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Kinetochore
Kinetochore
Kinetochore
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A kinetochore (/kɪˈnɛtəkɔːr/, /-ˈniːtəkɔːr/) is a flared oblique-shaped protein structure associated with duplicated chromatids in eukaryotic cells where the spindle fibers, which can be thought of as the ropes pulling chromosomes apart, attach during cell division to pull sister chromatids apart.[1] The kinetochore assembles on the centromere and links the chromosome to microtubule polymers from the mitotic spindle during mitosis and meiosis. The term kinetochore was first used in a footnote in a 1934 Cytology book by Lester W. Sharp[2] and commonly accepted in 1936.[3] Sharp's footnote reads: "The convenient term kinetochore (= movement place) has been suggested to the author by J. A. Moore", likely referring to John Alexander Moore who had joined Columbia University as a freshman in 1932.[4]

Monocentric organisms, including vertebrates, fungi, and most plants, have a single centromeric region on each chromosome which assembles a single, localized kinetochore. Holocentric organisms, such as nematodes and some plants, assemble a kinetochore along the entire length of a chromosome.[5]

Kinetochores start, control, and supervise the striking movements of chromosomes during cell division. During mitosis, which occurs after the amount of DNA is doubled in each chromosome (while maintaining the same number of chromosomes) in S phase, two sister chromatids are held together by a centromere. Each chromatid has its own kinetochore, which face in opposite directions and attach to opposite poles of the mitotic spindle apparatus. Following the transition from metaphase to anaphase, the sister chromatids separate from each other, and the individual kinetochores on each chromatid drive their movement to the spindle poles that will define the two new daughter cells. The kinetochore is therefore essential for the chromosome segregation that is classically associated with mitosis and meiosis.

Structure

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The kinetochore contains two regions:

  • an inner kinetochore, which is tightly associated with the centromere DNA and assembled in a specialized form of chromatin that persists throughout the cell cycle;
  • an outer kinetochore, which interacts with microtubules; the outer kinetochore is a very dynamic structure with many identical components, which are assembled and functional only during cell division.

Even the simplest kinetochores consist of more than 19 different proteins. Many of these proteins are conserved between eukaryotic species, including a specialized histone H3 variant (called CENP-A or CenH3) which helps the kinetochore associate with DNA. Other proteins in the kinetochore adhere it to the microtubules (MTs) of the mitotic spindle. There are also motor proteins, including both dynein and kinesin, which generate forces that move chromosomes during mitosis. Other proteins, such as Mad2, monitor the microtubule attachment as well as the tension between sister kinetochores and activate the spindle checkpoint to arrest the cell cycle when either of these is absent.[6] The actual set of genes essential for kinetochore function varies from one species to another.[7][8]

Kinetochore functions include anchoring of chromosomes to MTs in the spindle, verification of anchoring, activation of the spindle checkpoint and participation in the generation of force to propel chromosome movement during cell division.[9] On the other hand, microtubules are metastable polymers made of α- and β-tubulin, alternating between growing and shrinking phases, a phenomenon known as dynamic instability.[10] MTs are highly dynamic structures, whose behavior is integrated with kinetochore function to control chromosome movement and segregation. It has also been reported that the kinetochore organization differs between mitosis and meiosis and the integrity of meiotic kinetochore is essential for meiosis specific events such as pairing of homologous chromosomes, sister kinetochore monoorientation, protection of centromeric cohesin and spindle-pole body cohesion and duplication.[11][12]

In animal cells

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The kinetochore is composed of several layers, observed initially by conventional fixation and staining methods of electron microscopy,[13][14] (reviewed by C. Rieder in 1982[15]) and more recently by rapid freezing and substitution.[16]

Kinetochore structure and components in vertebrate cells. Based on Maiato et al. (2004).[9]

The deepest layer in the kinetochore is the inner plate, which is organized on a chromatin structure containing nucleosomes presenting a specialized histone (named CENP-A, which substitutes histone H3 in this region), auxiliary proteins, and DNA. DNA organization in the centromere (satellite DNA) is one of the least understood aspects of vertebrate kinetochores. The inner plate appears like a discrete heterochromatin domain throughout the cell cycle.

External to the inner plate is the outer plate, which is composed mostly of proteins. This structure is assembled on the surface of the chromosomes only after the nuclear envelope breaks down.[13] The outer plate in vertebrate kinetochores contains about 20 anchoring sites for MTs (+) ends (named kMTs, after kinetochore MTs), whereas a kinetochore's outer plate in yeast (Saccharomyces cerevisiae) contains only one anchoring site.

The outermost domain in the kinetochore forms a fibrous corona, which can be visualized by conventional microscopy, yet only in the absence of MTs. This corona is formed by a dynamic network of resident and temporary proteins implicated in the spindle checkpoint, in microtubule anchoring, and in the regulation of chromosome behavior.

During mitosis, each sister chromatid forming the complete chromosome has its own kinetochore. Distinct sister kinetochores can be observed at first at the end of G2 phase in cultured mammalian cells.[17] These early kinetochores show a mature laminar structure before the nuclear envelope breaks down.[18] The molecular pathway for kinetochore assembly in higher eukaryotes has been studied using gene knockouts in mice and in cultured chicken cells, as well as using RNA interference (RNAi) in C. elegans, Drosophila and human cells, yet no simple linear route can describe the data obtained so far.[citation needed]

Fluorescence microscopy micrographs, showing the endogenous human protein Mad1 (one of the spindle checkpoint components) in green, along the different phases in mitosis; CENP-B, in red, is a centromeric marker, and DAPI (in blue) stains DNA

The first protein to be assembled on the kinetochore is CENP-A (Cse4 in Saccharomyces cerevisiae). This protein is a specialized isoform of histone H3.[19] CENP-A is required for incorporation of the inner kinetochore proteins CENP-C, CENP-H and CENP-I/MIS6.[20][21][22][23][24] The relation of these proteins in the CENP-A-dependent pathway is not completely defined. For instance, CENP-C localization requires CENP-H in chicken cells, but it is independent of CENP-I/MIS6 in human cells. In C. elegans and metazoa, the incorporation of many proteins in the outer kinetochore depends ultimately on CENP-A.

Kinetochore proteins can be grouped according to their concentration at kinetochores during mitosis: some proteins remain bound throughout cell division, whereas some others change in concentration. Furthermore, they can be recycled in their binding site on kinetochores either slowly (they are rather stable) or rapidly (dynamic).

  • Proteins whose levels remain stable from prophase until late anaphase include constitutive components of the inner plate and the stable components of the outer kinetocore, such as the Ndc80 complex,[25][26] KNL/KBP proteins (kinetochore-null/KNL-binding protein),[27] MIS proteins[27] and CENP-F.[28][29] Together with the constitutive components, these proteins seem to organize the nuclear core of the inner and outer structures in the kinetochore.
  • The dynamic components that vary in concentration on kinetochores during mitosis include the molecular motors CENP-E and dynein (as well as their target components ZW10 and ROD), and the spindle checkpoint proteins (such as Mad1, Mad2, BubR1 and Cdc20). These proteins assemble on the kinetochore in high concentrations in the absence of microtubules; however, the higher the number of MTs anchored to the kinetochore, the lower the concentrations of these proteins.[30] At metaphase, CENP-E, Bub3 and Bub1 levels diminish by a factor of about three to four as compared with free kinetochores, whereas dynein/dynactin, Mad1, Mad2 and BubR1 levels are reduced by a factor of more than 10 to 100.[30][31][32][33]
  • Whereas the spindle checkpoint protein levels present in the outer plate diminish as MTs anchor,[33] other components such as EB1, APC and proteins in the Ran pathway (RanGap1 and RanBP2) associate to kinetochores only when MTs are anchored.[34][35][36][37] This may belong to a mechanism in the kinetochore to recognize the microtubules' plus-end (+), ensuring their proper anchoring and regulating their dynamic behavior as they remain anchored.

A 2010 study used a complex method (termed "multiclassifier combinatorial proteomics" or MCCP) to analyze the proteomic composition of vertebrate chromosomes, including kinetochores.[38] Although this study does not include a biochemical enrichment for kinetochores, obtained data include all the centromeric subcomplexes, with peptides from all 125 known centromeric proteins. According to this study, there are still about one hundred unknown kinetochore proteins, doubling the known structure during mitosis, which confirms the kinetochore as one of the most complex cellular substructures. Consistently, a comprehensive literature survey indicated that there had been at least 196 human proteins already experimentally shown to be localized at kinetochores.[39]

Function

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The number of microtubules attached to one kinetochore is variable: in Saccharomyces cerevisiae only one MT binds each kinetochore, whereas in mammals there can be 15–35 MTs bound to each kinetochore.[40] However, not all the MTs in the spindle attach to one kinetochore. There are MTs that extend from one centrosome to the other (and they are responsible for spindle length) and some shorter ones are interdigitated between the long MTs. Professor B. Nicklas (Duke University), showed that, if one breaks down the MT-kinetochore attachment using a laser beam, chromatids can no longer move, leading to an abnormal chromosome distribution.[41] These experiments also showed that kinetochores have polarity, and that kinetochore attachment to MTs emanating from one or the other centrosome will depend on its orientation. This specificity guarantees that only one chromatid will move to each spindle side, thus ensuring the correct distribution of the genetic material. Thus, one of the basic functions of the kinetochore is the MT attachment to the spindle, which is essential to correctly segregate sister chromatids. If anchoring is incorrect, errors may ensue, generating aneuploidy, with catastrophic consequences for the cell. To prevent this from happening, there are mechanisms of error detection and correction (as the spindle assembly checkpoint), whose components reside also on the kinetochores. The movement of one chromatid towards the centrosome is produced primarily by MT depolymerization in the binding site with the kinetochore. These movements require also force generation, involving molecular motors likewise located on the kinetochores.

Chromosome anchoring to MTs in the mitotic spindle

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Capturing MTs

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Chromosomes attach to the mitotic spindle through sister kinetochores, in a bipolar orientation

During the synthesis phase (S phase) in the cell cycle, the centrosome starts to duplicate. Just at the beginning of mitosis, both centrioles in each centrosome reach their maximal length, centrosomes recruit additional material and their nucleation capacity for microtubules increases. As mitosis progresses, both centrosomes separate to establish the mitotic spindle.[42] In this way, the spindle in a mitotic cell has two poles emanating microtubules. Microtubules are long proteic filaments with asymmetric extremes, a "minus"(-) end relatively stable next to the centrosome, and a "plus"(+) end enduring alternate phases of growing-shrinking, exploring the center of the cell. During this searching process, a microtubule may encounter and capture a chromosome through the kinetochore.[43][44] Microtubules that find and attach a kinetochore become stabilized, whereas those microtubules remaining free are rapidly depolymerized.[45] As chromosomes have two kinetochores associated back-to-back (one on each sister chromatid), when one of them becomes attached to the microtubules generated by one of the cellular poles, the kinetochore on the sister chromatid becomes exposed to the opposed pole; for this reason, most of the times the second kinetochore becomes attached to the microtubules emanating from the opposing pole,[46] in such a way that chromosomes are now bi-oriented, one fundamental configuration (also termed amphitelic) to ensure the correct segregation of both chromatids when the cell will divide.[47][48]

Scheme showing cell cycle progression between prometaphase and anaphase. (Chromosomes are in blue and kinetochores in light yellow).
Scheme showing cell cycle progression between prometaphase and anaphase. (Chromosomes are in blue and kinetochores in light yellow).

When just one microtubule is anchored to one kinetochore, it starts a rapid movement of the associated chromosome towards the pole generating that microtubule. This movement is probably mediated by the motor activity towards the "minus" (-) of the motor protein cytoplasmic dynein,[49][50] which is very concentrated in the kinetochores not anchored to MTs.[51] The movement towards the pole is slowed down as far as kinetochores acquire kMTs (MTs anchored to kinetochores) and the movement becomes directed by changes in kMTs length. Dynein is released from kinetochores as they acquire kMTs[30] and, in cultured mammalian cells, it is required for the spindle checkpoint inactivation, but not for chromosome congression in the spindle equator, kMTs acquisition or anaphase A during chromosome segregation.[52] In higher plants or in yeast there is no evidence of dynein, but other kinesins towards the (-) end might compensate for the lack of dynein.

Metaphase cells with low CENP-E levels by RNAi, showing chromosomes unaligned at the metaphase plate (arrows). These chromosomes are labeled with antibodies against the mitotic checkpoint proteins Mad1/Mad2. Hec1 and CENP-B label the centromeric region (the kinetochore), and DAPI is a specific stain for DNA.

Another motor protein implicated in the initial capture of MTs is CENP-E; this is a high molecular weight kinesin associated with the fibrous corona at mammalian kinetochores from prometaphase until anaphase.[53] In cells with low levels of CENP-E, chromosomes lack this protein at their kinetochores, which quite often are defective in their ability to congress at the metaphase plate. In this case, some chromosomes may remain chronically mono-oriented (anchored to only one pole), although most chromosomes may congress correctly at the metaphase plate.[54]

It is widely accepted that the kMTs fiber (the bundle of microtubules bound to the kinetochore) is originated by the capture of MTs polymerized at the centrosomes and spindle poles in mammalian cultured cells.[43] However, MTs directly polymerized at kinetochores might also contribute significantly.[55] The manner in which the centromeric region or kinetochore initiates the formation of kMTs and the frequency at which this happens are important questions,[according to whom?] because this mechanism may contribute not only to the initial formation of kMTs, but also to the way in which kinetochores correct defective anchoring of MTs and regulate the movement along kMTs.

Role of Ndc80 complex

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MTs associated to kinetochores present special features: compared to free MTs, kMTs are much more resistant to cold-induced depolymerization, high hydrostatic pressures or calcium exposure.[56] Furthermore, kMTs are recycled much more slowly than astral MTs and spindle MTs with free (+) ends, and if kMTs are released from kinetochores using a laser beam, they rapidly depolymerize.[41]

When it was clear that neither dynein nor CENP-E is essential for kMTs formation, other molecules should be responsible for kMTs stabilization. Pioneer genetic work in yeast revealed the relevance of the Ndc80 complex in kMTs anchoring.[25][57][58][59] In Saccharomyces cerevisiae, the Ndc80 complex has four components: Ndc80p, Nuf2p, Spc24p and Spc25p. Mutants lacking any of the components of this complex show loss of the kinetochore-microtubule connection, although kinetochore structure is not completely lost.[25][57] Yet mutants in which kinetochore structure is lost (for instance Ndc10 mutants in yeast[60]) are deficient both in the connection to microtubules and in the ability to activate the spindle checkpoint, probably because kinetochores work as a platform in which the components of the response are assembled.

The Ndc80 complex is highly conserved and it has been identified in S. pombe, C. elegans, Xenopus, chicken and humans.[25][26][57][61][62][63][64] Studies on Hec1 (highly expressed in cancer cells 1), the human homolog of Ndc80p, show that it is important for correct chromosome congression and mitotic progression, and that it interacts with components of the cohesin and condensin complexes.[65]

Different laboratories have shown that the Ndc80 complex is essential for stabilization of the kinetochore-microtubule anchoring, required to support the centromeric tension implicated in the establishment of the correct chromosome congression in high eukaryotes.[26][62][63][64] Cells with impaired function of Ndc80 (using RNAi, gene knockout, or antibody microinjection) have abnormally long spindles, lack of tension between sister kinetochores, chromosomes unable to congregate at the metaphase plate and few or any associated kMTs.

There is a variety of strong support for the ability of the Ndc80 complex to directly associate with microtubules and form the core conserved component of the kinetochore-microtubule interface.[66] However, formation of robust kinetochore-microtubule interactions may also require the function of additional proteins. In yeast, this connection requires the presence of the complex Dam1-DASH-DDD. Some members of this complex bind directly to MTs, whereas some others bind to the Ndc80 complex.[58][59][67] This means that the complex Dam1-DASH-DDD might be an essential adapter between kinetochores and microtubules. However, in animals an equivalent complex has not been identified, and this question remains under intense investigation.

Verification of kinetochore–MT anchoring

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During S-Phase, the cell duplicates all the genetic information stored in the chromosomes, in the process termed DNA replication. At the end of this process, each chromosome includes two sister chromatids, which are two complete and identical DNA molecules. Both chromatids remain associated by cohesin complexes until anaphase, when chromosome segregation occurs. If chromosome segregation happens correctly, each daughter cell receives a complete set of chromatids, and for this to happen each sister chromatid has to anchor (through the corresponding kinetochore) to MTs generated in opposed poles of the mitotic spindle. This configuration is termed amphitelic or bi-orientation.

However, during the anchoring process some incorrect configurations may also appear:[68]

Scheme showing different anchoring configurations between chromosomes and the mitotic spindle.[55]
  • monotelic: only one of the chromatids is anchored to MTs, the second kinetochore is not anchored; in this situation, there is no centromeric tension, and the spindle checkpoint is activated, delaying entry in anaphase and allowing time for the cell to correct the error. If it is not corrected, the unanchored chromatid might randomly end in any of the two daughter cells, generating aneuploidy: one daughter cell would have chromosomes in excess and the other would lack some chromosomes.
  • syntelic: both chromatids are anchored to MTs emanating from the same pole; this situation does not generate centromeric tension either, and the spindle checkpoint will be activated. If it is not corrected, both chromatids will end in the same daughter cell, generating aneuploidy.
  • merotelic: at least one chromatid is anchored simultaneously to MTs emanating from both poles. This situation generates centromeric tension, and for this reason the spindle checkpoint is not activated. If it is not corrected, the chromatid bound to both poles will remain as a lagging chromosome at anaphase, and finally will be broken in two fragments, distributed between the daughter cells, generating aneuploidy.

Both the monotelic and the syntelic configurations fail to generate centromeric tension and are detected by the spindle checkpoint. In contrast, the merotelic configuration is not detected by this control mechanism. However, most of these errors are detected and corrected before the cell enters in anaphase.[68] A key factor in the correction of these anchoring errors is the chromosomal passenger complex, which includes the kinase protein Aurora B, its target and activating subunit INCENP and two other subunits, Survivin and Borealin/Dasra B (reviewed by Adams and collaborators in 2001[69]). Cells in which the function of this complex has been abolished by dominant negative mutants, RNAi, antibody microinjection or using selective drugs, accumulate errors in chromosome anchoring. Many studies have shown that Aurora B is required to destabilize incorrect anchoring kinetochore-MT, favoring the generation of amphitelic connections. Aurora B homolog in yeast (Ipl1p) phosphorilates some kinetochore proteins, such as the constitutive protein Ndc10p and members of the Ndc80 and Dam1-DASH-DDD complexes.[70] Phosphorylation of Ndc80 complex components produces destabilization of kMTs anchoring. It has been proposed that Aurora B localization is important for its function: as it is located in the inner region of the kinetochore (in the centromeric heterochromatin), when the centromeric tension is established sister kinetochores separate, and Aurora B cannot reach its substrates, so that kMTs are stabilized. Aurora B is frequently overexpressed in several cancer types, and it is currently a target for the development of anticancer drugs.[71]

Spindle checkpoint activation

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Main article: Spindle checkpoint

The spindle checkpoint, or SAC (for spindle assembly checkpoint), also known as the mitotic checkpoint, is a cellular mechanism responsible for detection of:

  • correct assembly of the mitotic spindle;
  • attachment of all chromosomes to the mitotic spindle in a bipolar manner;
  • congression of all chromosomes at the metaphase plate.

When just one chromosome (for any reason) remains lagging during congression, the spindle checkpoint machinery generates a delay in cell cycle progression: the cell is arrested, allowing time for repair mechanisms to solve the detected problem. After some time, if the problem has not been solved, the cell will be targeted for apoptosis (programmed cell death), a safety mechanism to avoid the generation of aneuploidy, a situation which generally has dramatic consequences for the organism.

Whereas structural centromeric proteins (such as CENP-B), remain stably localized throughout mitosis (including during telophase), the spindle checkpoint components are assembled on the kinetochore in high concentrations in the absence of microtubules, and their concentrations decrease as the number of microtubules attached to the kinetochore increases.[30]

At metaphase, CENP-E, Bub3 and Bub1 levels decreases 3 to 4 fold as compared to the levels at unattached kinetochores, whereas the levels of dynein/dynactin, Mad1, Mad2 and BubR1 decrease >10-100 fold.[30][31][32][33] Thus at metaphase, when all chromosomes are aligned at the metaphase plate, all checkpoint proteins are released from the kinetochore. The disappearance of the checkpoint proteins out of the kinetochores indicates the moment when the chromosome has reached the metaphase plate and is under bipolar tension. At this moment, the checkpoint proteins that bind to and inhibit Cdc20 (Mad1-Mad2 and BubR1), release Cdc20, which binds and activates APC/CCdc20, and this complex triggers sister chromatids separation and consequently anaphase entry.

Several studies indicate that the Ndc80 complex participates in the regulation of the stable association of Mad1-Mad2 and dynein with kinetochores.[26][63][64] Yet the kinetochore associated proteins CENP-A, CENP-C, CENP-E, CENP-H and BubR1 are independent of Ndc80/Hec1. The prolonged arrest in prometaphase observed in cells with low levels of Ndc80/Hec1 depends on Mad2, although these cells show low levels of Mad1, Mad2 and dynein on kinetochores (<10-15% in relation to unattached kinetochores). However, if both Ndc80/Hec1 and Nuf2 levels are reduced, Mad1 and Mad2 completely disappear from the kinetochores and the spindle checkpoint is inactivated.[72]

Shugoshin (Sgo1, MEI-S332 in Drosophila melanogaster[73]) are centromeric proteins which are essential to maintain cohesin bound to centromeres until anaphase. The human homolog, hsSgo1, associates with centromeres during prophase and disappears when anaphase starts.[74] When Shugoshin levels are reduced by RNAi in HeLa cells, cohesin cannot remain on the centromeres during mitosis, and consequently sister chromatids separate synchronically before anaphase initiates, which triggers a long mitotic arrest.

On the other hand, Dasso and collaborators have found that proteins involved in the Ran cycle can be detected on kinetochores during mitosis: RanGAP1 (a GTPase activating protein which stimulates the conversion of Ran-GTP in Ran-GDP) and the Ran binding protein called RanBP2/Nup358.[75] During interphase, these proteins are located at the nuclear pores and participate in the nucleo-cytoplasmic transport. Kinetochore localization of these proteins seem to be functionally significant, because some treatments that increase the levels of Ran-GTP inhibit kinetochore release of Bub1, Bub3, Mad2 and CENP-E.[76]

Orc2 (a protein that belongs to the origin recognition complex -ORC- implicated in DNA replication initiation during S phase) is also localized at kinetochores during mitosis in human cells;[77] in agreement with this localization, some studies indicate that Orc2 in yeast is implicated in sister chromatids cohesion, and when it is eliminated from the cell, spindle checkpoint activation ensues.[78] Some other ORC components (such orc5 in S. pombe) have been also found to participate in cohesion.[79] However, ORC proteins seem to participate in a molecular pathway which is additive to cohesin pathway, and it is mostly unknown.

Force generation to propel chromosome movement

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See also: Microtubule

Most chromosome movements in relation to spindle poles are associated to lengthening and shortening of kMTs. One of the features of kinetochores is their capacity to modify the state of their associated kMTs (around 20) from a depolymerization state at their (+) end to polymerization state. This allows the kinetochores from cells at prometaphase to show "directional instability",[80] changing between persistent phases of movement towards the pole (poleward) or inversed (anti-poleward), which are coupled with alternating states of kMTs depolymerization and polymerization, respectively. This kinetochore bi-stability seem to be part of a mechanism to align the chromosomes at the equator of the spindle without losing the mechanic connection between kinetochores and spindle poles. It is thought that kinetochore bi-stability is based upon the dynamic instability of the kMTs (+) end, and it is partially controlled by the tension present at the kinetochore. In mammalian cultured cells, a low tension at kinetochores promotes change towards kMTs depolymerization, and high tension promotes change towards kMTs polymerization.[81][82]

Kinetochore proteins and proteins binding to MTs (+) end (collectively called +TIPs) regulate kinetochore movement through the kMTs (+) end dynamics regulation.[83] However, the kinetochore-microtubule interface is highly dynamic, and some of these proteins seem to be bona fide components of both structures. Two groups of proteins seem to be particularly important: kinesins which work like depolymerases, such as KinI kinesins; and proteins bound to MT (+) ends, +TIPs, promoting polymerization, perhaps antagonizing the depolymerases effect.[84]

  • KinI kinesins are named "I" because they present an internal motor domain, which uses ATP to promote depolymerization of tubulin polymer, the microtubule. In vertebrates, the most important KinI kinesin controlling the dynamics of the (+) end assembly is MCAK.[85] However, it seems that there are other kinesins implicated.
  • There are two groups of +TIPs with kinetochore functions.
    • The first one includes the protein adenomatous polyposis coli (APC) and the associated protein EB1, which need MTs to localize on the kinetochores. Both proteins are required for correct chromosome segregation.[86] EB1 binds only to MTs in polymerizing state, suggesting that it promotes kMTs stabilization during this phase.
    • The second group of +TIPs includes proteins which can localize on kinetochores even in absence of MTs. In this group there are two proteins that have been widely studied: CLIP-170 and their associated proteins CLASPs (CLIP-associated proteins). CLIP-170 role at kinetochores is unknown, but the expression of a dominant negative mutant produces a prometaphase delay,[87] suggesting that it has an active role in chromosome alignment. CLASPs proteins are required for chromosome alignment and maintenance of a bipolar spindle in Drosophila, humans and yeast.[88][89]

References

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Kinetochore

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The kinetochore is a large, multilayered that assembles on the centromeric region of eukaryotic chromosomes and serves as the primary site for attachment during and , facilitating the precise segregation of to daughter cells and thereby maintaining genomic stability. It functions as a dynamic interface that couples dynamics to chromosome movement, generates or transduces mechanical forces, and monitors attachment fidelity through the spindle assembly checkpoint (SAC) to prevent errors in division. Structurally, the kinetochore is organized into inner and outer layers: the inner kinetochore interacts directly with centromeric chromatin via the histone H3 variant CENP-A, while the outer kinetochore forms a fibrous corona and connects to spindle . The outer kinetochore's core is the KMN network, a conserved assembly comprising three subcomplexes—KNL1 (Knl1/Zwint), MIS12 (Mis12/Dsn1/Nsl1/Pmf1), and NDC80 (Ndc80/Nuf2/Spc24/Spc25)—that together form a rigid, prong-shaped scaffold approximately 300 long, as revealed by recent cryo-electron microscopy (cryo-EM) structures. This architecture positions microtubule-binding calponin-homology (CH) domains of NDC80 outward, enabling end-on attachments to 15–25 per kinetochore in human cells, while MIS12 and KNL1 anchor to the inner kinetochore via interactions with CENP-C and CENP-T. Accessory components, such as the Ska complex, Dam1 ring (in yeast), and motor proteins like and CENP-E, further stabilize attachments and contribute to congression and error correction. Functionally, kinetochores orchestrate biorientation by binding from opposite spindle poles, with Aurora B activity destabilizing improper attachments through of KMN components. The SAC, scaffolded by KNL1, recruits checkpoint proteins like Bub1 and Mad1 to halt until all kinetochores are properly attached, averting . In addition to force coupling and signaling, kinetochores regulate polymerization/depolymerization via associated enzymes such as MCAK and XMAP215, ensuring bi-oriented chromosomes align at the plate. Dysfunctions in kinetochore assembly or regulation are implicated in chromosomal instability and diseases like cancer.

Overview

Definition and Location

The kinetochore is a large proteinaceous structure composed of over 100 distinct proteins that assembles on the of eukaryotic to mediate their attachment to the mitotic spindle. This multiprotein complex functions as the primary site for binding during segregation in . The is the specialized chromosomal region that maintains sister cohesion until and serves as the locus for kinetochore formation to ensure proper segregation of sister to daughter cells. The kinetochore assembles specifically on this centromeric , forming a multi-layered disc perpendicular to the axis. Its inner layer is embedded within the centromeric , while the outer layer extends toward the to facilitate interactions with spindle components. In vertebrates, this disc-like structure typically measures approximately 145 nm (120-170 nm) in diameter and accommodates 15-25 per kinetochore.

Role in Cell Division

The kinetochore serves as the primary interface between chromosomes and the mitotic spindle, linking to to enable their bipolar attachment and subsequent equal segregation to cells during . This attachment ensures that each cell receives an identical set of chromosomes, maintaining genomic stability through the process of equatorial division. In , kinetochores facilitate chromosome segregation in a reductional manner during meiosis I, where homologous chromosomes separate, and in an equational manner during meiosis II, akin to , to produce haploid gametes. By coordinating with spindle components, kinetochores generate pulling forces through microtubule depolymerization, which can reach up to 700 pN per chromosome, while simultaneously sensing the status of microtubule attachments. This sensing mechanism activates the spindle assembly checkpoint (SAC) to halt cell cycle progression until all chromosomes achieve proper bipolar alignment at the metaphase plate, thereby preventing premature anaphase onset. Such coordination is crucial in both mitosis and meiosis to avoid errors in chromosome distribution. Dysfunction in kinetochore function leads to chromosomal instability, a hallmark of cancer, where improper attachments result in and missegregation events that promote tumorigenesis. For instance, defects in kinetochore-microtubule interactions have been observed to increase rates in cancer cells, contributing to disease progression. Similarly, in meiotic contexts, kinetochore errors can lead to aneuploid gametes, underscoring the structure's role in preventing heritable genomic imbalances.

Discovery and History

Early Observations

The term "kinetochore" was first introduced in 1934 by botanist Lester W. Sharp in his textbook Fundamentals of Cytology, derived from the Greek roots kinesis (motion) and choros (place or space), to describe the chromosomal region responsible for directed movement during mitosis as observed through light microscopy. Sharp's usage stemmed from contemporary studies visualizing chromosome congression and segregation, where the structure appeared as a distinct locus facilitating attachment to the mitotic spindle. In 1936, cytogeneticist Cyril D. Darlington further formalized the concept in his analysis of mechanics, explicitly describing the kinetochore as the precise "point of attachment" for spindle fibers, particularly in observations of spermatocytes where it enabled oriented pulling. Darlington's work built on prior light microscopy evidence, emphasizing the kinetochore's role in ensuring bipolar orientation and faithful segregation. Throughout the early , fixed and live-cell imaging techniques provided key evidence of spindle fiber connections to chromosomes, notably during when fibers shortened to draw poleward, as documented in studies of and cells. Pioneering observations, such as those by Franz Schrader in using light microscopy on cells, confirmed the physical continuity between spindle fibers and specific chromosomal sites, solidifying the kinetochore's implication in force transmission. Despite these advances, early light microscopy suffered from resolution limitations, often blurring the kinetochore with the broader centromeric region and hindering precise morphological delineation until electron microscopy emerged in the 1960s. This technological gap restricted initial descriptions to gross attachments rather than details.

Molecular and Structural Milestones

In the 1960s, electron microscopy provided the first detailed views of kinetochore in mammalian cells, revealing a trilaminar organization. Finnish biologist Pentti T. Jokelainen's 1967 study on mitotic cells described the kinetochore as a composite disk approximately 2000–2450 in diameter, comprising an electron-dense inner layer contiguous with the centromeric , a central dense plate, and an outer dome-like layer projecting toward the spindle poles. This trilaminar model was corroborated by subsequent work from Bill Brinkley and colleagues, who observed similar plate-like features in mammalian somatic cells, establishing the kinetochore as a multilayered proteinaceous structure essential for segregation. The 1980s and 1990s marked a shift toward molecular identification of kinetochore components, driven by autoantibodies from patients with scleroderma, particularly the CREST subset. In 1985, William Earnshaw and Neville Rothfield used these sera to identify a family of centromere proteins (CENPs), including CENP-A, a 17 kDa antigen localized exclusively to kinetochores and later confirmed as a centromere-specific variant of histone H3 that replaces conventional H3 in centromeric nucleosomes. This approach yielded additional CENPs, such as CENP-B (an 80 kDa DNA-binding protein) and CENP-C (involved in inner kinetochore organization), with over a dozen antigens mapped by the mid-1990s through immunofluorescence and biochemical fractionation, providing the first protein-level insights into kinetochore composition. Advancing into the 2000s, biochemical and genetic studies delineated modular networks within the outer kinetochore, culminating in the discovery of the KMN complex—a core assembly of the KNL1, Mis12, and Ndc80 subcomplexes that bridges centromeric to . The human Mis12 complex, a heterotetramer essential for kinetochore assembly, was characterized in 2006 for its role in recruiting Ndc80 and stabilizing attachments. Concurrent proteomic efforts, including of isolated kinetochores, identified approximately 125 centromeric and kinetochore-associated proteins by 2010, expanding from the initial CENPs to include regulators of attachment and checkpoint signaling. The 2020s have brought high-resolution structural breakthroughs via cryo-electron microscopy (cryo-EM), illuminating the inner kinetochore's architecture. In 2022, structures of the human constitutive centromere-associated network (CCAN)—a multi-subunit assembly including CENPs-L, M, N, T, and others—were resolved at near-atomic resolution when bound to CENP-A nucleosomes, revealing how CCAN clamps DNA and orients the kinetochore for outer layer recruitment. Building on this, a 2025 cryo-EM study in budding yeast delineated dual force transmission pathways through the inner kinetochore, with the Mif2 protein (orthologous to human CENP-C) and the Okp1/Ame1 heterodimer (part of the COMA complex) independently channeling mechanical loads from to centromeric , thereby enhancing stability under tension.

Structure

Inner Kinetochore

The inner kinetochore constitutes the stable, chromatin-proximal layer of the kinetochore, serving as a foundational platform embedded within centromeric chromatin. It is primarily composed of the constitutive centromere-associated network (CCAN), a multi-subunit complex comprising 16 centromere proteins (CENPs) that remain associated with the centromere throughout the cell cycle. Key components include CENP-A nucleosomes, which form the epigenetic mark defining centromeric chromatin, as well as CENP-C, which acts as a central scaffold for CCAN assembly, and the CENP-H/I/K/L/M/N/O subcomplex, which stabilizes interactions with centromeric DNA. These proteins collectively ensure the inner kinetochore's persistence across interphase and mitosis, providing a constitutive interface for higher-order kinetochore structures. Structurally, the inner kinetochore organizes into an approximately 70 nm thick inner plate that integrates directly with the underlying centromeric . This plate is anchored by arrays of CENP-A octamers, which replace canonical in nucleosomes to create a specialized environment that recruits and positions CCAN components. Cryo-electron studies reveal that CCAN modules, such as the CENP-L/N and CENP-T/W/S/X complexes, encircle and grip the emerging from CENP-A nucleosomes, forming robust, edge-on attachments that embed the structure within the fiber. This organization not only tethers the inner kinetochore to the but also orients it for load-bearing during chromosome segregation. The inner kinetochore interfaces with specific modifications that contribute to specification and maintenance. In particular, trimethylation of at 9 (H3K9me3) in pericentromeric helps delineate the boundaries of centromeric domains, promoting the focused deposition of CENP-A and stabilizing CCAN occupancy. This epigenetic landscape ensures the inner kinetochore's fidelity in defining kinetochore assembly sites amid repetitive α-satellite DNA sequences. Recent insights from cryo-electron tomography have highlighted how centromeric forms distinct "clearings"—regions depleted of dense nucleosomes—that precisely demarcate sites for inner kinetochore assembly. These clearings, spanning 20-25 nm and containing nucleosome-associated CCAN complexes, are maintained by CENP-C and CENP-N, which organize fibers to create accessible platforms for kinetochore formation during . This mechanism underscores the inner kinetochore's role in translating architecture into precise attachment points for microtubule-binding components.

Outer Kinetochore

The outer kinetochore constitutes the dynamic, microtubule-interacting layer of the kinetochore, built upon the stable inner kinetochore platform. It primarily comprises the conserved KMN network, a ten-subunit assembly divided into three subcomplexes: the KNL1 complex (including Knl1 and Zwint1), the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C). The Mis12C serves as a bridging element that connects the KMN network to the inner kinetochore, while the Ndc80C provides the primary interface for binding through its calponin-homology (CH) domains at the N-terminal ends of Ndc80 and Nuf2 subunits, enabling end-on attachments. Additional proteins enrich the outer kinetochore's functionality, including the kinesin-like motor CENP-E, which localizes to the fibrous corona and aids in initial capture, and cytoplasmic , which is recruited via the RZZ complex and contributes to poleward transport. In vertebrates, the outer kinetochore organizes into a fibrous corona—a transient meshwork extending from the outer plate—and features 15–35 attachment sites per kinetochore, allowing for multiple end-on connections. The outer plate itself forms a flexible network of fibers that embed plus-ends, while the fibrous corona, prominent on unattached kinetochores, spans approximately 100 nm and facilitates initial lateral interactions before maturation to end-on attachments. This layer undergoes dynamic assembly and disassembly throughout : it expands in early to form the extended fibrous corona, enhancing capture efficiency, and compacts upon attachment in . Recent studies from 2025 have revealed the outer kinetochore's intricate, flexible architecture, incorporating over 100 proteins whose interactions are finely tuned by events, such as those on Mis12C components that modulate corona projection and stability.

Assembly

Cell Cycle Regulation

The assembly of kinetochores is tightly synchronized with the cell cycle to ensure accurate chromosome segregation. In the G1 phase, new CENP-A nucleosomes are deposited at centromeres to establish the foundation for the inner kinetochore, a process mediated by the specific histone chaperone HJURP, which forms a complex with CENP-A and H4 for targeted chromatin incorporation. This deposition is restricted to early G1 and inhibited during S, G2, and M phases by cyclin-dependent kinases (CDKs) such as CDK1 and CDK2, maintaining centromeric identity across cell divisions. As cells progress into and , outer kinetochore components are rapidly recruited to the inner kinetochore, peaking during to facilitate interactions. This recruitment is predominantly driven by phosphorylation from the cyclin B-CDK1 complex, which modifies key proteins like CENP-T at specific and serine residues (e.g., Thr11, Thr85, and Ser201), enabling the binding of up to three NDC80 complexes and one MIS12 complex per CENP-T molecule. Polo-like kinase 1 () further contributes by phosphorylating the Mis18 complex in G1 to prime this mitotic assembly, ensuring timely maturation. Kinetochore disassembly commences at anaphase onset, triggered by the ubiquitin-mediated degradation of cyclin B and securin by the anaphase-promoting complex/cyclosome (APC/C), which inactivates CDK1 and allows the spindle assembly checkpoint (SAC) to be silenced, permitting progression beyond metaphase. This leads to dephosphorylation of CDK1 substrates by protein phosphatase 2A (PP2A), particularly the B55α subunit, which reverses mitotic phosphorylations and promotes the dissociation of outer kinetochore proteins like NDC80. A 2022 review highlights additional layers of regulation, including Ran-GTP gradients that generate spatial cues for protein localization around kinetochores during , and PLK1's role in coordinating timing through targeted s that prevent ectopic assembly. Feedback loops integrate attachment status to modulate kinetochore maturation; for instance, unstable attachments sustain states that exclude premature outer kinetochore stabilization via competitive protein binding, thereby preventing erroneous segregation until bi-orientation is achieved.

Key Protein Networks

The constitutive centromere-associated network (CCAN) forms the foundational inner kinetochore scaffold, comprising multiple proteins that anchor the kinetochore to centromeric DNA and recruit outer kinetochore components. Central to this network, CENP-C acts as a key recruiter by directly binding the Mis12 complex (Mis12C), which in turn facilitates the attachment of the Ndc80 complex (Ndc80C), thereby bridging the inner and outer kinetochore domains. Complementing this pathway, the CENP-T/W/X/S subcomplex provides an alternative tethering mechanism, with CENP-T directly interacting with histone-fold proteins CENP-W and CENP-X to establish stable connections to centromeric and nucleosomes, independent of CENP-A. These interactions ensure the CCAN's role as a persistent platform for kinetochore assembly throughout the . The KMN network, consisting of the Knl1, Mis12, and Ndc80 complexes, represents the core outer kinetochore assembly that interfaces with . This ten-subunit structure adopts an elongated, oligomeric configuration, where the Ndc80 complex's calponin-homology-like domains and internal loops enable multivalent binding to microtubule protofilaments, promoting stable attachment through cooperative oligomerization along lattices. Mis12C serves as a critical adaptor within the KMN, linking CENP-C from the CCAN to both Knl1 and Ndc80, while its phosphorylation-sensitive interactions fine-tune assembly dynamics. The oligomeric of Ndc80C, forming arrays that track microtubule ends, underscores its primary role in load-bearing connections. Beyond the conserved CCAN and KMN, additional protein networks contribute to kinetochore functionality, particularly in microtubule coupling and chromosome movement. In yeast, the DASH (Dam1) complex assembles into oligomeric rings that encircle , enhancing processivity and force transmission in coordination with Ndc80, thereby facilitating end-on attachments during segregation. In metazoans, the kinesin-like CENP-E forms a distinct network at kinetochores, driving congression by transporting mono-oriented along toward the plate. Regulatory interactions, such as Aurora B of Ndc80's N-terminal tail, modulate these networks by destabilizing erroneous attachments and promoting dynamic remodeling. Overall, the kinetochore incorporates over 100 proteins across these networks, with many of their functions still poorly understood, highlighting ongoing challenges in dissecting their contributions.

Microtubule Interaction

Attachment Mechanisms

The search-and-capture model describes the initial interaction between kinetochores and spindle , where dynamic explore the intracellular space to locate and bind kinetochores on . In this process, kinetochores first form lateral attachments to sides, facilitated by plus-end tracking proteins (+TIPs) such as EB1 and CLIP-170, which accumulate at growing plus ends and promote initial contacts. These lateral interactions then transition to stable end-on attachments at plus ends, enabling force generation for alignment. The Ndc80 complex, a key component of the outer kinetochore, plays a central role in mediating these end-on attachments by directly binding microtubule plus ends. Its calponin-homology domains in the Hec1 subunit, along with flexible loop structures, allow the complex to grip and track depolymerizing microtubule ends, stabilizing attachments through multivalent interactions. Each kinetochore can support varying numbers of , typically one in budding yeast but 20 or more in mammalian cells, reflecting differences in kinetochore size and across species. Initial capture of often involves motor proteins to enhance efficiency. , recruited to the kinetochore corona, mediates sliding of laterally attached toward the plus end, transporting chromosomes poleward to facilitate subsequent end-on binding. Additionally, the kinesin-like protein CENP-E captures chromosomes from the spindle periphery, using its motor domain to congress them toward the equator via interactions. These attachments generate mechanical forces essential for chromosome movement, with each microtubule-kinetochore connection producing approximately 1 pN of tension under load. This force arises from microtubule coupled to kinetochore gripping, balancing attachment stability across species where kinetochores handle single while mammalian ones manage multiple attachments to achieve similar per-fiber tension.

Bi-orientation and Tension Sensing

Bi-orientation refers to the stable attachment of sister kinetochores to emanating from opposite spindle poles, ensuring proper segregation during . This configuration generates pulling forces that stretch the kinetochores and centromeric , with intra-kinetochore distances increasing by approximately 20 nm between inner components like CENP-A and outer elements such as Ndc80 under tension. This tension stabilizes amphitelic attachments while distinguishing them from erroneous configurations, such as syntelic (both sisters to one pole) or merotelic (one kinetochore to both poles) orientations. The primary mechanism for sensing this tension involves the spatial separation of Aurora B kinase, localized at the inner , from its substrates in the outer kinetochore. In the absence of tension, Aurora B remains proximal to these substrates (e.g., Ndc80 complex proteins), promoting their and thereby weakening binding affinity. Upon bi-orientation, the applied tension elongates the kinetochore structure, increasing the distance—estimated at around 80 nm based on the INCENP tether length—between Aurora B and its targets, which reduces levels and stabilizes correct attachments. This "dog leash" model underscores how mechanical forces directly regulate enzymatic activity without requiring additional signaling cascades. When tension is low, as in erroneous syntelic or merotelic attachments, sustained Aurora B proximity leads to persistent of outer kinetochore components, which decreases microtubule plus-end rates and enhances . This promotes the detachment and turnover of incorrectly bound , facilitating error correction and the search for bi-oriented configurations. Experimental from and micromanipulation studies confirms that artificially reducing tension stabilizes such errors, while restoring it triggers rapid destabilization. A recent study in budding yeast has revealed that force transmission through the inner kinetochore occurs via two parallel pathways—the Mif2-dependent route and the Okp1/Ame1 (OA) complex route—both of which are crucial for bi-orientation stability. Using chimeric centromeric DNA constructs, researchers demonstrated that centromeric sequences in Cse4 nucleosomes specifically enhance OA-mediated force propagation, leading to stronger microtubule attachments and reduced detachment under load. This dual-pathway mechanism ensures robust tension generation and maintenance, minimizing segregation errors.

Regulatory Roles

Spindle Assembly Checkpoint

The spindle assembly checkpoint (SAC) is a critical surveillance mechanism at kinetochores that delays the onset of until all achieve proper bipolar attachments, thereby ensuring accurate chromosome segregation during . Unattached kinetochores serve as the primary signal generators for SAC activation, recruiting checkpoint proteins to initiate a diffusible inhibitory signal that propagates throughout the cell. This process prevents premature separation of , which could lead to genomic instability. SAC activation begins when unattached kinetochores recruit the Mad1-Mad2 complex via the KMN network component KNL1. Specifically, the kinase Mps1 phosphorylates MELT motifs on KNL1, enabling binding of Bub1-Bub3, which in turn recruits the Mad1-Mad2 core complex to the kinetochore. This kinetochore-localized Mad1-Mad2 acts as a template to catalyze the conversion of cytosolic open-Mad2 (O-Mad2) to closed-Mad2 (C-Mad2), which binds Cdc20 to form an intermediate complex. This intermediate then associates with BubR1-Bub3 to assemble the mitotic checkpoint complex (MCC), consisting of Mad2, BubR1, Bub3, and Cdc20. The MCC diffuses from the kinetochore to inhibit the anaphase-promoting complex/cyclosome (APC/C) in the cytoplasm, a E3 ubiquitin ligase that targets securin and cyclin B for degradation; this inhibition blocks the activation of separase and the degradation of cyclin B1, respectively, thereby maintaining high cyclin B-Cdk1 activity and arresting the cell in metaphase. Even a single unattached kinetochore can generate sufficient diffusible MCC to sustain the wait-anaphase signal across the cell. SAC silencing occurs upon microtubule attachment to kinetochores, which displaces the Mad1-Mad2 complex and halts MCC production. Microtubule occupancy leads to the recruitment of protein phosphatase 1 (PP1), which dephosphorylates KNL1's MELT motifs, stripping Bub1-Bub3 and Mad1-Mad2 from the kinetochore; additionally, dynein-mediated transport and intra-kinetochore stretching contribute to this disassembly. With the loss of the Mad1-Mad2 template, MCC levels decline, allowing APC/C activation, securin degradation, separase-mediated cohesin cleavage, and anaphase progression. This attachment-dependent silencing ensures the checkpoint is satisfied only when all kinetochores are properly engaged. The SAC is essential for preventing , as its dysfunction allows missegregation and genomic instability. Defects in SAC components, such as mutations in BUB1B (encoding BubR1) or TRIP13, are associated with chromosomal instability syndromes like mosaic variegated and increased tumorigenesis risk in various cancers, including colorectal and breast tumors. For instance, partial loss of SAC function promotes -driven cancer progression by permitting cells with unbalanced genomes to proliferate.

Error Correction

Error correction in kinetochores ensures the fidelity of chromosome segregation by destabilizing improper attachments, such as syntelic or merotelic orientations, while stabilizing bi-oriented attachments that generate inter-kinetochore tension. This process relies on spatial and tension-dependent of and activities at the kinetochore, preventing during . The primary effector of error correction is Aurora B kinase, a component of the chromosomal passenger complex (CPC) localized to the inner centromere. Aurora B phosphorylates key outer kinetochore proteins, including the Ndc80 complex and KNL1, which reduces their affinity for microtubules and promotes detachment of low-tension attachments. These phosphorylation events occur preferentially at attachments lacking tension, as the distance between the inner centromere-localized Aurora B and outer kinetochore substrates increases under tension, limiting kinase access. Opposing this, protein phosphatases PP1 and PP2A dephosphorylate these sites when tension is applied, stabilizing correct bi-oriented attachments by enhancing microtubule-binding affinity. Additional contributors include microtubule-depolymerizing enzymes like MCAK (mitotic centromere-associated ), a -13 family member that localizes to kinetochores and accelerates of incorrectly attached ends. MCAK's activity complements Aurora B by directly shortening , facilitating detachment and recycling of for new attachment attempts. A 2025 study in revealed that the Spc105/Kre28 complex recruits Ipl1 (the homolog of Aurora B) and its activator Sli15 to the outer kinetochore, enhancing local and error correction efficiency independent of inner localization. Error correction operates through iterative cycles of attachment formation, tension sensing, destabilization of errors, and reattachment until stable bi-orientation is achieved. This process is gated by a tension threshold of approximately 4–6 pN per kinetochore-microtubule attachment, below which erroneous configurations persist and are corrected. Recent insights from 2025 highlight how inner kinetochore complexes, such as the COMA network (including Mif2 and Okp1/Ame1), enhance force transmission from centromeric DNA to the outer kinetochore, improving the sensitivity and efficiency of tension-dependent error correction. This structural reinforcement ensures that even subtle tension differences effectively modulate Aurora B activity, reducing the time required for bi-orientation.

Comparative Aspects

In Yeast

In budding yeast (Saccharomyces cerevisiae), kinetochores assemble at point centromeres, which are compact DNA sequences of approximately 125 base pairs that specify a single site for kinetochore formation on each chromosome. These point centromeres are defined by a single nucleosome containing the centromere-specific histone variant Cse4 (the yeast homolog of CENP-A), which wraps the central 80 base pairs of the centromeric DNA and serves as the foundational platform for kinetochore assembly. Unlike more complex centromeres in other organisms, this minimalist configuration results in each kinetochore attaching to just one microtubule, facilitating precise chromosome segregation in the yeast's small mitotic spindle, which measures about 1-2 micrometers in length. The kinetochore structure in budding is notably simpler than in higher eukaryotes, lacking a fibrous corona layer and relying on a streamlined set of protein complexes for interaction. Key outer kinetochore components include the conserved Ndc80 complex, which binds through its calponin-homology domains in the Ndc80 and Nuf2 subunits, and the Mtw1-containing MIND complex (with Mtw1 as the yeast homolog of Mis12), which bridges the inner and outer kinetochore layers to recruit Ndc80. A distinctive feature is the Dam1/ complex, a ten-subunit ring that oligomerizes to encircle lattices, enabling processive kinetochore movement along depolymerizing and stable attachment under tension. This ring structure, coupled with Ndc80, allows for rapid capture and attachment in the confined space of the yeast spindle, supporting efficient biorientation and segregation of the 16 chromosomes. Error correction in budding yeast kinetochores is mediated by the Aurora B Ipl1, which phosphorylates outer kinetochore proteins like Dam1 and Ndc80 to destabilize improper attachments lacking tension. A recent discovery revealed that Ipl1 is recruited to the outer kinetochore via direct interaction between its activator Sli15 and the Spc105/Kre28 complex (a component of the KMN network), enabling tension-sensitive error correction at the attachment site. This outer recruitment mechanism enhances the kinase's local activity, promoting detachment of syntelic or merotelic attachments and reorientation toward stable bi-orientation. In fission yeast (Schizosaccharomyces pombe), kinetochores form at regional centromeres spanning 35-110 kilobases, which incorporate multiple Cse4 nucleosomes (typically 3-5 per ) and attach to 1-3 , providing a slightly more elaborate but still simplified model compared to multicellular organisms. Core components like Ndc80 and the Mtw1 homolog Mis12 contribute to binding, while Ipl1/Aurora B performs analogous error correction roles, though lacking an essential Dam1 ring; processivity is achieved through Ndc80 complex oligomerization and plus-end tracking proteins such as Mal3 (the EB1 homolog), with the Sim4 complex contributing to inner kinetochore assembly. Both species offer exceptional advantages for kinetochore due to their genetic tractability, allowing precise manipulations via temperature-sensitive mutants and editing, and their absence of a corona layer, which simplifies visualization and dissection of core assembly pathways.

In Metazoans and Plants

In metazoans, particularly vertebrates, kinetochores assemble on regional centromeres characterized by repetitive DNA sequences spanning 10-100 kb, which facilitate the recruitment of centromeric proteins like CENP-A. These kinetochores typically attach to 15-35 per kinetochore, enabling robust chromosome-to-spindle connections during . A distinctive feature is the fibrous corona, a transient outer layer visible on unattached kinetochores that aids in initial capture through proteins such as CENP-E and ZW10. In plants, kinetochore organization varies between monocentric and holocentric types; for instance, exhibits monocentric kinetochores where CENH3, the plant-specific histone H3 variant analogous to CENP-A, localizes to centromeric regions for precise attachment. Holocentric kinetochores, observed in species like , distribute CENH3 along the length, allowing diffuse spindle attachments without a single constriction point. As of 2025, structural studies have defined the plant KMN network, revealing adaptations in CENH3 structure and anastral spindle geometry lacking centrosomes, which differ from astral spindles in metazoans and support flexible alignment in diverse plant architectures; evolutionary analyses indicate divergence through KMN component expansions enhancing tolerance. Key differences between metazoan and plant kinetochores include the absence of a prominent fibrous corona in , compensated by unique plus-end tracking proteins (+TIPs) such as EB1 homologs that stabilize plus ends at attachment sites. correction mechanisms in rely on Aurora-like kinases, which destabilize improper attachments similar to metazoan Aurora B, but with adaptations for polyploid genomes. Functionally, kinetochores often manage higher numbers—typically 8–18 per kinetochore in meiotic spindles of species like —reflecting the demands of expansive spindles in tissues. This scalability supports tolerance, where robust kinetochore- interfaces prevent during rapid cell divisions in development and stress responses.

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