Kinetochore-microtubule Interactions Research Articles

Slk19 clusters kinetochores and facilitates chromosome bipolar attachment

In all eukaryotic cells, DNA is packaged into multiple chromosomes that are linked to microtubules through a large protein complex called a kinetochore. Previous data show that the kinetochores are clustered together during most of the cell cycle, but the mechanism and the biological significance of kinetochore clustering are unknown. As a kinetochore protein in budding yeast, the role of Slk19 in the stability of the anaphase spindle has been well studied, but its function in chromosome segregation has remained elusive. Here we show that Slk19 is required for kinetochore clustering when yeast cells are treated with the microtubule-depolymerizing agent nocodazole. We further find that slk19Δ mutant cells exhibit delayed kinetochore capture and chromosome bipolar attachment after the disruption of the kinetochore-microtubule interaction by nocodazole, which is likely attributed to defective kinetochore clustering. In addition, we show that Slk19 interacts with itself, suggesting that the dimerization of Slk19 may mediate the interaction between kinetochores for clustering. Therefore Slk19 likely acts as kinetochore glue that clusters kinetochores to facilitate efficient and faithful chromosome segregation.

Direct binding between BubR1 and B56–PP2A phosphatase complexes regulate mitotic progression

BubR1 is a central component of the spindle assembly checkpoint that inhibits progression into anaphase in response to improper kinetochore-microtubule interactions. In addition, BubR1 also helps stabilize kinetochore-microtubule interactions by counteracting the Aurora B kinase but the mechanism behind this is not clear. Here we show that BubR1 directly binds to the B56 family of protein phosphatase 2A (PP2A) regulatory subunits through a conserved motif that is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and polo-like kinase 1 (Plk1). Two highly conserved hydrophobic residues surrounding the serine 670 Cdk1 phosphorylation site are required for B56 binding. Mutation of these residues prevents the establishment of a proper metaphase plate and delays cells in mitosis. Furthermore, we show that phosphorylation of serines 670 and 676 stimulates the binding of B56 to BubR1 and that BubR1 targets a pool of B56 to kinetochores. Our data suggest that BubR1 counteracts Aurora B kinase activity at improperly attached kinetochores by recruiting B56-PP2A phosphatase complexes.

Ndc80 Loop as a protein-protein interaction motif

Our understanding of the structure and function of kinetochores has advanced dramatically over the past 10 years, yet how the plus end of spindle microtubules interacts with the kinetochore and establishes amphitelic attachment for proper sister chromatid segregation remains unresolved. However, several recent reports from different organisms have shed new light on this issue. A key player in microtubule-kinetochore interaction is the conserved Ndc80 outer kinetochore complex. In both yeast and human cells in particular, a ubiquitous internal ‘loop’ found in the Ndc80 molecule interrupting its C-terminal coiled-coil domain plays critical roles in protein-protein interaction, by recruiting microtubule-binding proteins to ensure proper kinetochore-microtubule attachment. In this commentary, we summarise the recent progress made and discuss the evolutionary significance of this loop’s role in microtubule dynamics at the kinetochore for accurate chromosome segregation.

Development of a Drosophila Cell-Based Error Correction Assay

Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT) interactions before anaphase onset results in chromosomal instability (CIN), which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F) is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5). Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due, in part, to kinesin-14 (Ncd) activity when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC). Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and CIN.

Mitosis-Specific Regulation of Nuclear Transport by the Spindle Assembly Checkpoint Protein Mad1p

Nuclear pore complexes (NPCs) and kinetochores perform distinct tasks, yet their shared ability to bind several proteins suggests their functions are intertwined. Among these shared proteins is Mad1p, a component of the yeast spindle assembly checkpoint (SAC). Here we describe a role for Mad1p in regulating nuclear import that employs its ability to sense a disruption of kinetochore-microtubule interactions during mitosis. We show that kinetochore-microtubule detachment arrests nuclear import mediated by the transport factor Kap121p through a mechanism that requires Mad1p cycling between unattached, metaphase kinetochores and binding sites at the NPC. This signaling pathway requires the Aurora B-like kinase Ipl1p, and the resulting transport changes inhibit the nuclear import of Glc7p, a phosphatase that acts as an Ipl1p antagonist. We propose that a distinct branch of the SAC exists in which Mad1p senses unattached kinetochores and, by altering NPC transport activity, regulates the nuclear environment of the spindle.

Csi1 links centromeres to the nuclear envelope for centromere clustering

In the fission yeast Schizosaccharomyces pombe, the centromeres of each chromosome are clustered together and attached to the nuclear envelope near the site of the spindle pole body during interphase. The mechanism and functional importance of this arrangement of chromosomes are poorly understood. In this paper, we identified a novel nuclear protein, Csi1, that localized to the site of centromere attachment and interacted with both the inner nuclear envelope SUN domain protein Sad1 and centromeres. Both Csi1 and Sad1 mutants exhibited centromere clustering defects in a high percentage of cells. Csi1 mutants also displayed a high rate of chromosome loss during mitosis, significant mitotic delays, and sensitivity to perturbations in microtubule-kinetochore interactions and chromosome numbers. These studies thus define a molecular link between the centromere and nuclear envelope that is responsible for centromere clustering.

Looping in on Ndc80 – How does a protein loop at the kinetochore control chromosome segregation?

Segregation of chromosomes during mitosis requires the interaction of dynamic microtubules with the kinetochore, a large protein structure established on the centromere region of sister chromatids. The core microtubule-binding activity of the kinetochore resides in the KMN network, an outer kinetochore complex. As part of the KMN network, the Ndc80 complex, which is composed of Ndc80, Nuf2, Spc24, and Spc25, is able to bind directly to microtubules and has the ability to track with depolymerizing microtubules to produce chromosome movement. The Ndc80 complex binds directly to microtubules through a calponin homology domain and an unstructured tail in the N terminus of the Ndc80 protein. A recent flurry of papers has highlighted the importance of an internal loop region in Ndc80 in establishing end-on attachment to microtubules. Here I discuss these recent findings that suggest that the Ndc80 internal loop functions as a binding site for proteins required for kinetochore-microtubule interactions.

Integration of Kinase and Phosphatase Activities by BUBR1 Ensures Formation of Stable Kinetochore-Microtubule Attachments

Maintenance of chromosomal stability depends on error-free chromosome segregation. The pseudokinase BUBR1 is essential for this, because it is a core component of the mitotic checkpoint and is required for formation of stable kinetochore-microtubule attachments. We have identified a conserved and highly phosphorylated domain (KARD) in BUBR1 that is crucial for formation of kinetochore-microtubule attachments. Deletion of this domain or prevention of its phosphorylation abolishes formation of kinetochore microtubules, which can be reverted by inhibiting Aurora B activity. Phosphorylation of KARD by PLK1 promotes direct interaction of BUBR1 with the PP2A-B56α phosphatase that counters excessive Aurora B activity at kinetochores. As a result, removal of BUBR1 from mitotic cells or inhibition of PLK1 reduces PP2A-B56α kinetochore binding and elevates phosphorylation of Aurora B substrates on the outer kinetochore. We propose that PLK1 and BUBR1 cooperate to stabilize kinetochore-microtubule interactions by regulating PP2A-B56α-mediated dephosphorylation of Aurora B substrates at the kinetochore-microtubule interface.

EB1 acetylation by P300/CBP-associated factor (PCAF) ensures accurate kinetochore–microtubule interactions in mitosis

In eukaryotes, microtubules are essential for cellular plasticity and dynamics. Here we show that P300/CBP-associated factor (PCAF), a kinetochore-associated acetyltransferase, acts as a negative modulator of microtubule stability through acetylation of EB1, a protein that controls the plus ends of microtubules. PCAF acetylates EB1 on K220 and disrupts the stability of a hydrophobic cavity on the dimerized EB1 C terminus, which was previously reported to interact with plus-end tracking proteins (TIPs) containing the SxIP motif. As determined with an EB1 acetyl-K220-specific antibody, K220 acetylation is dramatically increased in mitosis and localized to the spindle microtubule plus ends. Surprisingly, persistent acetylation of EB1 delays metaphase alignment, resulting in impaired checkpoint silencing. Consequently, suppression of Mad2 overrides mitotic arrest induced by persistent EB1 acetylation. Thus, our findings identify dynamic acetylation of EB1 as a molecular mechanism to orchestrate accurate kinetochore-microtubule interactions in mitosis. These results establish a previously uncharacterized regulatory mechanism governing localization of microtubule plus-end tracking proteins and thereby the plasticity and dynamics of cells.

Dynamic regulation of kinetochore-microtubule interaction during mitosis

Chromosome segregation is carried out by dynamic interplay between kinetochores and microtubules. First, kinetochores are efficiently captured by microtubules. Then, flexible interactions between kinetochores and microtubules allow proper orientation of chromosomes aligned on the centre of the spindle. Finally, microtubules stably attached to kinetochores drag the chromosomes towards the spindle poles. During these processes, the mode of interaction of kinetochores with microtubules changes from lateral to end-on attachment, accompanied by changes in kinetochore structure/composition and microtubule dynamics. The molecular mechanisms for stable kinetochore-microtubule attachment have been progressively revealed in recent years. On the other hand, the mechanism of dynamic regulation of kinetochore-microtubule interaction in early mitosis, which is crucial for faithful chromosome segregation, continues to be elusive. Here I focus on this early step of chromosome segregation and discuss how kinetochores establish proper attachments to microtubules.

Complications dawn for kinetochore regulation by Aurora

Organisms must faithfully segregate their chromosomes during cell division; mistakes in this process can be costly and even fatal to the organism (1, 2). During mitosis, replicated chromosomes attach to the spindle, a dynamic system of microtubules organized around two poles. Chromosomes attach to the spindle via kinetochores, structures that form on centromeres and bind the ends of microtubules. For accurate segregation, kinetochores on sister chromosomes must attach to microtubules from opposite poles; incorrect attachments lead to missegregation (3). In PNAS, Umbreit et al. (4) expand our understanding of how kinetochore–microtubule interactions can be regulated to correct improper attachments. The authors use in vitro studies to demonstrate that a component of the kinetochore, the Ndc80 complex, can directly influence the dynamics of the microtubules it is bound to and how the complex can be regulated to correct errors in chromosome attachment.

Sos7, an Essential Component of the Conserved Schizosaccharomyces pombe Ndc80-MIND-Spc7 Complex, Identifies a New Family of Fungal Kinetochore Proteins

Chromosome segregation is powered by the kinetochore, a large macromolecular structure assembled on centromeric chromatin. Attachment of sister chromatids to microtubules is mediated by the highly conserved tripartite KMN (acronym for KNL-1-Mis12-Ndc80) kinetochore network. In the fission yeast Schizosaccharomyces pombe, the equivalent complex is called NMS (Ndc80-MIND-Spc7). Here, we show that not all components of the NMS complex had been identified previously. A 10th NMS component exists, the essential Sos7 protein, which is a genetic and physical interaction partner of Spc7. The analysis of sos7 kinetochore-null mutant yeast strains demonstrated that Sos7 is central to NMS function. In particular, Sos7 is required for kinetochore targeting of Spc7 as well as components of the MIND complex. sos7 mutant strains show severe chromosome missegregation phenotypes and have compromised microtubule-kinetochore interactions. Sos7 is the founding member of a functionally conserved fungal kinetochore family not present in the point centromere carrying Saccharomycotina clusters, suggesting that the new Sos7 family might be a signature motif of fungi with regional centromeres.

Regulatory mechanisms of kinetochore-microtubule interaction in mitosis.

Interaction of microtubules with kinetochores is fundamental to chromosome segregation. Kinetochores initially associate with lateral surfaces of microtubules and subsequently become attached to microtubule ends. During these interactions, kinetochores can move by sliding along microtubules or by moving together with depolymerizing microtubule ends. The interplay between kinetochores and microtubules leads to the establishment of bi-orientation, which is the attachment of sister kinetochores to microtubules from opposite spindle poles, and subsequent chromosome segregation. Molecular mechanisms underlying these processes have been intensively studied over the past 10 years. Emerging evidence suggests that the KNL1-Mis12-Ndc80 (KMN) network plays a central role in connecting kinetochores to microtubules, which is under fine regulation by a mitotic kinase, Aurora B. However, a growing number of additional molecules are being shown to be involved in the kinetochore-microtubule interaction. Here I overview the current range of regulatory mechanisms of the kinetochore-microtubule interaction, and discuss how these multiple molecules contribute cooperatively to allow faithful chromosome segregation.

Human papillomavirus type 16 E7 oncoprotein engages but does not abrogate the mitotic spindle assembly checkpoint

The mitotic spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during mitosis by censoring kinetochore–microtubule interactions. It is frequently rendered dysfunctional during carcinogenesis causing chromosome missegregation and genomic instability. There are conflicting reports whether the HPV16 E7 oncoprotein drives chromosomal instability by abolishing the SAC. Here we report that degradation of mitotic cyclins is impaired in cells with HPV16 E7 expression. RNAi-mediated depletion of Mad2 or BubR1 indicated the involvement of the SAC, suggesting that HPV16 E7 expression causes sustained SAC engagement. Mutational analyses revealed that HPV16 E7 sequences that are necessary for retinoblastoma tumor suppressor protein binding as well as sequences previously implicated in binding the nuclear and mitotic apparatus (NuMA) protein and in delocalizing dynein from the mitotic spindle contribute to SAC engagement. Importantly, however, HPV16 E7 does not markedly compromise the SAC response to microtubule poisons.

Structural and Functional Organization of the Ska Complex, a Key Component of the Kinetochore-Microtubule Interface

The Ska complex is an essential mitotic component required for accurate cell division in human cells. It is composed of three subunits that function together to establish stable kinetochore-microtubule interactions in concert with the Ndc80 network. We show that the structure of the Ska core complex is a W-shaped dimer of coiled coils, formed by intertwined interactions between Ska1, Ska2, and Ska3. The C-terminal domains of Ska1 and Ska3 protrude at each end of the homodimer, bind microtubules in vitro when connected to the central core, and are essential in vivo. Mutations disrupting the central coiled coil or the dimerization interface result in chromosome congression failure followed by cell death. The Ska complex is thus endowed with bipartite and cooperative tubulin-binding properties at the ends of a 350 Å-long molecule. We discuss how this symmetric architecture might complement and stabilize the Ndc80-microtubule attachments with analogies to the yeast Dam1/DASH complex.

Localization and function of the Ska complex during mouse oocyte meiotic maturation

The Ska (spindle and kinetochore-associated) complex is composed of three proteins: Ska1, Ska2 and Ska3. It is required for stabilizing kinetochore-microtubule (KT-MT) interactions and silencing spindle checkpoint during mitosis. However, its roles in meiosis remain unclear. The present study was designed to investigate the localization and function of the Ska complex during mouse oocyte meiotic maturation. Our results showed that the localization and function of Ska complex in mouse oocyte meiosis differ in part from those in mitosis. Injection of low dose exogenous Myc-Ska mRNA showed that, instead of localizing to the kinetochores (KTs) and mediating KT-MT interactions from pro-metaphase to mid-anaphase stages as in mitosis, the members of the Ska complex were only localized on spindle microtubules from the Pro-MI to MII stages in mouse oocyte meiosis. Time-lapse live imaging analysis showed that knockdown of any member of the Ska complex by Morpholino injection into mouse oocytes resulted in spindle movement defects and enlarged polar bodies. Depletion of the whole Ska complex disrupted the stability of the anaphase spindle and influenced the extrusion of the first polar body. Taken together, these results show that the Ska complex plays an important role in meiotic spindle migration and anaphase spindle stability during mouse oocyte maturation.

Reconstituting the kinetochore–microtubule interface: what, why, and how

The kinetochore is the proteinaceous complex that governs the movement of duplicated chromosomes by interacting with spindle microtubules during mitosis and meiosis. Faithful chromosome segregation requires that kinetochores form robust load-bearing attachments to the tips of dynamic spindle microtubules, correct microtubule attachment errors, and delay the onset of anaphase until all chromosomes have made proper attachments. To understand how this macromolecular machine operates to segregate duplicated chromosomes with exquisite accuracy, it is critical to reconstitute and study kinetochore–microtubule interactions in vitro using defined components. Here, we review the current status of reconstitution as well as recent progress in understanding the microtubule-binding functions of kinetochores in vivo.

CENP-E Kinesin Interacts with SKAP Protein to Orchestrate Accurate Chromosome Segregation in Mitosis

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochore. Although previous studies show that the mitotic kinesin CENP-E forms a link between attachment of the spindle microtubule to the kinetochore and the mitotic checkpoint signaling cascade, the molecular mechanism underlying dynamic kinetochore-microtubule interactions in mammalian cells remains elusive. Here, we identify a novel interaction between CENP-E and SKAP that functions synergistically in governing dynamic kinetochore-microtubule interactions. SKAP binds to the C-terminal tail of CENP-E in vitro and is essential for an accurate kinetochore-microtubule attachment in vivo. Immunoelectron microscopic analysis indicates that SKAP is a constituent of the kinetochore corona fibers of mammalian centromeres. Depletion of SKAP or CENP-E by RNA interference results in a dramatic reduction of inter-kinetochore tension, which causes chromosome mis-segregation with a prolonged delay in achieving metaphase alignment. Importantly, SKAP binds to microtubules in vitro, and this interaction is synergized by CENP-E. Based on these findings, we propose that SKAP cooperates with CENP-E to orchestrate dynamic kinetochore-microtubule interaction for faithful chromosome segregation.

Kinetochore-Dependent Microtubule Rescue Ensures Their Efficient and Sustained Interactions in Early Mitosis

SummaryHow kinetochores regulate microtubule dynamics to ensure proper kinetochore-microtubule interactions is unknown. Here, we studied this during early mitosis in Saccharomyces cerevisiae. When a microtubule shrinks and its plus end reaches a kinetochore bound to its lateral surface, the microtubule end attempts to tether the kinetochore. This process often fails and, responding to this failure, microtubule rescue (conversion from shrinkage to growth) occurs, preventing kinetochore detachment from the microtubule end. This rescue is promoted by Stu2 transfer (ortholog of vertebrate XMAP215/ch-TOG) from the kinetochore to the microtubule end. Meanwhile, microtubule rescue distal to the kinetochore is also promoted by Stu2, which is transported by a kinesin-8 motor Kip3 along the microtubule from the kinetochore. Microtubule extension following rescue facilitates interaction with other widely scattered kinetochores, diminishing long delays in collecting the complete set of kinetochores by microtubules. Thus, kinetochore-dependent microtubule rescue ensures efficient and sustained kinetochore-microtubule interactions in early mitosis.

Small GTPase Rab5 participates in chromosome congression and regulates localization of the centromere-associated protein CENP-F to kinetochores

Rab5 is a small GTPase known to regulate vesicular trafficking during interphase. Here, we show that Rab5 also plays an unexpected role during mitotic progression. RNAi-mediated silencing of Rab5 caused defects in chromosome congression and extensive prometaphase delay, and it correlated with a severe reduction in the localization of the centromere-associated protein CENP-F to kinetochores. CENP-F is a component of the nuclear matrix required for chromosome congression that, at mitotic entry, localizes to the nuclear envelope and assembles on kinetochores, contributing to the establishment of kinetochore microtubule interactions. We found that Rab5 forms a complex with a subset of CENP-F in mitotic cells and regulates the kinetics of release of CENP-F from the nuclear envelope and its accumulation on kinetochores. Simultaneous depletion of both Rab5 and CENP-F recapitulated the mitotic defects caused by silencing of either Rab5 or CENP-F alone, indicating epistatic roles for these two proteins in the pathway that orchestrates chromosome congression. These results reveal the involvement of Rab5 in the proper execution of mitotic programs whose deregulation can undermine chromosomal stability.