21st Century Directions in Biology
The Kinetochore Moves Ahead:
Contributions of Molecular and
Genetic Techniques to Our
Understanding of Mitosis
MARY KATHRINE JOHNSON AND DWAYNE A. WISE
Cell division is necessary to the building of a soma from the single-celled zygote during development, as well as the sine qua non, in the form of
meiosis, for the evolutionary success of species. Here we review recent progress in our understanding of a key player, the kinetochore, in these
processes. The kinetochore is both the anchor to the mitotic spindle for chromosomes at division and the motor for distribution of chromosomal units
to daughter cells. In addition, the kinetochore plays a key role in the molecular checkpoints of cell-cycle progression. Although the nucleation of the
kinetochore at a chromosomal site is under epigenetic control, the underlying base sequence of the DNA at the centromere is not critical: The
assembly of the kinetochore occurs at exactly the same place on the same chromosomes at every division cycle. We discuss recent advances in our
understanding of how the kinetochore is organized and assembled, as well as how it contributes to critical cell-cycle checkpoints and to chromosome
movement.
Keywords: kinetochore, molecular methods, history of the kinetochore, mitosis
C
ell division is a cornerstone of evolutionary success in
higher organisms: It provides for a body, the soma, and
for the gametes necessary to produce the next generation. In
this review, we focus on a critical component of karyokinesis, the partitioning of the genome contained in the nucleus.
This is accomplished by the mitotic spindle, a highly conserved
feature of karyokinesis in the cells of multitudes of organisms
that have been examined by light or electron microscopy.
After 125 years of study, we know much about the “mitotic
machine,” but as we hope to demonstrate here, much remains to be discovered. To the continual bafflement of many,
both the terms “centromere” and “kinetochore” are used for,
variously, the constriction in chromosomes that marks the
location of spindle attachment, the underlying DNA base
sequence at this site, the heterochromatin observable at this
location in many organisms, and the multiprotein complex
that provides attachment to the spindle and the motor
molecules that move the chromosomes at anaphase.
To be clear, we define the kinetochore as a highly conserved
structure localized at the primary constriction on chromosomes (Maney et al. 2000). When we use the term “centromere,” we mean the underlying base sequence at this site, and
we are aware of the irony that this structure (by definition)
cannot be seen in the light microscope (Earnshaw 1991). The
kinetochore is a multiprotein chromatin complex at which
the forces of mitosis work to congress, and later to separate,
chromosomes into daughter cells. We will treat the meiotic
and mitotic kinetochore identically, specifying only when it
is relevant, because a significant difference between the two
has never been observed. However, it is worth keeping in mind
that the back-to-back orientation of sister kinetochores at
mitosis and at meiosis II is rearranged to the side-by-side
arrangement seen in meiosis I cells. Further, the accurate
separation of homologues (reduction) depends on this
rearrangement (figure 1; Nicklas 1986). Kinetochore structure and function have been well studied and many reviews
are available (e.g., Rieder 1982, Brinkley 1990, Cleveland et
al. 2003, Maiato and Sunkel 2004, Chan et al. 2005, Carroll
and Straight 2006). Therefore, our presentation will not be
comprehensive. Instead, we will detail how traditional microscopy and modern molecular techniques have merged to
produce new insight into kinetochore structure and function.
The history of kinetochore research:
Light microscopy
The light microscope was the first, and arguably is the most
significant, tool in the study of cellular division. Images of
mitosis in living cells have illuminated the basic mechanisms
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Figure 1. (a) Structure of the vertebrate kinetochore at the light (left) and electron (right) microscope levels. The centromere
region is shown by virtue of binding to antibodies specific for kinetochore proteins. The two sister kinetochores have back-toback orientation and are attached to the outer aspect of the chromatid. (b) The kinetochore has a trilaminar structure with
outer and inner electron-opaque layers and a middle electron-translucent layer. The outer lamina binds spindle
microtubules during prometaphase. Rearranged from figure 17-36 in Alberts and colleagues (2008).
of mitosis, such as the shape of the spindle, kinetochore
attachment to spindle poles by fibers (later known to be
microtubules constructed from tubulin polymers), condensation of genetic material into chromosomes, congression of
chromatids, and the separation of sister chromatids in
anaphase (Mitchison and Salmon 2001).
With the advent of chromosome banding, centromeric
heterochromatin became observable. Heterochromatin (as
opposed to euchromatin) is highly condensed, which makes
it bind firmly to certain dye molecules (Mitchison and Salmon
2001). Chromosome banding techniques use a variety of
treatments and stains to produce patterns of differentiation
on the chromosome. These patterns allowed for identification
and early homology studies (Sumner et al. 1971). One technique, C-banding, allows Giemsa stain binding for visualization of heterochromatic regions, which can be found near
most kinetochores. C-banding was observed first in the light
microscope, and then in the transmission electron microscope
(TEM; Jack et al. 1985).
In addition to observation, light microscopy has been
useful for elucidation of the physical properties of kinetochores. Micromanipulation studies showed that kinetochores are physically attached to the spindle. When
chromosomes in live cells were pulled with a micromanipulation needle, the kinetochore was stretched, but it
remained attached to microtubules (Nicklas et al. 1982). A
combination of light microscopy, the TEM, and micromanipulation allowed Nicklas and colleagues (1982) to pull
chromosomes out of the spindle. They found that after
being removed, chromosomes reattached and moved back
into the spindle, where they were reoriented. Light microscopy
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was one of the earliest tools used in kinetochore research and
it remains one of the most powerful, especially with the
advent of the laser scanning confocal microscope, which
can produce a three-dimensional image.
A powerful addition: Transmission
electron microscopy
Transmission electron microscopy is an important tool in determining kinetochore structure, given the size of the structure (roughly 1 to 2 micrometers [μm] in diameter; Brinkley
and Stubblefield 1966). Use of the TEM allowed for the elucidation of the trilaminar structure of the kinetochore (figure 1). The kinetochore has a coarsely granular inner plate that
is 20 to 40 nanometers (nm) wide; an outer plate, 30 to 40 nm
wide, composed of fibrillar components; and a middle zone,
15 to 35 nm wide, made of loosely organized fibrillar material that appears electron-translucent in the TEM (McEwen
et al. 1993). What appear to be hairlike projections on the outer
plate are most conspicuous on kinetochores that have not
made contact with microtubules (Brinkley 1990). This area
was termed the “corona” by Jokelainen (1967). The TEM allowed for accurate measurement of kinetochore size: from 0.4
to 0.6 μm in width in PtK (rat kangaroo) cells, but as large as
1.45 μm in length in Indian muntjac cells (Earnshaw 1991).
In prophase, the kinetochore appears to be a ball inserted
into a dense cup. In vertebrates, kinetochores appear soon
after the nuclear envelope breaks down, and the cell enters
prometaphase. Microtubules connect with kinetochores, and
the ball-and-cup structure begins to differentiate into a platelike structure. The corona is then observable until the
microtubules bind, after which it is less visible. By late
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prometaphase, the fully developed trilaminar plate is apparent. The kinetochore plate remains visible until the nuclear
envelop re-forms in telophase (Maiato and Sunkel 2004).
A diverse group of organisms, from diatoms to humans,
has kinetochores that are trilaminar in structure, but not all
kinetochores fit this description (Wise 1988, Brinkley 1990).
Insects and higher plants have a ball-and-cup kinetochore
(Rufas et al. 1994). Even so, Maiato and colleagues (2006)
argued that the kinetochores of Drosophila, an insect, are
very similar to those of vertebrates.
Microtubule attachment to kinetochores was first observed
in detail using TEM (Brinkley and Stubblefield 1966). Kinetochore fibers (K-fibers) are bundles of microtubules that connect to kinetochores. There appears to be a rough correlation
between evolutionary complexity and the number of kinetochore microtubules. The budding yeast, Saccharomyces
cerevisiae, has one microtubule attached to the kinetochore,
whereas the fission yeast, Schizosaccharomyces pombe, has
two to four; Drosophila melanogaster has four to six; and
mammals have 20 or more microtubules. The packing of
microtubules in the kinetochore plate seems constant, with
an intermicrotubule spacing of 50 to 60 nm (Maiato and
Sunkel 2004).
Live-cell light microscope imaging and TEM used in tandem have been successful tools in understanding K-fibers.
McEwen and colleagues (1997) determined that in PtK cells,
the number of microtubules attached to each kinetochore increases from prometaphase to anaphase. This finding indicates
that the fibers are more stable in later mitotic stages than in
earlier stages (McEwen et al. 1997), and leads to the notion
of kinetochore maturation, the idea that kinetochores become
more stable as mitosis progresses.
Major technological advances such as the discovery of restriction endonucleases, recombinant DNA technology, and
advanced confocal laser scanning microscopy have revolutionized the study of mitosis (Macgregor 1993). The merger
of modern technology with microscopic techniques has enabled researchers to ask questions that had not been possible
before. Here, we detail some of the results relevant to kinetochore structure and function.
Centromeric DNA
Centromeric DNA cloning and sequencing techniques are
complex, and will not be detailed in this review; however, the
progression of DNA sequence to kinetochore and protein
function is an important point, and thus is covered briefly (for
a review, see Carroll and Straight 2006). The kinetochore
forms in the same location on each chromosome at each
mitotic event. However, the location is not always determined by the specific DNA sequence. In budding yeast,
the centromeric sequence is 125 base pairs (bp) in length,
divided into three domains (Fitzgerald-Hayes et al. 1982).
These sequences are not chromosome specific; sequences
can be switched between chromosomes to no ill effect.
Carbon and Clarke (1984) altered the nucleotide sequence
of the DNA surrounding the centromere of yeast chromosome
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III and found that domains two and three are necessary for
normal centromere function. Therefore, a specific region is
required for segregation. The fission yeast contains a central
nonrepetitive region, flanked by centromere-specific innermost repeats that form the site of kinetochore assembly (Carroll and Straight 2006). Surrounding this central domain are
long, tandem arrays of outer repeats, common to all centromeres in S. pombe, for a total length of 40 to 100 kilobase
pairs. No single DNA sequence is necessary for kinetochore
assembly, indicating an epigenetic mechanism. How these
factors work is not yet evident.
Centromeres of human chromosomes contain large arrays
of tandem repeated 170 bp a-satellite monomers that can span
several megabases (Carroll and Straight 2006). Masumoto and
colleagues (1989) used immunoblots and gel electrophoresis
in HeLa cells to show that many of these repeats contain a
binding site for a sequence specific protein, CENP-B. Therefore, a-satellite DNA can promote the binding of sequencespecific DNA binding proteins. Neocentromeres, at which a
new centromere is activated on a noncentromeric region,
have been observed in several organisms. Even though some
known kinetochore proteins do not bind to these neocentromeres, a functional kinetochore is assembled. Neocentromeres are heritable through many generations, so specific
DNA sequences are not necessary for kinetochore assembly,
but could be favorable (Carroll and Straight 2006). The discovery of neocentromeres strengthened the idea that epigenetic factors determine kinetochore placement.
Human antikinetochore antibodies
Serum from human patients with scleroderma (CREST)
contains autoantibodies that bind specifically to kinetochore
proteins (Moroi et al. 1980, Tan et al. 1980). These antibodies
have been localized to kinetochores during mitosis, but
also reveal “prekinetochores” present during interphase. In
early interphase, prekinetochores are single and become
doubled in later interphase (figure 2). This suggests that there
is a presumptive, or preliminary, kinetochore when the
chromatin is decondensed. However, there is no structural similarity to the mitotic kinetochore, as discrete mitotic kinetochore structure does not appear in interphase in animal cells
(Brenner et al. 1981). CREST staining showed for the first time
that kinetochore proteins are present throughout the cell
cycle.
While the cytology and ultrastructure of kinetochores
were relatively well understood before these experiments, little was known about the biochemical composition of the
kinetochore (Valdivia and Brinkley 1995). The use of CREST
antiserum to label the kinetochore was responsible for initiating a period of molecular and biochemical experiments that
led to discoveries about kinetochore proteins and an understanding of the kinetochore assembly and checkpoint pathways. To date, more than 60 kinetochore proteins have been
detected in budding yeast, and more than 100 in vertebrate
cells (figure 3; Okada et al. 2006, Musacchio and Salmon
2007).
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Figure 2. Prekinetochores in Chinese hamster ovary cultured cells revealed by binding to kinetochore-specific antibodies
found in human CREST serum. (a) Single structures in early interphase; (b) double structures in late interphase.
Magnification approximately 3000X.
The kinetochore assembly pathway
and CENP-A analysis
A variety of kinetochore assembly proteins was examined
in parallel from the CREST breakthrough, but we will use
only one, the centromere-associated protein A (CENP-A),
as an example of how modern techniques unite with
microscopy to provide information about a protein or
pathway. Earnshaw and Rothfield (1985) and others (Valdivia and Brinkley 1985) used SDS-PAGE (sodium dodecyl
sulfate polyacrylamide gel electrophoresis) or fractionation and immunoblots (Western blots) to discover several
discrete antibodies in CREST serum. Using these antibodies, the first centromere-associated proteins (CENPs)
were identified (Craig et al. 1999). Palmer and colleagues
(1991) used reverse-phase, high-performance liquid chromatography (HPLC) to purify CENP-A (a 17 kD protein
identified by Earnshaw and Rothfield [1985]) from bull
spermatozoa, and determined a partial gene sequence.
The sequence was found to be similar to that of the histone H3, which is involved in the structure of chromatin.
These results suggested that CENP-A had a role in centromeric chromatin packing and function. The sequence
and function of CENP-A was revealed by the isolation of
partial cDNA using reverse-transcriptase PCR (polymerase chain reaction), chimeric proteins, crystal structure studies, and the creation of mutants. Through these
experiments and others, highly conserved CENP-A
histone H3–like variants have been found in all eukaryotes observed (Maney et al. 2000, Kitagawa and Heiter
2001).
Immunofluorescence, the labeling of antibodies that
bind to a protein in the cell with fluorescent dyes, allowed
for easier protein localization than did previous methods. CENP-A antibodies allowed for immunofluorescence, and gave information about the localization of the
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Figure 3. A diagram of kinetochore structure, including many of
the protein complexes known to be involved in metazoan kinetochore structure or function. The precipitating event in kinetochore
assembly is the binding of the modified histone, CENP-A, to
nucleosomes in the centromere region. Source: Figure 4 from
Musacchio and Salmon (2007); see that article for a full
definition of protein acronyms.
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21st Century Directions in Biology
protein. Comparisons between immunofluorescence and
immunoeletcron microscopy determined that the protein is
found on the inner kinetochore plate (Maney et al. 2000).
Specific genes can be silenced (“knocked out”) using
genetic engineering techniques. Knockouts enabled further
analysis of CENP-A in vivo. Howman and colleagues (2000)
found that in CENP-A heterozygous knockout mice, there
was no obvious effect; the mice were fertile and apparently
healthy. However, all homozygous knockout mutants died
within a few days. Mutant embryos had severe mitotic defects
that were specifically characterized. In addition, other CENP
proteins were affected by the loss of CENP-A, indicating that
CENP-A was necessary for kinetochore targeting of these
proteins. RNA interference (RNAi) inhibits gene expression
by affecting transcription of the gene of interest. RNA interference technology was used to deplete CENP-A in HEp-2
(human) cells. Depletion of CENP-A caused a high percentage of defects in mitosis. A budding yeast CENP-A homolog,
Cse4p, was introduced after CENP-A depletion, and it was
determined that the homolog could rescue CENP-A depleted
cells, and therefore was functionally similar (Wieland et al.
2004). Thus, through a variety of molecular techniques, it was
discovered that CENP-A localization depends on various
other proteins (Hayashi et al. 2004, Okada et al. 2006, Kwon
et al. 2007). Together, these experiments permitted a better
understanding of the centromeric chromatin and the complicated kinetochore assembly pathways (figure 2). To date,
at least two pathways are known: the Mis12 pathway, independent of CENP-A, and a CENP-A assembly pathway (Chan
et al. 2005). In addition, pathways that connect inner kinetochore proteins (such as CENP-E) with outer kinetochore
proteins (motor and checkpoint proteins) have also been
found.
The kinetochore fiber
All spindles are bipolar, and in animal cells, the centrosomes
form the spindle poles. In budding yeast, spindle pole
bodies are embedded in the nuclear envelope, which does
not break down during mitosis. Higher plants and certain
other organisms do not have centrosomes; they rely on microtubules and associated proteins to create a bipolar spindle.
Microtubules have intrinsic polarity, with the plus end located
at the equatorial plane and the minus end at the spindle
poles (Gadde and Heald 2004). Microtubules are nucleated
from centrosomes to create kinetochore fibers. It has been
known for decades that only the bipolar orientation of sister
kinetochores is stable (figure 4). Unstable orientations are most
often converted to the stable one. When this does not happen,
nondisjunction occurs.
Although it is true that kinetochore microtubules are
nucleated at the poles in most spindles, it is a fact that the
kinetochore itself can nucleate microtubule assembly under
some circumstances (Rieder 2005). In early experiments,
when isolated HeLa mitotic chromosomes were incubated
with isolated chick brain tubulin, the kinetochores could act
as initiation sites for microtubule polymerization (Telzer et
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al. 1975, Maiato and Sunkel 2004). These results were further
confirmed using lysed cells incubated with exogenous tubulin (Snyder and McIntosh 1975). Mitchison and Kirschner
(1984) found that microtubules are dynamic and have two
phases, a “growing” and a “shrinking” phase, which was confirmed by experiments using immunofluorescence and light
microscopy (Maiato and Sunkel 2004). These results led to the
search-and-capture model of kinetochore-microtubule interaction proposed by Mitchison and colleagues (1986), which
posited that in early stages of mitosis, microtubules grow
from the centrosomes and probe the cytoplasmic space
until they contact a kinetochore. When Hayden and colleagues (1990) used light microscopy to show that chromosome attachment in newt cells results from astral microtubules
interacting with a kinetochore, the search-and-capture model
became the most widely accepted model of kinetochoremicrotubule interaction (figure 5; Rieder 2005).
Recent experiments (Compton 2000) show that microtubule nucleation is enhanced by centrosome-associated gtubulin and the ring complex it forms (Job et al. 2003). Also,
several microtubule-associated proteins have been shown to
have various functions such as stabilizing, anchoring, and nucleating microtubules (Schuyler and Pellman 2001, Maiato and
Sunkel 2004). These studies provide support for the searchand-capture model, and indicate that the basic requirement
to create a kinetochore fiber in vitro seems to be microtubules
and a functioning kinetochore to stabilize polymerized microtubules (Maiato and Sunkel 2004).
Congression
Congression refers to the back-and-forth movements of chromosomes, with final arrival at the spindle equator (figure 5).
Figure 4. A diagram of the ways in which two sister
kinetochores can attach to the spindle. Each orientation
can be converted to the only stable (bipolar) orientation
by changes in spindle microtubules binding. A molecular
mechanism, the spindle assembly checkpoint, monitors
stable attachment of all chromosomes, and perturbations
of attachment or the tension generated across the sister
kinetochores can delay anaphase onset. Source: Diagram
from figure 17-39 in Alberts and colleagues (2008).
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Rieder and Alexander (1990) demonstrated in newt lung
cells that chromosomes can interact with a spindle microtubule through lateral interactions in early prometaphase. The
kinetochore is then transported poleward on the surface of
the microtubule without microtubule depolymerization,
suggesting that motor proteins on the surface of the kinetochore or on the surface of the microtubules could be responsible for early chromosome movement. It has been suggested
that chromosomes that are closer to one spindle pole than
to the other undergo polar ejection forces, which push the
chromosomes toward the midzone of the spindle. Rieder
and colleagues (1986) used laser microsurgery in conjunction
with light microscopy and TEM to create mono-oriented
chromosomes with one kinetochore that moved away from
the pole. Optical trapping microscopy has recently indicated
that motor proteins located in or on the chromosome arms
(such as the protein Kid, a kinesin-like DNA-binding
protein) may interact with astral microtubules and may be
responsible for these forces (Antonio et al. 2000, Brouhard and
Hunt 2005).
In living newt lung cells, Skibbens and colleagues (1993)
made use of high-resolution video microscopy and computer-assisted tracking techniques to measure the positions
of individual kinetochores with respect to the poles through
time. These experiments showed that kinetochores oscillate,
switching between movements toward one pole (termed “P”)
and the other (termed “AP”). This phenomenon was termed
“directional instability.” Later, video light microscopy and
TEM determined that the trailing kinetochore (moving away
from the pole) always is bound to at least twice as many
microtubules, and thus may require more force than the
leading kinetochore (moving toward the pole; Maiato and
Sunkel 2004). Sister kinetochores are attached to both
spindle poles, and Mitchison and colleagues (1986) determined, using microinjected biotinylated tubulin and the
TEM, that one kinetochore fiber must polymerize while
the other depolymerizes. These results have been supported
by other experiments (Wise et al. 1991, Maiato and Sunkel
2004).
Maddox and colleagues (2003) proposed a “slip-clutch”
mechanism for congression movement. High-resolution
fluorescent speckle microscopy and direct labeling of
kinetochores used in Xenopus extracts showed that at high
tensions, microtubule polymerization is promoted in order
to prevent detachment, and that the polymerization state
of the kinetochore is determined by tension. Therefore, kinetochores oscillate from poleward (pulling forces) to antipoleward because of spindle forces that produce flux (Maiato
and Sunkel 2004). Plus-end–directed motor proteins,
chromokinesins (Nod and Kid), also play an important part
in the congression of chromosomes. Immunolocalization and
overexpression of Nod in Drosophila showed that in meiotic
cells that have not undergone recombination, Nod is necessary for the proper alignment of chromosomes. Antibodyinduced inhibition of Kid was shown to block chromosome
oscillations, and immunodepletion prevented proper
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chromosome alignment (Cleveland et al. 2003). Molecular
techniques, protein identification, and function information
have all aided in understanding chromosome congression.
The spindle assembly checkpoint
Progression through mitosis is controlled by a series of checkpoints. The spindle assembly checkpoint (SAC) operates to
assure proper distribution of sister chromatids. In mitosis,
kinetochore pairs attach to microtubules, align at the
metaphase plate, and migrate to opposite poles to be separated
into the daughter cells (figure 5). The SAC prevents the transition from metaphase to anaphase until proper alignment and
tension are detected by a series of proteins. One unattached
or ill-aligned kinetochore causes metaphase arrest and will
prevent aneuploidy.
The SAC functions by inhibiting the anaphase promoting
complex (APC), an E3 ubiquitin ligase that targets regulating
proteins for degradation by the 26S proteasome, turning off
the checkpoint (Chan et al. 2005). When active, the APC
targets the protein securin, and securin then triggers the
release of separase, leading to the degradation of cohesin.
Cohesin is a protein that binds sister chromatids together, and
its degradation allows the sisters to be pulled apart in anaphase.
One of the APC regulators is the protein Cdc20 (or Slp1 in
fission yeast). Many proteins are involved in the pathway,
with more being discovered regularly. However, since this is
not a checkpoint review, we will forgo comprehensive analysis here, and instead will demonstrate how molecular tools
contributed to this research.
The attachment component of the SAC
When the human gene, mitotic arrest deficient 2 (MAD2),
was sequenced, the protein was found to be homologous
with budding yeast Mad2 and Xenopus Mad2, implying that
the protein is evolutionarily conserved. Li and Benezra (1996)
used electropermeabilization, a technique that increases the
permeability of the cell membrane, to incorporate anti-Mad2
antibodies into cells, and found that Mad2 was necessary to
activate the SAC. Another study (Gorbsky et al. 1998) confirmed these results in other mammalian cells.
The yeast two-hybrid technique, which allows the investigation of protein-to-protein interactions, has been instrumental in understanding mitotic checkpoint mechanisms.
Through yeast two-hybrid interactions, it was discovered
that Mad1, Mad2, and Mad3 interact with Cdc20 (Hwang
et al. 1998). These experiments initiated a series of others
that investigated the mitotic checkpoint and the involvement
of Mad2. Experiments using immunofluorescence showed
Mad2 to be concentrated on unattached kinetochores in
prometaphase, and to delocalize after chromosomes were
properly attached to the spindle (Chan et al. 2005).
The use of the drug taxol, which decreases tension at the
kinetochore by promoting microtubule assembly, was used to
determine whether or not Mad2 monitors for tension or for
alignment of the kinetochore in PtK1 cells (Waters et al.
1998). Taxol was added and Mad2 relocalized to only a few
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Figure 5. Activities of the kinetochore during the course of mitosis. Top: Attachment to the plus ends of spindle microtubules
and alignment at the equator (congression). The force necessary to move the chromosome during congression is probably
provided by motor proteins embedded in the kinetochore itself. Bottom: Separation of sister chromatids and movement to
opposite spindle poles (anaphase A). Sliding of overlapping midzone microtubules to move apart the spindle poles (anaphase
B). The kinetochore is probably not involved in the latter. Source: Rearranged from figures 17-28 and 17-46 in Alberts and
colleagues (2008).
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kinetochores, probably because loss of tension can lead to loss
of microtubule attachment. Also, when microtubules were
depolymerized using the drug nocodazole, Mad2 returned,
indicating that the protein detects unattached kinetochores
rather than tension (Maney et al. 2000). It was recently determined that the protein exists in two conformations, open
(O-Mad2) and closed (C-Mad2) forms (figure 3). At least two
models of Mad2 regulation emerged from these discoveries,
both of which implicate the protein Cdc20 (Chan et al. 2005).
Molecular, genetic, and biochemical methods have illuminated
possible mechanisms of action of Mad2 and other proteins
involved in the attachment component of the checkpoint
(Chan et al. 2005).
The tension component of the SAC
The tension component of the checkpoint, the 3F3 phosphoepitope, was originally detected in studies of antibodies
directed against Xenopus egg extracts (Cyert et al. 1988).
Later, Gorbsky and Ricketts (1993) used cell fractionation, immunoblots, and immunofluorescence to determine that the
3F3 phosphoepitope binds to kinetochores in prophase and
prometaphase, and in midprometaphase in misaligned kinetochores. The phosphoepitope brightly stained both sister
kinetochores near the poles, but the leading kinetochore was
more brightly stained near the metaphase plate. These and
other workers concluded that the phosphoepitope may have
a checkpoint function (Maney et al. 2000).
The microinjection of antibodies against the 3F3 phosphoepitope in PtK1 cells showed that cells remained labeled
at prophase and prometaphase, but anaphase was delayed.
In the same series of experiments, Campbell and Gorbsky
(1995) demonstrated that microtubule-destabilizing drugs
(which reduce tension at the kinetochore) added during
metaphase caused the 3F3 phosphoepitope to relocalize to the
kinetochore, providing additional evidence that the 3F3
phosphoepitope is involved in the SAC. In addition, they
used the TEM to determine that the 3F3 phosphoepitope
was found in the middle layer of the kinetochore. Nicklas and
colleagues (1995) showed that the 3F3 phosphoepitope monitors tension at the kinetochore. Using micromanipulation,
light microscopy, and immunofluorescence in grasshopper
spermatocytes, these authors determined that the longer
chromosomes remained off the spindle, the more strongly they
stained. A properly attached kinetochore was dislodged from
the spindle and reattached to one pole, resulting in loss of
tension at the kinetochore. Here, the kinetochores stained
brightly, indicating that attachment was not sufficient to lose
the 3F3 phosphoepitope. Other experiments confirmed these
results (Waters et al. 1998, Maney et al. 2000).
Various other experiments demonstrated that the 3F3
phosphoepitope was created by the enzyme polo-like kinase
1 (Plk1). Depletion of Plk1 led to a loss of 3F3 phosphoepitope
activity, while purified recombinant Plk1 could generate the
3F3 phosphoepitope. Small interfering RNA experiments
and rephosphorylation assays on Plk1 confirmed these results.
Recently, Wong and Fang (2007) used recombinant proteins,
940 BioScience • December 2009 / Vol. 59 No. 11
immunodepletion, and immunofluorescence to demonstrate
that phosphorylation of the protein BubR1 by the Xenopus
polo-like kinase 1 (Plx1) generates the 3F3 phosphoepitope.
Motor proteins
Of the various motor proteins located at the kinetochore, we
will focus on centromere protein E (CENP-E), since it has been
well studied and is involved in SAC activation (figure 3).
Yen and colleagues (1991) identified CENP-E as a protein
necessary for the metaphase to anaphase transition. In the
same series of experiments, immunofluorescence was used to
determine that CENP-E was first localized at the kinetochore
during prometaphase, and relocalized to the spindle midzone
at the start of anaphase. When sequenced, CENP-E was
determined to be a member of the kinesin family, with a
motor domain located at the amino terminus. The protein is
fairly well conserved, with homologues detected in a variety
of eukaryotes, budding yeast being an interesting exception
(Maney et al. 2000).
Yeast two-hybrid experiments showed that CENP-E interacts with CENP-F (found on the kinetochore) and the SAC
protein, BubR1. It was suggested that these proteins create a
binding site for CENP-E on the kinetochore. Other experiments determined that CENP-E was necessary for normal
mitosis, and defects in the protein resulted in severe mitotic
defects or mitotic arrest (Chan et al. 2005). These and other
results confirmed that CENP-E is a kinetochore-microtubule
sensor that relays information to the SAC.
Anaphase
After the SAC is satisfied, anaphase is initiated. Anaphase
occurs in two stages, anaphases A and B (figure 5). Anaphase
A is defined as chromosome movement toward the poles;
anaphase B occurs when the spindle elongates. Here, we will
discuss only anaphase A, since it is the component of anaphase
in which the kinetochore clearly plays a major role. Anaphase
B consists principally of spindle elongation by sliding of
overlapping midzone microtubules.
Although many models have been proposed for anaphase
A movement (Pickett-Heaps et al. 1982), two major models
are now being debated: the pac-man and the traction-fiber
models. The latter does not directly involve the kinetochore
and therefore will not be discussed here. The pac-man model
is currently the most accepted one, and proposes that poleward forces are generated near or at the kinetochore by
motor proteins (Nicklas 1989). As a by-product, the kinetochore microtubules disassemble, and the kinetochore “chews”
its way toward the pole (Gadde and Heald 2004). Protein functional studies have revealed new information about anaphase
movement and the function of motor proteins. Rogers and
colleagues (2004) discovered two microtubule-destabilizing
kinesin-like enzymes in Drosophila that are responsible for
chromosome-to-pole movement. One of these proteins
contributes to motility, as in the pac-man mechanism, while
the other is involved in poleward microtubule flux. Work
with molecular techniques such as RNAi and immunofluowww.biosciencemag.org
21st Century Directions in Biology
rescence microscopy have further clarified these models
(Zhang et al. 2007). The current state of affairs indicates that
both kinetochore motor molecules and microtubule flux
contribute to chromosome movement.
Experimental models
Attempts to create a model spindle have provided additional
insight into the function of kinetochores. Brinkley and colleagues (1988) found that when hydroxyurea and caffeine were
added to Chinese hamster ovary cells, the cells entered a premature mitosis without replicating their DNA. The chromatin was fractured and scattered throughout the cell and
kinetochores were detached from the chromosomes, producing single kinetochore fragments with small amounts of
chromatin attached. Wise and Brinkley (1997) found that these
fragments achieved prometaphase congression and metaphase
alignment, and interacted with microtubules in orthodox
ways. These experiments showed that even single kinetochore fragments are able to attach to the spindle, to congress
to the metaphase plate, and to be distributed equally to the
daughter cells.
Recent experiments using laser microsurgery and maize
meiotic mutants have suggested that a single kinetochore
can act in the same manner as two sister kinetochores
during congression, and that kinetochores are redundant
structures able to carry out their functions in duplicate
(Zinkowski et al. 1991, Khodjakov et al. 1997, Yu and Dawe
2000).
The future
Pickett-Heaps and colleagues (1982) wrote that “after 40
years of intense investigative work, biologists hardly seem
closer to a solution” for the mechanism of chromosome
movement. Modern molecular and genetic technology, merged
with microscopy, has improved our ability to probe, but
much is left to be determined about kinetochore assembly, the
SAC, and the role of the kinetochore in accurate chromosome
orientation and movement. We also have much to learn about
the intricacies of epigenetic factors on the centromere and on
the placement of the kinetochore. The analysis of protein
function, coupled with light and electron microscopy, will play
an important role in discovering more about the kinetochore’s structure and function.
Acknowledgments
The authors would like to thank Lisa Evans for her assistance and Charles Matyi for his advice. We are also grateful
for the sage advice of the reviewers of this manuscript. We
regret that many important papers could not be cited because
of space limitations; we have chosen those we deem to be most
relevant to the subject at hand. This article is dedicated to
Professor Bruce Nicklas for his abiding interest in and landmark work on the kinetochore and its various functions.
www.biosciencemag.org
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Mary Kathrine Johnson ([email protected]) and Dwayne A. Wise
([email protected]) are with the Department of Biological Sciences at
Mississippi State University.
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