* Laboratory of Cell Biology, Ludwig Institute for Cancer Research, Division of Cellular and Molecular Medicine, School of
Medicine, University of California, San Diego, La Jolla, California 92093-0660
Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Using immunoelectron microscopy, CENP-E is shown to be an integral component of the kinetochore corona fibers that tether centromeres to the spindle. Immediately upon nuclear envelope fragmentation, an associated plus end motor trafficks cytoplasmic CENP-E toward chromosomes along astral microtubules that enter the nuclear volume. Before or concurrently with initial lateral attachment of spindle microtubules, CENP-E targets to the outermost region of the developing kinetochores. After stable attachment, throughout chromosome congression, at metaphase, and throughout anaphase A, CENP-E is a constituent of the corona fibers, extending at least 50 nm away from the kinetochore outer plate and intertwining with spindle microtubules. In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement. Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.
CHROMOSOME movements during mitosis are orchestrated by the interaction of spindle microtubules
with a specialized chromosome domain located
within the centromere. This specialized region, called the
kinetochore (Luykx, 1965 A generally accepted idea is that microtubule motors located at or near the kinetochore power chromosome
movement during mitosis (Nicklas, 1989 For CENP-E, whose cell cycle-dependent accumulation
yields a maximum of ~5,000 molecules per HeLa cell in
G2/M, (i.e., about 50 molecules per kinetochore; Brown et
al., 1994 Antibodies
Antibodies against a part of the coiled-coil domain of CENP-E (amino acids 955-1571) were raised in rabbits as described by Brown et al. (1996) To test the specificity of the CENP-E antibody, mitotic cells were harvested by shake-off followed by sedimentation and solubilization in RIPA
buffer (25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.5% SDS, and 1% deoxycholate). The extracts were then sonicated and centrifuged to remove the
residual insoluble materials. The chromosome scaffolds were prepared as
described below. Before electrophoresis, an appropriate amount of extract was diluted with 4× sample buffer and boiled for 2 min. After separation in SDS-PAGE, the proteins were transferred onto a nitrocellulose
membrane (Micron Separation Inc., Westborough, MA) and incubated
with anti-CENP-E antibody followed by 125I-protein A. Immunoreactive
signals were visualized by autoradiography on Kodak BioMAX MS film
(Rochester, NY) for 6-8 h at Cell Culture
HeLa cells, from American Type Culture Collection (Rockville, MD),
were maintained as subconfluent monolayers in RPMI 1640 media
(GIBCO BRL; Life Technologies, Gaithersburg, MD) with 10% FCS
(Gemini Bio-Products, Inc. Calabasas, CA) and 100 U/ml penicillin plus
100 µg/ml streptomycin (GIBCO BRL; Life Technologies).
Chromosome Scaffold Preparation
Logarithmically growing HeLa cells were treated with 10 ng/ml nocodazole (Sigma Chemical Co.) for 18 h. After arrest, mitotic HeLa cells were
harvested by mitotic shake-off and washed with ice-cold PBS. Chromosomes were isolated by the protocol described by Mitchison and Kirschner
(1985) After centrifugation, a visible, flocculent band migrating at the 50-60%
sucrose interphase was harvested and suspended in 3 vol of PEM buffer.
A subsequent chromosome scaffold preparation was performed according
to the protocol described by Lewis and Laemmli (1982) Immunofluorescence Microscopy
For immunolabeling, cells were trypsinized and seeded onto acid-treated
sterile 18-mm coverslips in six-well dishes (Corning Glass Works, Corning, NY). After reaching 75% confluence in ~36 h, cells were rinsed for 1 min with PHEM buffer (100 mM Pipes, 20 mM Hepes, pH 6.9, 5 mM
EGTA, 2 mM MgCl2, and 4 M glycerol) and permeabilized for 1 min with
PHEM plus 0.1% Triton X-100 as described (Compton et al., 1992 For visualization of cytoplasmic CENP-E distribution in the interphase
cells, HeLa cells were fixed in 4% paraformaldehyde plus 0.05% glutaraldehyde before the detergent extraction. After fixation, the cells were permeabilized with 0.2% NP-40 in PBS. The visualization of CENP-B and
CENP-E was achieved by using rhodamine-conjugated goat anti-human
IgG and FITC-linked goat anti-rabbit IgG, respectively. The slides were
examined with a fluorescent microscope (model Axiophot; Carl Zeiss,
Inc., Thornwood, NY), and the images were collected and analyzed with
MetaMorph software (Universal Imaging Co., West Chester, PA).
Electron Microscopy
After fluorescent examination to verify fixation and antibody binding,
CENP-E was visualized by 10-nm colloidal gold by modification of the
protocol described by Yao et al. (1996) In some cases, the immunogold labeling was monitored by using a bifunctional probe, FluoroNanogold, in which rabbit IgG was conjugated
with fluorescein and 1.4-nm gold particles (Nanoprobe Inc.). The advantage of using Nanoprobe enabled monitoring the efficiency of immunolabeling and observation of the same specimen with light and electron microscopic analyses. Immunostained coverslips were incubated with a silver
enhancement kit for ~6 min (Ted Pella, Inc., Redding, CA) followed by
uranyl acetate staining.
Production of Microtubule-depolymerized Cells
Using Nocodazole
To examine CENP-E position in the absence of kinetochore microtubules,
nocodazole treatment was used to depolymerize microtubules proximal to
kinetochores. Nocodazole treatment (100 ng/ml) was incubated with
HeLa cells for 12 h followed by the standard extraction and fixation protocol mentioned above.
Upon Nuclear Envelope Fragmentation in Late
Prophase, an Associated Plus End Motor Trafficks
CENP-E to Centromeres
Previous studies revealed cell cycle-regulated distribution
of CENP-E and localization of CENP-E near the kinetochore region of mitotic chromosomes. To define more
closely the location of CENP-E during kinetochore maturation into a trilaminar structure, a polyclonal antibody
against a bacterially expressed portion of the rod domain
of CENP-E (amino acids 955-1571, designated as HpX, illustrated in Fig. 1 A) was generated and affinity purified using an antigen-coupled Sepharose matrix. Protein immunoblot analysis revealed that the affinity-purified CENP-E
antibody specifically recognized a single protein band of
~310 kD in whole mitotic HeLa cell extracts and isolated
chromosome scaffolds (Fig. 1 B, lanes 2 and 4, respectively).
This 310-kD band was not recognized by preimmune serum. To verify further the specificity of this HpX antibody,
CENP-E localization was visualized in HeLa cells using HpX antibody and a fluorescein-conjugated goat anti-rabbit secondary antibody (Fig. 1 C, upper left), while a human CREST anticentromere antibody that reacts primarily with CENP-B followed by a rhodamine-conjugated goat anti-human secondary antibody was used to identify
the actual centromere (Fig. 1 C, upper right). This revealed
that, in accord with previous reports (Yen et al., 1992
To examine CENP-E localization as it first associates
with chromosomes and/or spindle microtubules, we carried out immunoelectron microscopy on a cell in late
prophase/earliest prometaphase, just as the nuclear envelope had started to disassemble (Fig. 2 A). At this point,
astral microtubules emanate from centrioles, reach the remaining nuclear envelope (Fig. 2 C, bracket), and in some instances pass though gaps in the envelope, coming in
close proximity to newly condensing chromosomes (Fig. 2,
D and E). Even at these earliest times, gold particles representing the labeling of CENP-E are found almost exclusively along astral microtubules or at developing kinetochores adjacent to microtubules that have penetrated into the nuclear volume. CENP-E bound to astral microtubules
was often closely associated with electron-dense structures
(Fig. 2 C, upper left, arrowhead). No similar structures
were found in cells before onset of mitosis, suggesting the
assembly of a CENP-E-containing complex just at the onset
of mitosis. Some CENP-E was found localized to domains
of the condensing chromatin (Fig. 2, D and E, arrowhead). Careful examination of serial sections of chromosomes did
not reveal any trilaminar kinetochore structures, nor were
any microtubules obviously attached laterally or end on.
However, in light of CENP-E's association with more mature kinetochores (see below), we infer that these areas of
chromosome-bound CENP-E represent the immature kinetochores. All chromosomes with more than one gold
particle representing bound CENP-E did have astral microtubules within 200 ± 40 nm (e.g., Fig. 2, D and E, arrowhead). Virtually no gold particles were found on other
structures (i.e., vesicular membranes or at other surface
regions of the chromosomes) (Fig. 2 D).
These findings indicate that even by earliest prometaphase,
CENP-E binds to astral microtubules and apparently accumulates at immature kinetochores, before stable chromosome association with microtubules.
Early in Prometaphase CENP-E Binds along the
Outermost Surface of Kinetochores as Chromosomes
Initially Attach Laterally to Microtubules
Serial micrographs (Fig. 3, A, C, and E) from cells in
prometaphase revealed numerous apparently monoorientated chromosomes attached laterally to spindle microtubules. At higher magnification of one telocentric chromosome pair (Fig. 3, B, D, and F), the gold particles marking
CENP-E position were seen at the interfaces of the developing kinetochores with their laterally associated spindle
microtubules (Fig. 3, B, D, and F), with virtually no gold
found on other microtubules or at the surface regions of the chromosome. While we cannot be absolutely certain in
this example that all of the microtubules are from the adjacent pole, it is likely that these chromosomes are monooriented. Moreover, it is clear that in this example and in 13 other cells examined, CENP-E is found in a fibrous network extending 30-60 nm from the not fully developed
outer kinetochore surfaces of the sister kinetochores. In
addition, CENP-E surrounds the semicircular, immature
kinetochores, both those with obvious lateral attachment
to microtubules and without associated microtubules.
These findings demonstrate that at the earliest stages of
microtubule-chromosome interaction, CENP-E is highly
concentrated at the surface of the centromere as a fibrillar
component extending up to 60 nm. Thus, the prometaphase kinetochore outermost surface is surrounded by a collar of
CENP-E molecules.
The Earliest Chromosomes to Become
Bioriented Always Have CENP-E on the Outer
Kinetochore Surface, Although They Are
Still Morphologically Immature
To probe for the localization of CENP-E in chromosomes
as they congress toward the spindle equator, we examined
bioriented chromosomes in prometaphase cells. Two different examples from a single cell are highlighted in Fig. 4,
A-C and D-F. At higher magnifications, nine gold particles representing specific labeling of CENP-E can be
clearly seen on one leading kinetochore (i.e., defined here
to be the one closer to the midzone; Fig. 4 C, arrow), while
five particles are found on the other (trailing kinetochore; Fig. 4 C, arrowhead). Again, there was virtually no gold
staining on other microtubules or at other surface regions
of the chomosome (Fig. 4, B and E). In a second example
(Fig. 4, D-F), seven gold particles were associated with the
trailing sister kinetochore (Fig. 4 E), while the leading one
reveals 14 particles, plus two adjacent clusters of CENP-E
associated with a kinetochore microtubule. By examining
23 serial sections from five bioriented chromosomes, we
determined that the leading kinetochore always displayed
more intense CENP-E reactivity (12 ± 4 gold particles for
the leading vs 7 ± 3 for the trailing), demonstrating a difference in abundance, conformation, or accessibility of kinetochore-bound CENP-E during chromosome congression. The increased immunoreactivity on the apparently
leading kinetochore was confirmed at the light microscopic level. While CENP-B showed comparable staining
on leading and trailing kinetochores of a lagging chromosome pair (Fig. 4 G, arrowhead and arrow, respectively),
CENP-E staining was more intense on the kinetochore
closest to the midzone (Fig. 4 H, arrowhead). Furthermore, in serial sections, none displayed the clear trilaminar structure seen at metaphase (e.g., see Fig. 5 B), demonstrating that kinetochore assembly is both multistep and incomplete even as late as bipolar microtubule attachment.
It should be emphasized that since chromosomes oscillate toward and away from the pole during congression,
we cannot be certain that the kinetochore closest to the
midzone was actually leading movement in any of these
examples. Nevertheless, since chromosomes do spend
most of their time during congression moving toward the midzone (Skibbens et al., 1993 At Metaphase CENP-E Extends from the
Mature Kinetochore Outer Plate at Least 50 nm along Spindle Microtubules
HeLa cells with aligned chromosomes were examined
(Fig. 5 A) to track CENP-E at bioriented kinetochores
that have completed congression but are still under tension exerted by opposing spindle microtubules. Higher
magnification views (Fig. 5, B and C) revealed that CENP-E,
evidenced by 10-nm gold particles, is readily apparent adjacent to six spindle microtubules that are attached through a fuzzy layer of corona fibers to the electron-dense kinetochore outer plate (Fig. 5 B, top arrow). Serial sections revealed an equivalent level of CENP-E on the sister kinetochores of fully congressed chromosomes. Furthermore,
the 10-nm gold particles are uniformly distributed in the fibrous corona and lie an average distance of 50 nm from
the outer kinetochore plate (Fig. 5 C). This is in contrast
with the previously reported position of CENP-B and
CENP-C, localized to heterochromatin and the inner plate, respectively (Cooke et al., 1990 CENP-E Is an Integral Component of
Kinetochore Corona Fibers Extending
30-60 nm from the Outer Plate
The evidence outlined above implicates CENP-E as a potential linking protein for chromosome attachment to
spindle microtubules. To study the nature of the interaction of CENP-E with kinetochore substructures, we examined CENP-E location on condensed prometaphase chromosomes in the absence of microtubules. To this end,
nocodazole was used to disassemble microtubules. Immunoelectron microscopy showed that kinetochores appear
curved and elongated, becoming three to four times their
normal length along the chromosome surface (Fig. 6, A
and B), with an easily identifiable trilaminar structure readily apparent in higher magnifications (Fig. 6 B). An
electron-translucent zone between the inner and outer
plates of the kinetochore was evident, as reported earlier
(e.g., Rieder, 1982
Kinetochore-associated CENP-E Leads as Sister
Chromatids Move toward the Poles in Anaphase A,
Dissociating Gradually from Corona Fibers and
Redistributing to the Midzone in Late Anaphase
To verify if CENP-E is located in the kinetochore corona
during anaphase chromosome movement toward the poles,
CENP-E positioning was examined in cells early in anaphase
(anaphase A; Fig. 7, A and B). The deposition of gold particles demonstrated that CENP-E remains a kinetochore corona component, extending along spindle microtubules (Fig.
7 B, arrow). Later in anaphase, when chromosomes have
moved most of the way to the poles and pole separation
has been initiated (anaphase B), CENP-E is still localized
to the kinetochore outer plate (Fig. 7 C, boxed area, D, arrows), but a significant number of gold particles are now
found redistributed to the interzonal microtubules (Fig. 7
C, arrow; E). Examination of 32 anaphase kinetochore sections (from both randomly picked and serial stacks) revealed
that the number of gold particles located to kinetochores
is reduced by about half compared with metaphase chromosomes (8 ± 3 for metaphase vs 5 ± 2 for anaphase). Again,
very few gold particles were found elsewhere in the cytoplasm.
CENP-E Cross-Links the
Interzonal Microtubules Beginning in anaphase
B and Continuing through Telophase
To test if detectable CENP-E remains at telophase centromeres, when the functional kinetochores are disassembled, we examined cells in telophase. Fig. 8 A displays a
cell in which chromosomes are decondensing and a nuclear
lamina has begun to reform around the DNA. No CENP-E
was found chromosome associated. Rather, CENP-E was
restricted to bundles of antiparallel microtubules in the midzone. For example (Fig. 8 B), in each of five microtubule bundles formed by antiparallel microtubules, CENP-E
was found microtubule associated, but only in the electron
dense region of overlapping microtubule plus ends (Fig. 8 C).
CENP-E Links Spindle Microtubules to Kinetochores
From these ultrastructural experiments, we can develop a
kinetic picture of CENP-E integration into functional kinetochore assembly (summarized in Fig. 9). The evidence
presented here provides proof that CENP-E is localized to
developing kinetochores of condensing chromosomes before, or concurrently with, astral microtubule attachment after initial nuclear envelope disassembly (Fig. 2). By
prometaphase, CENP-E binds along the outermost surface of monooriented kinetochores that are attached laterally to spindle microtubules (Fig. 3). During congression,
CENP-E is asymmetrically localized, with more present
(or accessible) at the leading kinetochore (Fig. 4). Before microtubule attachment, during congression, at metaphase,
and during anaphase A, CENP-E is a component of kinetochore corona fibers, extending at least 50 nm along spindle microtubules from the mature kinetochore outer plate
(Figs. 3-7). Along with CENP-E's structural features (Fig.
1 A), including an amino-terminal kinesin-like motor domain and a 2,069-amino acid coiled-coil domain (Yen et al., 1992
A question not fully settled experimentally is the orientation of CENP-E while kinetochore associated. We had
hoped that amino- and carboxy-terminal peptide antibodies to CENP-E would allow us unambiguously to identify
which end is extended along microtubules and which is
embedded in the kinetochore; however, despite efficient reactivity in immunoblots, both terminal antibodies we
produced have failed (not shown) with the immunoelectron microscopic protocols successfully used here for antibody HpX. Nevertheless, with an amino-terminal motor
domain, the most pleasing view would be that the carboxy-terminal tail of CENP-E is embedded in the kinetochore, leaving the motor domain flexibly tethered for attachment
to, and extension along, spindle microtubules, consistent
with the identification of a centromere-targeting domain
at the carboxy-terminal tail of CENP-E (Chan, G.K.T.,
and T.J. Yen. 1996. Mol. Biol. Cell. 7:565a). While it remains formally possible that either or both ends of CENP-E
could be exposed for microtubule-binding at the kinetochore, CENP-E's ATP-independent microtubule-binding
domain present near the extreme carboxy terminus (Yen
et al., 1992 One important question addressed by our studies is the
function and nature of the corona fibers. Using real time
light microscopy coupled with subsequent electron microscopic analyses, Rieder and Alexander (1990) A Model of CENP-E in Kinetochore Assembly and
Chromosome Movement
Our finding that CENP-E extends at least 50 nm from the
kinetochore outer surface (modeled in Fig. 9) reinforces
several lines of evidence showing that altering CENP-E
action can affect chromosome movements: antibodies to
CENP-E do inhibit poleward chromosome movements
powered by microtubule disassembly in vitro (Lombillo et
al., 1995a Dynamic Kinetochore Assembly and Structure
It has been shown that the kinetochores undergo dramatic
structural and morphological changes as mitosis progresses
(Rieder, 1982 Recent studies from Wilson and his colleagues (Thrower
et al., 1996 Spindle Checkpoints, Tension, and Asymmetric
Localization of CENP-E during Congression
That CENP-E antibodies preferentially label the leading
kinetochore of a congressing chromosome pair provides
direct support for the view that tension exerted on (or generated by) the leading kinetochore triggers either a change
in the abundance or conformation of kinetochore-associated CENP-E. Moreover, elimination of spindle microtubules results in increased CENP-E accessibility at the kinetochore (Fig. 6 B), reinforcing the view that tension, or at least microtubule binding, affects CENP-E distribution
or epitope availability. As initially proposed by McIntosh
(1991); Brinkley and Stubblefield, 1966
),
is the site for spindle microtubule-centromere association. Structurally, the kinetochore is composed of four layers:
an innermost plate that apparently consists of a specialized
layer of chromatin, an interzone, an outer plate that has
been argued to consist of tightly packed fibers (Ris and
Witt, 1981
; Rattner, 1986
), and an outermost fuzzy, fibrous
corona that is most clearly seen after microtubule disassembly (e.g., Wordeman et al., 1991
). Although kinetochore morphology has been documented in numerous ultrastructural studies (e.g., Brinkley and Stubblefield, 1966
;
Jokelainen, 1967
; Comings and Okada, 1973
; Roos, 1973
;
Rieder, 1982
; McEwen et al., 1993
), there is little information about kinetochore composition and the respective localization of known kinetochore proteins except for three
initially identified as human autoantigens (centromere-associated protein A [CENP-A]1 [attached to centromeric heterochromatin; Palmer et al., 1991
; Pluta et al., 1995
],
CENP-B [underneath the inner plate; Cooke et al., 1990
],
and CENP-C [a component of the inner plate; Saitoh et al.,
1992
]).
; Rieder and Alexander, 1990
; Hyman and Mitchison, 1991
). To date, fluorescence microscopy has been used to localize three microtubule motor proteins to the centromere/kinetochore: cytoplasmic dynein (Pfarr et al., 1990
; Steuer et al., 1990
), CENP-E (Yen et al., 1992
), and MCAK/XKCM1 (Wordeman and Mitchison, 1995
; Walzak et al., 1996). Although
cytoplasmic dynein has been implicated in transient association with kinetochores (Pfarr et al., 1990
; Steuer et al.,
1990
), microinjection of specific antibodies has resulted instead in spindle collapse (Vaisberg et al., 1993
), rather
than a direct effect on chromosome attachment to spindles, disruption of chromosome congression, or movement
at anaphase. Dynein has also been shown to be involved in
aster formation and spindle pole assembly in Xenopus
(Verde et al., 1991
; Heald et al., 1996
; Merdes et al., 1996
)
and HeLa cell (Gaglio et al., 1996
) extracts, while evidence
from budding yeast has proven its role in spindle positioning (Eshel et al., 1993
; Li et al., 1993
) with a possible involvement in anaphase chromosome segregation (Saunders et al., 1995
). Echeverri et al. (1996)
have localized a
proportion of p50, a component of the dynactin complex
that can activate cytoplasmic dynein (Steuer et al., 1990
),
to prometaphase kinetochores followed by release at or
after bipolar attachment to spindles. Overexpression of
p50 using DNA transfection disrupts spindle assembly and
eliminates kinetochore-associated cytoplasmic dynein but
does not block microtubule attachment to centromeres. Rather, the aberrant spindles generally display monopolar
attachment of chromosomes near microtubule plus ends,
findings demonstrating that initial kinetochore attachment
to microtubules is mediated, at least in part, by components other than dynein.
), there is evidence that altering its action can affect chromosome movements: (a) Antibodies to CENP-E
do inhibit poleward chromosome movements powered by
microtubule disassembly in vitro (Lombillo et al., 1995a
);
(b) antibody injection into cells slows the metaphase- to-anaphase transition (Yen et al., 1992
); (c) antibody injection into mouse eggs completely blocks meiosis I at
prometaphase/metaphase (Duesbery et al., 1997
); and (d)
immunodepletion of CENP-E from Xenopus egg extracts
blocks chromosome congression but not attachment to
spindles assembled in vitro (Wood et al., 1997
). The sum
of this evidence suggests that CENP-E functions as a kinetochore-associated microtubule motor, but to better understand the exact molecular function of the motor, it is important to know in which of the four layers of the kinetochore
CENP-E is located, and whether or how CENP-E distribution changes during the various phases of chromosome
movement in mitosis. Using immunoelectron microscopy, we now show that CENP-E binds to the outer surface of
the immature kinetochores early in prometaphase, consistent with CENP-E function during the earliest stages of
initial microtubule capture. From earliest prometaphase
through anaphase A, CENP-E extends from the kinetochore at least 50 nm along spindle microtubules. Thus, CENP-E is one component of the corona fibers that represents the linkers that connect spindle microtubules to kinetochores.
Materials and Methods
.
The rabbit IgG fraction was purified using the affinity beads in which
CENP-E955-1571 was immobilized to CNBr-activated Sepharose 4B beads
(Sigma Chemical Co., St. Louis, MO). The affinity purification procedure
was carried out as described by Harlow and Lane (1988)
. The IgG fraction
was eluted with 0.1 M glycine, pH 2.8, followed by a immediate desalting (Bio-Rad Labs, Hercules, CA) into PBS.
80°C with an intensifying screen.
. Briefly, mitotic HeLa cells were hypotonically swollen for 5 min at
room temperature in 10 vol of PEM buffer containing 5 mM Pipes, pH 7.2, 0.5 mM EDTA, 5 mM MgCl2, 5 mM NaCl, and a protease inhibitor cocktail (1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml pepstatin A, and 2 µg/ml leupeptin). The hypotonically swollen cells were harvested by centrifugation and homogenized in PEM buffer containing 0.1% digitonin (Sigma Chemical Co.). The homogenates were clarified to remove nuclei and the supernatant was loaded onto a stepwise gradient containing 30, 40, 50, and 60%
sucrose in PEM buffer and centrifuged (2,500 g for 15 min) at 4°C.
.
). In
some instances, 5 µM Taxol (Sigma Chemical Co.) was included to minimize the depolymerization of microtubules during extraction. Extracted
cells were then fixed in 4% freshly made paraformaldehyde (Polysciences,
Inc., Warrington, PA) plus 0.05% glutaraldehyde (Tousimis Research
Corp., Rockville, MD) in PHEM and rinsed three times in PBS. The coverslips were blocked with 0.05% Tween-20 in PBS (TPBS) containing 1%
BSA (Sigma Chemical Co.). The cells were incubated with CENP-E antibody in a humidified chamber for 1 h followed by three washes of TPBS. To visualize microtubules simultaneously, an antitubulin antibody (YL1/2;
Kilmartin et al., 1982
) was incubated with cells in a humidified chamber
for 1 h followed by three washes of TPBS. Visualization of CENP-E location was achieved by rhodamine-conjugated goat anti-rabbit IgG, while
labeling of tubulin was achieved by fluorescein and 1.4-nm gold-conjugated goat anti-rat IgG (Nanoprobes, Inc., Stony Brook, NY).
. Coverslips processed as above
were rinsed with TPBS (3× 5 min) and fixed with 1% glutaraldehyde (Tousimis) in PBS followed by three washes in PBS. Cells were then postfixed in 2% osmium tetroxide (Electron Microscopy Sciences, Fort Washington, PA), dehydrated in a graded alcohol series followed by 100% acetone, and embedded in Epoxy (Ernest F. Fullam, Inc., Latham, NY). The
cells were detached from coverslips using hydrofluoric acid, and the designated areas were excised and glued to blocks. Thin serial sections (silver-
gold) were then cut, placed on copper grids, and stained with uranyl acetate and lead citrate. The sections were examined by a JEOL 1200 EM
(Peabody, MA). Serial sections were examined to document CENP-E
deposition on both sister kinetochores.
Results
;
Brown et al., 1995), CENP-E accumulates in the cytoplasm of G2 cells (Fig. 1 C, upper left, arrows) but is absent
from most interphase cells (arrowheads). At mitosis,
CENP-E staining appears as pairs of clearly resolved double dots (lower left, arrows), while CREST centromere antigens are present as pairs of unresolved dots (Fig. 1 C,
lower right, arrowhead).
Fig. 1.
Characterization of the affinity-purified CENP-E antibody HpX. (A) Schematic drawing of CENP-E denoting the region used
to generate HpX, a fragment of 70 kD recombinant polypeptide expressed in bacteria. (B) Specificity of affinity-purified HpX antibody. Immunoblots of mitotic whole cell lysates (lane 2, 50 µg) and isolated chromosome scaffold (lane 4, 25 µg). The same materials were
separated in SDS-PAGE and stained with Coomassie blue (lanes 1 and 3). (C) Upper panels: CENP-E is accumulated in the cytoplasm just before nuclear envelope breakdown. Indirect immunofluorescence image of HeLa cells stained with HpX antibody (upper left),
DAPI (upper middle) and human CREST sera (upper right). CREST sera stained centromeres in both early interphase cell (arrowheads) and late interphase cells (arrows). CENP-E signal appeared only in the late interphase cells (upper left, arrows). Interphase nuclei lack CENP-E staining (arrowheads). Lower panels: CENP-E is located to kinetochores as pairs of clearly resolved double dots
(lower left, arrows), while CREST sera mark centromeres as unresolved dots (lower right, arrowhead). Bars: (upper panels) 20 µm;
(lower panels) 10 µm.
[View Larger Versions of these Images (78 + 64 + 4K GIF file)]
Fig. 2.
An associated plus
end motor activity trafficks
CENP-E along newly assembled astral microtubules into
the nuclear domain after nuclear envelope fragmentation. HeLa cells were processed as described in
Materials and Methods. (A)
Low magnification view of a
prophase/prometaphase HeLa
cell bearing condensed chromosomes and a partially
fragmented nuclear envelope. One spindle pole is
readily apparent (asterisk). Examination of serial sections did not reveal another
pole, consistent with a prophase cell before centriole
separation. (B) Magnified view of boxed region in A
shows that astral microtubules emanating from the
centriole come in close proximity to the nuclear envelope. (C) Higher magnification
view reveals that CENP-E is
microtubule-associated along
astral microtubules adjacent
to the remaining nuclear envelope (bracket). Arrowheads
point to microtubule-bound
gold particles reporting
CENP-E location. (D) Magnified view of the dashed box in A and highlighting astral
microtubules passing through
the fragmented lamina and
lying in close proximity to
a chromosome. (E) Higher
magnification of the area
boxed in D, revealing that some CENP-E is found along
the microtubules, but additional CENP-E is associated
with a localized domain on the chromosome, presumably the developing kinetochore. Note the chromosome is not yet attached to microtubules. Bars: (A) 2 µm; (B) 400 nm; (C) 200 nm; (D) 800 nm; (E) 140 nm.
[View Larger Version of this Image (150K GIF file)]
Fig. 3.
At early prometaphase, CENP-E binds all
long the outermost surface of
monooriented kinetochores
attached laterally to spindle microtubules. HeLa cells
grown on coverslips were
preextracted and fixed. The
visualization of CENP-E was
achieved by 10-nm gold-conjugated goat anti-rabbit IgG.
(A, C, and E) Low magnification serial sections of an early
prometaphase HeLa cell. Asterisks denote the two poles of the developing bipolar
spindle. An apparently monooriented chromosome is
boxed, and higher power
views are shown in B, D, and
F. 10-nm gold particles representing CENP-E position
decorate the interface between immature kinetochore
and the laterally attached spindle microtubules. Note
the labeling of CENP-E on
the kinetochore appears as a
crescent (C) shape. Bars: (A,
C, and E) 2 µm; (B) 160 nm;
(D and F) 110 nm.
[View Larger Version of this Image (163K GIF file)]
Fig. 4.
The leading kinetochore of a congressing chromosomes pair has increased
level or accessibility of
CENP-E. HeLa cells were processed as described in Fig.
2. (A and D) Low magnification views of a prometaphase
HeLa cell (poles of the bipolar spindle are labeled with
asterisks). (B and E) Intermediate magnification of two
examples of a bioriented chromosome. (B) Boxed area of A showing chromosomes pair
partially congressed from spindle poles toward the equator
of spindle poles, but not yet
aligned at the equator. (C
and F) High magnification
of the two bioriented chromosomes in B and E. 10-nm
gold particles representing
CENP-E decorate the outer
kinetochore surface. A trilaminar structure of the kinetochore is not yet apparent,
indicating that these kinetochores are not fully mature.
(G-I) Double immunofluorescence demonstrating preferential CENP-E staining on
the kinetochore closest to the
midzone on a chromosome
not yet congressed to the metaphase plate. (G) CENP-B, (H) CENP-E, and (I) DAPI
to display chromosome positioning. Bars: (A and D) 2 µm;
(B and E) 230 nm; (C) 90 nm;
(F) 110 nm; (H-I) 10 µm.
[View Larger Version of this Image (125K GIF file)]
Fig. 5.
At metaphase CENP-E extends from the kinetochore
outer plate at least 50 nm along spindle microtubules. Low magnification view of a metaphase HeLa cell with chromosomes
aligned at the equator between the spindle poles (asterisks). (B)
Magnified view of one metaphase chromosome showing that
spindle microtubules indeed associate with a kinetochore with a
trilaminar structure. Five 10-nm gold particles are located to each
sister kinetochore (arrows). Five additional gold particles just to
the right of the boxed area represent CENP-E associated with the
kinetochore of another chromosome (more clearly seen in adjacent
sections). (C) Higher magnification view shows that CENP-E is located to the corona fibers of the kinetochore. op, outer plate; ip,
inner plate; cf, corona fibers. Bars: (A) 2 µm; (B) 170 nm; (C) 70 nm.
[View Larger Version of this Image (84K GIF file)]
; Waters et al., 1996
), this is the most likely case. Since the leading kinetochore produces the pulling force that apparently governs chromosome congression (Khodjakov and Rieder, 1996
), the increased labeling of CENP-E on this kinetochore reflects
the functional status of congressing kinetochores.
; Saitoh et al., 1992
). We
conclude that CENP-E is located in the fibrous corona
connecting kinetochores to spindle microtubules.
), indicating significant structural preservation during sample preparation. The outer kinetochore plate appearance was consistent with tightly packed
fibers as described earlier (Ris and Witt, 1981
; Rattner, 1986
). Most interestingly, at the tips of those organized fibers, there are numerous 10-nm gold particles (33 particles
in total in the example in Fig. 6 B) denoting the measure of
CENP-E evenly dispersed to the outermost region of the
outer plate and to corona fibers and lying an average of 90 nm (90 ± 17 nm, n = 33) from the electron-translucent
zone. Again, very few gold particles were found in the cell
cytoplasm or other regions of chromosomes. The relatively uniform distance of these gold particles from the translucent zone strongly suggests that CENP-E is unidirectionally oriented, extending in a fibrous pattern at least
100 nm away.
Fig. 6.
CENP-E is a integral component of kinetochore corona
fibers extending 50 nm from the outer plate. HeLa cells were initially treated with a low dose of nocodazole for 12 h to disassemble microtubules and processed as described in Fig. 2. Nocodazole treatment bulges the kinetochore outward from the surface
of the chromosome. (A) Low magnification view of a nocodazole-treated prometaphase HeLa cell. The asterisk denotes one
spindle pole. Note that not all spindle microtubules are depolymerized. Microtubule-free kinetochores appeared swollen and
crescent shaped. (B) Magnified view of boxed area in A shows
that 33 gold particles representing CENP-E position are located
to the tip of the enlarged kinetochore outer plate, which appears
to consist of tightly packed fibers. Bars: (A) 2 µm; (B) 85 nm.
[View Larger Version of this Image (82K GIF file)]
Fig. 7.
CENP-E remains
a component of the kinetochore fibers as sister chromatids move toward the poles in
anaphase. HeLa cells were processed as described in Fig.
2. (A) Low magnification
view of an early anaphase
HeLa cell. The two spindle
pole positions are marked
with asterisks (one is apparent
while another is in a different section). (B) Magnified view
of a kinetochore-microtubule
interface shows that CENP-E
is located between the kinetochore outer plate and the
spindle microtubules (arrow). No gold particles are
seen in other regions on
chromosomes or on microtubules. (C) Low magnification view of a late anaphase HeLa
cell bearing elongated spindle poles, labeled with asterisks; one is apparent while
another is in a different section. Interzonal microtubules
are readily seen (arrow). (D)
Magnified view of the upper
boxed region in C, showing
that CENP-E is located between a kinetochore and its
associated spindle microtubules (arrow). Examination
of the number of particles revealed a reduction relative to
metaphase. (E) Magnified
view of area pointed by the
arrow in C. Some CENP-E is
now localized to the interzonal microtubules. Bars:
(A) 2 µm; (B) 120 nm; (C)
2 µm; (D) 70 nm; (E) 90 nm.
[View Larger Version of this Image (132K GIF file)]
Fig. 8.
CENP-E cross-links the interzonal microtubules during
telophase. HeLa cells were processed as described in Fig. 2. (A)
Low magnification view of a late telophase HeLa cell. Lamin
deposition to reform nuclei is partially complete. (B) Magnified
view of boxed area in A shows that CENP-E is located along and/
or between the interzonal microtubules (boxed). (C) Higher magnification of interzonal microtubules shows that gold particles are
primarily located between the microtubule bundles. Bars: (A) 2 µm; (B) 500 nm; (C) 90 nm.
[View Larger Version of this Image (79K GIF file)]
Discussion
) that could extend as much as 300 nm (i.e., 2,069 amino acids × 0.15 nm per residue in an
-helical coiled-coil = 310 nm), the collective evidence strongly implicates
CENP-E as a major linker protein (possibly the major
linker protein) that mediates centromere-microtubule interactions and is appropriately positioned to power chromosome congression and/or poleward anaphase movement. Moreover, since the epitopes recognized by the
antibody we have used lie only about half of the way from
the carboxy-terminal end of the helical rod (Fig. 1 A), it is
very likely that the remainder of the rod and amino-terminal motor project much further along spindle microtubules
(possibly up to 200-300 nm from the kinetochore outer
plate).
Fig. 9.
Model for CENP-E function in chromosome movements. CENP-E is recruited to the immature kinetochore as soon
as the nuclear envelope disassembles. CENP-E localizes to kinetochores before stable microtubule attachment, apparently by
trafficking to the kinetochore by movement over astral microtubules. CENP-E is situated on the outermost surface of the kinetochore during initial lateral microtubule attachment. After biorientation, CENP-E remains on the corona fibers that link
kinetochores in an apparent end-on interaction with spindle microtubules. CENP-E abundance, conformation, or accessibility is
increased on the leading kinetochore of a congressing pair of
chromosomes. Upon the sister chromatid separation, CENP-E
remains kinetochore-associated and leads the poleward-moving chromosome. Once the chromosomes have reached the poles,
CENP-E releases and redistributes to midzone where it may stabilize interzonal microtubules or power microtubule sliding that
leads to pole-to-pole separation. op, outer plate; ip, inner plate;
cf, corona fibers; sp, spindle microtubules.
[View Larger Version of this Image (27K GIF file)]
; Liao et al., 1994
) remains inactive until midanaphase through cdc2 kinase-dependent phosphorylation
(Liao et al., 1994
). Both this view and the precedent of kinesin itself, a dimer of parallel heavy chains (Hisanaga et
al., 1989
), makes it seem most likely that CENP-E is oriented
with the motor extending away from the kinetochore.
concluded
that corona fibers are the first kinetochore component to
interact with spindle microtubules. In addition, they also
concluded that the fibers undergo a dynamic size change
during the prometaphase-metaphase transition. Given the
size of corona fibers, estimated at 250 nm in length (e.g., Brinkley and Stubblefield, 1966
) and the calculated length
for CENP-E, we hypothesize that CENP-E may be in fact
a major component of the corona fibers. This is fully consistent with the notion proposed by Rieder and Alexander
(1990)
that the motor(s) for prometaphase chromosome
movement is on the surface of the kinetochore (i.e., within
the corona, but not the plate), or distributed along the surface of kinetochore microtubules, or both.
), and microinjection of CENP-E antibody efficiently blocks progression past meiotic metaphase I (Duesbery et al., 1997
) and slows the metaphase-anaphase
transition in mitosis (Yen et al., 1991
). Furthermore, immunodepletion of CENP-E from Xenopus egg extracts
blocks chromosome congression without affecting spindle assembly. Moreover, a bacterially expressed fragment containing the motor domain has revealed that the CENP-E
motor is an ATPase capable of walking toward microtubule plus ends (i.e., away from the spindle poles) at 5 µm/
min (Wood et al., 1997
). An earlier demonstration with a
partially purified CENP-E complex isolated from HeLa
cells arrested in prometaphase showed that CENP-E is also
associated with a minus end-directed motor activity whose motility is eliminated by immunodepletion with CENP-E
antibodies (Thrower et al., 1995
). The initial transit of an
apparent CENP-E complex toward the plus ends of astral
microtubules at earliest prometaphase, presumably mediated by CENP-E's intrinsic plus end motor activity, adds
additional weight to the idea of such a complex. Perhaps
even more intriguing for the mechanism of chromosome
movement, given that the plus end motor kinesin has been
shown to have the capacity to couple cargoes to microtubule depolymerization-driven, minus end-directed movement (Lombillo et al., 1995b
), it is plausible by analogy
that kinetochore-bound CENP-E mediates both antipoleward movement (using its ATP-dependent plus end motor)
and poleward movement (by coupling movement to energy
liberated from disassembly of the microtubule lattice).
). Jokelainen (1967)
found that immature
"ball and cup" kinetochore of mammalian prophase chromosomes matures, after nuclear breakdown, into a dense-staining, platelike structure, which appears to be organized,
at least in part, from components associated with prophase kinetochores (Roos, 1973
). Fully differentiated trilaminar
kinetochores are never reported in the prometaphase cells,
consistent with our ultrastructural observation in which
CENP-E is initially located in the fibrous corona of kinetochores without an apparent trilaminar structure (Figs.
3-5). By metaphase, kinetochores have frequently been
observed as trilaminar structures (Jokelainen, 1967
; Roos, 1973
; Rieder, 1982
).
) documented that kinetochore structure, as reported by CENP-E staining, appears as a collarlike shape
in early prometaphase chromosomes, becoming a pair of
separated dots in later prometaphase or metaphase. Both
our electron microscopic and immunofluorescent (not
shown) data confirm this and extend it to show that
CENP-E is appropriately positioned to mediate lateral kinetochore attachment to spindle microtubules, appearing
as a crescent ("C") shape in early prometaphase (Fig. 3).
The C-shaped kinetochores were even more prominent in
the absence of microtubule-kinetochore association (Fig.
6), where the kinetochores, with abundant CENP-E, bulge outward from the surface of the centromeres.
, several recent reports have pointed to an intrinsic
role of the kinetochore in signaling successful capture and
congression of each chromosome as a necessary prelude to
triggering the transition to anaphase (Rieder et al., 1994
,
1995
; Chen et al., 1996
; Li and Benezra, 1996
; Wells and
Murray, 1996
; for review see Rudner and Murray, 1996
).
Manipulation with microneedles has demonstrated that in some meiotic cells, this checkpoint is sensitive to tension
on the kinetochore (Li and Nicklas, 1995
). Among the
changes in kinetochore chemistry is the tension-dependent
diminution of one kinetochore phosphoepitope (3F3/2;
Nicklas et al., 1995
). To these earlier efforts, our finding
that the kinetochore-associated, plus end motor CENP-E
also displays an asymmetric distribution (or accessibility) concurrent with the release of the spindle assembly checkpoint component MAD2 (Chen et al., 1996
; Li and Benezra, 1996
) implicates regulation of CENP-E activity as an
important contributor to this tension-dependent, kinetochore-mediated mitotic checkpoint.
Received for publication 20 June 1997 and in revised form 31 July 1997.
Address all correspondence to Dr. Don W. Cleveland, 3080 CMM-East, University of California, 9500 Gilman Drive, La Jolla, CA 92093-0660. Tel.: (619) 534-7811. Fax: (619) 534-7659.We thank Drs. K. Wood, K. Sullivan, and J. Kilmartin for providing CENP-E antiserum, CREST autosera, and antitubulin antibody, respectively. We also thank Drs. A. Merdes (Ludwig Institute), K. Wood (Ludwig Institute), K. McDonald (University of California, Berkeley, CA), and K. Sullivan (Scripps Research Institute, La Jolla, CA) for critical reading of this manuscript.
This work was supported by a National Institutes of Health grant (GM 29513) to D.W. Cleveland. Salary support for D.W. Cleveland is provided by the Ludwig Institute for Cancer Research. X. Yao was supported by postdoctoral fellowships from the Bank of America Giannini Foundation and from American Cancer Society California Division.
1. | Brinkley, B.R., and E. Stubblefield. 1966. The fine structure of the kinetochore of a mammalian cell in vitro. Chromosoma (Berl.). 19: 28-43 |
2. | Brown, K.D., R.M. Coulson, T.J. Yen, and D.W. Cleveland. 1994. Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis. J. Cell Biol. 125: 1303-1312 [Abstract]. |
3. |
Brown, K.D.,
K.W. Wood, and
D.W. Cleveland.
1996.
The kinesin-like protein
CENP-E is kinetochore-associated thoughout poleward chromosome segregation during anaphase-A.
J. Cell Sci.
109:
961-969
|
4. |
Chen, R.-H.,
J.C. Waters,
E.D. Salmon, and
A.W. Murray.
1996.
Association of
spindle assembly checkpoint component XMAD2 with unattached kinetochores.
Science (Wash. DC).
274:
242-246
|
5. | Comings, D.E., and T.A. Okada. 1973. Holocentric chromosomes in oncopettus kinetochore plates are present in mitosis but absent in meiosis. Chromosoma (Berl.). 37: 177-192 . |
6. | Compton, D.A., T.J. Yen, and D.W. Cleveland. 1992. Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds. J. Cell Biol. 112: 1083-1097 [Abstract]. |
7. | Cooke, C.A., R.L. Bernat, and W.C. Earnshaw. 1990. CENP-B: a major human centromere protein located beneath the kinetochore. J. Cell Biol. 110: 1475-1488 [Abstract]. |
8. |
Duesbery, N.S.,
T. Choi,
K.D. Brown,
K.W. Wood,
J. Resau,
K. Fukasawa,
D.W. Cleveland,
G.F. Vande, and
Woude.
1997.
CENP-E is an essential kinetochore motor in meiosis and is masked in Mos-dependent, cell cycle arrest at metaphase II.
Proc. Natl. Acad. Sci. USA.
94:
9165-9170
|
9. | Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J. Cell Biol. 132: 617-633 [Abstract]. |
10. | Eshel, D., L.A. Urrestarazu, S. Vissers, J.C. Jauniaux, J.C. van Vliet-Reedijk, R.J. Planta, and I.R. Gibbons. 1993. Cytoplasmic dynein is required for normal nuclear segregation in yeast. Proc. Natl. Acad. Sci. USA. 90: 11172-11176 [Abstract]. |
11. | Gaglio, T., A. Saredi, J.B. Bingham, M.J. Hasbani, S.R. Gill, T.A. Schroer, and D.A. Compton. 1996. Opposing motor activities are required for the organization of the mammalian mitotic spindle pole. J. Cell Biol. 135: 399-414 [Abstract]. |
12. | Harlow, E., and D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 726 pp. |
13. | Hayden, J.H., S.S. Bower, and C.L. Rieder. 1990. Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells. J. Cell Biol. 111: 1039-1045 [Abstract]. |
14. | Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature (Lond.). 382: 420-425 |
15. | Hisanaga, S., H. Murofushi, K. Okuhara, R. Sato, Y. Masuda, H. Sakai, and N. Hirokawa. 1989. The molecular structure of adrenal medulla kinesin. Cell Motil. Cytoskel. 12: 264-272 |
16. | Hyman, A.A., and T.J. Mitchison. 1991. Two different microtubule-based motor activities with opposite polarities in kinetochores. Nature (Lond.). 351: 206-211 |
17. | Jokelainen, P.T.. 1967. The ultrastructure and spatial organization of the metaphase kinetochore in mitotic rat cells. J. Ultrastruct. Res. 19: 19-44 |
18. | Khodjakov, A., and C.L. Rieder. 1996. Kinetochores moving away from their associated pole do not exert a significant pushing force on the chromosome. J. Cell Biol. 135: 315-327 [Abstract]. |
19. | Kilmartin, J.V., B. Wright, and C. Milstein. 1982. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J. Cell Biol. 93: 576-582 [Abstract]. |
20. | Lewis, C.D., and U.K. Laemmli. 1982. Higher order metaphase chromosome structure:evidence for metal-protein interaction. Cell. 29: 171-181 |
21. | Li, X., and R.B. Nicklas. 1995. Mitotic forces control a cell cycle checkpoint. Nature (Lond.). 373: 630-632 |
22. |
Li, Y., and
R. Benezra.
1996.
Identification of a human mitotic checkpoint
gene: hsMAD2.
Science (Wash. DC).
274:
246-249
|
23. | Li, Y.Y., E. Yeh, T. Hays, and K. Bloom. 1993. Disruption of mitotic spindle orientation in a yeast dynein mutant. Proc. Natl. Acad. Sci. USA. 90: 10096-10100 [Abstract]. |
24. | Liao, H., G. Li, and T.J. Yen. 1994. Mitotic regulation of microtubule cross-linking activity of CENP-E kinetochore protein. Science (Wash. DC). 265: 394-398 |
25. | Lombillo, V.A., C. Nislow, T.J. Yen, V.I. Gelfand, and R. McIntosh. 1995a. Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro. J. Cell Biol. 128: 107-115 [Abstract]. |
26. | Lombillo, V.A., R.J. Stewart, and R. McIntosh. 1995b. Minus-end-directed motion of kinesin-coated microspheres driven by microtubule depolymerization. Nature (Lond.). 373: 161-164 |
27. | Luykx, P.. 1965. The structure of the kinetochore in meiosis and mitosis Urechis eggs. Exp. Cell Res. 39: 643-657 |
28. | McEwen, B.F., J.T. Arena, J. Frank, and C.L. Rieder. 1993. Structure of the colcemid-treated PtK1 kinetochore outer plate as determined by high voltage electron microscopic tomography. J. Cell Biol. 120: 301-312 [Abstract]. |
29. | McIntosh, J.R.. 1991. Structural and mechanical control of mitotic progression. Cold Spring Harbor Symp. Quant. Biol. 56: 613-619 |
30. | Merdes, A., K. Ramyar, J.D. Vechio, and D.W. Cleveland. 1996. A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly. Cell. 87: 447-458 |
31. | Mitchison, T.J., and M.W. Kirschner. 1985. Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding. J. Cell Biol. 101: 755-765 [Abstract]. |
32. | Nicklas, R.B.. 1989. The motor for poleward chromosome movement in anaphase is in or near the kinetochore. J. Cell Biol. 109: 2245-2255 [Abstract]. |
33. | Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint. J. Cell Biol. 130: 929-939 [Abstract]. |
34. | Palmer, D.K., K. O'Day, H.L. Trong, H. Charbonneau, and R.L. Margolis. 1991. Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone. Proc. Natl. Acad. Sci. USA. 88: 3734-3738 [Abstract]. |
35. | Pfarr, C.M., M. Coue, P.M. Grissom, T.S. Hays, M.E. Porter, and J.R. McIntosh. 1990. Cytoplasmic dynein is localized to kinetochores during mitosis. Nature (Lond.). 345: 263-265 |
36. | Pluta, A.F., A.M. Mackay, A.M. Ainsztein, I.G. Goldberg, and W.C. Earnshaw. 1995. The centromere: hub of chromosomal activities. Science (Wash. DC). 270: 1591-1594 [Abstract]. |
37. | Rattner, J.B.. 1986. Organization within the mammalian kinetochore. Chromosoma (Berl.). 93: 515-520 |
38. | Rieder, C.L.. 1982. The formation, structure and composition of the mammalian kinetochore and kinetochore fiber. Int. Rev. Cytol. 79: 1-58 |
39. | Rieder, C.L., and S.P. Alexander. 1990. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J. Cell Biol. 110: 81-95 [Abstract]. |
40. | Rieder, C.L., A. Schultz, R. Cole, and G. Sluder. 1994. Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle. J. Cell Biol. 127: 1301-1310 [Abstract]. |
41. | Rieder, C.L., R.W. Cole, A. Khodjakov, and G. Sluder. 1995. The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. J. Cell Biol. 130: 941-948 [Abstract]. |
42. | Ris, H., and P.L. Witt. 1981. Structure of the mammalian kinetochore. Chromosoma (Berl.). 82: 153-170 |
43. | Roos, U.-P.. 1973. Light and electron microscopy of rat kangaroo cells in mitosis. I. Formation and breakdown of the mitotic apparatus. Chromosoma (Berl.). 40: 43-82 |
44. | Rudner, A.D., and A.W. Murray. 1996. The spindle assembly checkpoint. Curr. Opin. Cell Biol. 8: 773-780 |
45. | Saitoh, H., J. Tomkiel, C.A. Cooke, H. Ratrie III, M. Maurer, N.F. Rothfield, and W.C. Earnshaw. 1992. CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate. Cell. 70: 115-125 |
46. | Saunders, W.S., D. Koshland, D. Eshel, I.R. Gibbons, and M.A. Hoyt. 1995. Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation. J. Cell Biol. 128: 617-624 [Abstract]. |
47. | Skibbens, R.V., V.P. Skeen, and E.D. Salmon. 1993. Directional instability of kinetochore motility during chromosome congression and segregation in mitotic newt lung cells: a push-pull mechanism. J. Cell Biol. 122: 859-875 [Abstract]. |
48. | Steuer, E.R., L. Wordeman, T.A. Schroer, and M.P. Sheetz. 1990. Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature (Lond.). 345: 266-268 |
49. | Thrower, D.A., M.A. Jordan, B.R. Schaar, T.J. Yen, and L. Wilson. 1995. Mitotic HeLa cells contain a CENP-E-associated minus end-directed microtubule motor. EMBO (Eur. Mol. Biol. Organ.) J. 14: 918-926 [Abstract]. |
50. | Thrower, D.A., M.A. Jordan, and L. Wilson. 1996. Modulation of CENP-E organization at kinetochores by spindle microtubule attachment. Cell Motil. Cytoskel. 35: 121-133 |
51. | Vaisberg, E.A., M.P. Koonce, and J.R. McIntosh. 1993. Cytoplasmic dynein plays a role in mammalian mitotic spindle formation. J. Cell Biol. 123: 849-858 [Abstract]. |
52. | Verde, F., J.M. Berrez, C. Antony, and E. Karsenti. 1991. Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: requirement for phosphorylated factors and cytoplasmic dynein. J. Cell Biol. 112: 1177-1187 [Abstract]. |
53. | Walczak, C.E., T.J. Mitchison, and A. Desai. 1996. XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly. Cell. 84: 37-47 |
54. |
Waters, J.C.,
R.V. Skibbens, and
E.D. Salmon.
1996.
Oscillating mitotic newt
lung cell kinetochores are, on average, under tension and rarely push.
J. Cell
Sci.
109:
2823-2831
|
55. | Wells, W.A, and A.W. Murray. 1996. Aberrantly segregating centromeres activate the spindle assembly checkpoint in budding yeast. J. Cell Biol. 133: 75-84 [Abstract]. |
56. | Wood, K.W., R. Sakowics, L.S.B. Goldstein, and D.W. Cleveland. 1997. CENP-E is a plus end-directed kinetochore motor required for chromosomes congression. Cell. In press. |
57. | Wordeman, L., and T.J. Mitchison. 1995. Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis. J. Cell Biol. 128: 95-104 [Abstract]. |
58. | Wordeman, L., E.R. Steuer, M.P. Sheetz, and T.J. Mitchison. 1991. Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes. J. Cell Biol. 114: 285-294 [Abstract]. |
59. |
Yao, X.,
L. Cheng, and
J.G. Forte.
1996.
Biochemical characterization of ezrin-actin interaction.
J. Biol. Chem.
271:
7224-7229
|
60. | Yen, T.J., D.A. Compton, D. Wise, R.P. Zinkowski, B.R. Brinkley, W.C. Earnshaw, and D.W. Cleveland. 1991. CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase. EMBO (Eur. Mol. Biol. Organ.) J. 10: 1245-1254 [Abstract]. |
61. | Yen, T.J., G. Li, B.T. Schaar, I. Szilak, and D.W. Cleveland. 1992. CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature (Lond.). 359: 536-539 |