L., and Hartwell, L. H. (1993). MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae. J. Cell Biol. 123: 387-403. Medline abstract: 8408221

Cheeseman, I. M., Chappie, J. S., Wilson-Kubalek, E. M. and Desai, A. (2006). The conserved KMN network constitutes the core microtubule-binding site of the kinetochore. Cell 127: 983-997. Medline abstract: 17129783

Ciferri, C., et al. (2005). Architecture of the human ndc80-hec1 complex, a critical constituent of the outer kinetochore. J. Biol. Chem. 280: 29088-29095. Medline abstract: 15961401

Deluca, J. G., et al. (2006). Kinetochore microtubule dynamics and attachment stability are regulated by hec1. Cell 127: 969-982. Medline abstract: 17129782

Erhardt, S., et al. (2008). Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation. J. Cell Biol. 183: 805-818. PubMed Citation: 19047461

Foltz, D. R., et al. (2006) The human CENP-A centromeric nucleosome-associated complex. Nat Cell Biol 8: 458-469. Medline abstract: 16622419

Fukagawa, T. and Brown, W.R. (1997). Efficient conditional mutation of the vertebrate CENP-C gene. Hum. Mol. Genet. 6: 2301-2308. Medline abstract: 9361037

Fukagawa, T., Pendon, C., Morris, J., and Brown, W. (1999). CENP-C is necessary but not sufficient to induce formation of a functional centromere. EMBO J. 18: 4196-4209. Medline abstract: 10428958

Fukagawa, T., Regnier, V., and Ikemura, T. (2001). Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells. Nucleic Acids Res. 29: 3796-3803. Medline abstract: 11557811

Heeger, S., et al. (2005). Genetic interactions of Separase regulatory subunits reveal the diverged Drosophila Cenp-C homolog. Genes Dev 19: 2041-2053. Medline abstract: 16140985

Izuta, H., et al. (2006). Comprehensive analysis of the ICEN (interphase centromere complex) components enriched in the CENP-A chromatin of human cells. Genes Cells 11: 673-684. Medline abstract: 16716197

Kalitsis, P., Fowler, K. J., Earle, E., Hill, J. and Choo, K. H. (1998). Targeted disruption of mouse centromere protein C gene leads to mitotic disarray and early embryo death. Proc. Natl. Acad. Sci. 95(3): 1136-41. PubMed citation: 9448298

Klein, U. R., Nigg, E. A. and Gruneberg, U. (2006). Centromere targeting of the chromosomal passenger complex requires a ternary subcomplex of Borealin, Survivin, and the N-terminal domain of INCENP. Mol. Biol. Cell 17(6): 2547-58. Medline abstract: 16571674

Kline, S. L., Cheeseman, I. M., Hori, T., Fukagawa, T. and Desai, A. (2006). The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation. J. Cell Biol 173: 9-17. Medline abstract: 16585270

Kwon, M. S., Hori, T., Okada, M., and Fukagawa, T. (2007). CENP-C is involved in chromosome segregation, mitotic checkpoint function, and kinetochore assembly. Mol. Biol. Cell 18: 2155-2168. Medline abstract: 17392512

Maiato, H., et al. (2006). The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells. Chromosoma 115: 469-480. Medline abstract: 16909258

Mellone, B. G., et al. (2011). Assembly of Drosophila centromeric chromatin proteins during mitosis. PLoS Genet. 7(5): e1002068. PubMed Citation: 21589899

Meluh, P.B. and Koshland, D. (1995). Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol. Biol. Cell 6: 793-807. Medline abstract: 7579695

Moore, L. L. and Roth, M. B. (2001). HCP-4, a CENP-C-like protein in Caenorhabditis elegans, is required for resolution of sister centromeres. J. Cell Biol. 153: 1199-1208. Medline abstract: 11402064

Oegema, K., Desai, A., Rybina, S., Kirkham, M., and Hyman, A. A. (2001). Functional analysis of kinetochore assembly in Caenorhabditis elegans. J. Cell Biol. 153: 1209-1226. Medline abstract: 11402065

Okada, M,. et al. (2006). The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. Nat Cell Biol 8: 446-457. Medline abstract: 16622420

Petrovic, A., et al. (2010). The MIS12 complex is a protein interaction hub for outer kinetochore assembly. J. Cell Biol. 190: 835-852. PubMed Citation: 20819937

Politi, V., Perini, G., Trazzi, S., Pliss, A., Raska, I., Earnshaw, W.C., and Della Valle, G. (2002). CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains. J. Cell Sci. 115: 2317-2327. Medline abstract: 12006616

Przewloka, M. R., et al. (2011). CENP-C is a structural platform for kinetochore assembly. Curr. Biol. 21(5): 399-405. PubMed Citation: 21353555

Schittenhelm, R. B., et al. (2007). Spatial organization of a ubiquitous eukaryotic kinetochore protein network in Drosophila chromosomes. Chromosoma 116(4): 385-402. Medline abstract: 17333235

Schittenhelm, R. B., Althoff, F., Heidmann, S. and Lehner, C. F. (2010). Detrimental incorporation of excess Cenp-A/Cid and Cenp-C into Drosophila centromeres is prevented by limiting amounts of the bridging factor Cal1. J. Cell Sci. 123(Pt 21): 3768-79. PubMed Citation: 20940262

Screpanti, E., De Antoni, A., Alushin, G.M., Petrovic, A., Melis, T., Nogales, E. and Musacchio, A. (2011). Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore. Curr. Biol. 21(5): 391-8. PubMed Citation: 21353556

Talbert, P. B., Bryson, T. D., and Henikoff, S. (2004). Adaptive evolution of centromere proteins in plants and animals. J. Biol. 3: 18. Medline abstract: 15345035

Tanaka, K., Chang, H. L., Kagami, A. and Watanabe, Y. (2009). CENP-C functions as a scaffold for effectors with essential kinetochore functions in mitosis and meiosis. Dev. Cell 17(3): 334-43. PubMed Citation: 19758558

Tomkiel, J., Cooke, C. A., Saitoh, H., Bernat, R. L., and Earnshaw, W. C. (1994). CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase. J. Cell Biol. 125: 531-545. Medline abstract: 8175879

Vos, L. J., Famulski, J. K. and Chan, G. K. (2006). How to build a centromere: from centromeric and pericentromeric chromatin to kinetochore assembly. Biochem Cell Biol 84: 619-639. Medline abstract: 16936833

Wei, R. R., Sorger, P. K. and Harrison, S. C. (2005). Molecular organization of the Ndc80 complex, an essential kinetochore component. Proc. Natl. Acad. Sci. 102: 5363-5367. Medline abstract: 15809444

Wong, L. H., et al. (2007). Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere. Genome Res. 17(8): 1146-60. Medline abstract: 17623812

Yang, C. H., Tomkiel, J., Saitoh, H., Johnson, D. H. and Earnshaw, W. C. (1996). Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C. Mol. Cell. Biol. 16: 3576-3586. Medline abstract: 8668174

date revised: 25 October 2011

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