Journal of Cell Science 109, 961-969 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
JCS1192
961
The kinesin-like protein CENP-E is kinetochore-associated throughout
poleward chromosome segregation during anaphase-A
Kevin D. Brown1,*, Kenneth W. Wood1,2 and Don W. Cleveland1,2,†
1Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
2Ludwig Institute for Cancer Research, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
*Present address: NCHGR, National Institutes of Health, Building 49 Room 3A14, 49 Convent Drive, Bethesda, MD 20892-470, USA
†Author for correspondence at address 2
SUMMARY
The kinesin-like protein CENP-E transiently associates
with kinetochores following nuclear envelope breakdown
in late prophase, remains bound throughout metaphase,
but sometime after anaphase onset it releases and by
telophase becomes bound to interzonal microtubules of
the mitotic spindle. Inhibition of poleward chromosome
movement in vitro by CENP-E antibodies and association
of CENP-E with minus-end directed microtubule motility
in vitro have combined to suggest a key role for CENPE as an anaphase chromosome motor. For this to be
plausible in vivo depends on whether CENP-E remains
kinetochore associated during anaphase. Using Indian
muntjac cells whose seven chromosomes have large,
easily tracked kinetochores, we now show that CENP-E
is kinetochore-associated throughout the entirety of
anaphase-A (poleward chromosome movement), relocating gradually during spindle elongation (anaphase-B) to
the interzonal microtubules. These observations support
roles for CENP-E not only in the initial alignment of
chromosomes at metaphase and in spindle elongation in
anaphase-B, but also in poleward chromosome
movement in anaphase-A.
INTRODUCTION
ality of movement (with respect to microtubule polarity) is
dependent upon ATP concentration and phosphatase inhibition
(Hyman and Mitchison, 1991). Although these findings
demonstrate an active role for the kinetochore in microtubulemediated chromosome movement, we still know little of the
molecular components of the kinetochore responsible for this
movement.
CENP-E is a transient kinetochore component that binds to
chromosomes immediately after the breakdown of the nuclear
envelope during late prophase and remains fully bound
throughout chromosome congression to the metaphase plate.
At some point following the onset of anaphase chromosome
migration, CENP-E relocalizes to the interzonal microtubules
of the mitotic spindle (Yen et al., 1991) and during the latter
stages of mitosis is quantitatively degraded (Brown et al.,
1994). Molecular characterization of the CENP-E molecule
shows it to have a tri-partite structure comprised of aminoand carboxy-terminal globular domains separated by a
~1,500 residue α-helical domain predicted to form coiledcoils (Yen et al., 1992). The amino-terminal domain contains
striking homology to the microtubule-dependent motor
protein kinesin, thus demonstrating CENP-E to be a member
of the growing family of kinesin-like proteins (see Goldstein,
1993). Based on its homology to other known microtubuledependent motor proteins and its association with the kin-
Mitosis is a complex process during which chromosomes
undergo carefully orchestrated movements: duplicated chromosome pairs first align at a metaphase plate and then, in
anaphase-A, one copy of each chromosome translocates along
microtubules toward each spindle pole. Chromosome segregation is further advanced by spindle elongation (termed
anaphase-B). Several years of intense investigation have
uncovered several important clues as to the molecular mechanisms responsible for chromosome motion. First, chromosome
segregation is mediated by microtubules of the mitotic spindle
(e.g. Gorbsky et al., 1987; Koshland et al., 1988). A subset of
the spindle microtubules attaches to kinetochores, specialized
structures located at the centromere of each chromosome
(Euteneur and McIntosh, 1981). Kinetochores have been
shown in vitro and in vivo to capture microtubules originating
from the spindle poles (Mitchison and Kirschner, 1985b;
Hayden et al., 1990). Second, the kinetochore plays an active
role in chromosome migration during mitosis, as kinetochorebound microtubule motors have been shown to translocate
chromosomes poleward in vivo (Nicklas, 1989; Rieder and
Alexander, 1990). Other in vitro experiments have shown that
chromosomes can translocate along individual microtubules
through interactions mediated by kinetochores; the direction-
Key words: Mitosis, CENP-E, Kinetochore, Anaphase-A, AnaphaseB, Microtubule motor
962
K. D. Brown, K. W. Wood and D. W. Cleveland
etochore during mitosis, CENP-E is a possible candidate
molecule for facilitating chromosome migration during
mitosis.
While the function(s) of CENP-E are largely unidentified,
several findings have raised the possibility that CENP-E participates actively in chromosome migration. Microinjection
of a CENP-E monoclonal antibody during prometaphase significantly delayed the onset of anaphase (Yen et al., 1991).
More recently, some CENP-E antibodies were shown to
inhibit poleward chromosome migration driven by microtubule depolymerization in an in vitro assay (Lombillo et al.,
1995), while antibodies to, or u.v. induced cleavage of, cytoplasmic dynein (another motor suspected to play a role in
anaphase chromosome movement; Steuer et al., 1990; Pfarr
et al., 1990) had no effect on this chromosome movement in
vitro. Moreover, a CENP-E associated, minus-end
(poleward) microtubule motor activity has been detected
(Thrower et al., 1995). Taken together, these in vitro observations strongly suggest that CENP-E may be a minus-end
motor for powering chromosome segregation during
anaphase-A.
One key uncertainty that bears on this hypothesis is the
question of whether CENP-E is actually chromosome-associated in anaphase-A. While previous efforts have proven that
CENP-E dissociates sometime between anaphase onset and
telophase, the exact timing of CENP-E disassociation from the
kinetochore is unknown. To address this issue, we have
exploited the unusually large kinetochores and low chromosome number (male, 2n=7; female, 2n=6) of Indian muntjac
cells to track CENP-E dissociation from kinetochores. This
reveals that CENP-E remains kinetochore bound throughout
anaphase-A, gradually relocating to the interzonal microtubules during anaphase-B. Thus, CENP-E is appropriately
located to participate in all phases of mitotic chromosome
movement.
centrifugation (3,000 g, 4°C, 15 minutes) and rinsed twice in cold
(4°C) Tris-buffered saline (TBS: 10 mM Tris-HCl, pH 7.2, 0.9%
NaCl). The accumulated fusion protein was completely insoluble
after cell disruption in physiologic buffer conditions; consequently,
the washed cells were lysed by addition of buffer A (10 mM TrisHCl, pH 8.0, 100 mM NaH2PO4) containing 6 M guanidine-HCl. The
suspension was then sonicated vigorously and cleared by centrifugation (3,000 g, 4°C, 30 minutes). The supernatant was then added
to buffer A-washed Ni2+-NTA-agarose (Quiagen) (the fusion protein
adsorbs to this matrix due to the presence of the amino-terminally
located 6-His tag). After overnight incubation at room temperature
with end-over-end rocking, the column matrix was washed extensively with buffer B (buffer A containing 8 M urea), followed by
extensive washing with buffer B adjusted to pH 6.3. The fusion
protein was subsequently eluted with buffer B adjusted to pH 4.5.
The eluted protein fractions were then dialyzed against a 500-fold
volume of TBS (at which point the fusion protein precipitated out of
solution), the precipitate collected by centrifugation (15,000 rpm, 10
minutes, room temperature) and stored at −80°C. Electrophoretic
analysis revealed that the CENP-E fusion protein (designated His 6CE 955-1571) was greater than 50% of the total protein present in
the final fraction.
MATERIALS AND METHODS
Electrophoresis and immunoblotting
Cells were harvested by scraping from culture dishes, concentrated by
centrifugation, rinsed twice in cold (4°C) phosphate-buffered saline
(PBS: 10 mM Na2HPO4, 10 mM NaH2PO4, 0.9 % NaCl, pH 7.2) and
lysed by the addition of SDS-solubilization solution (25 mM TrisHCl, pH 7.5, 5 mM EDTA, 1% SDS). The extracts were then placed
in a boiling water bath for 5 minutes, briefly sonicated and centrifuged
to remove the residual insoluble material. The supernatants were
removed and stored at −80°C. Protein concentrations were determined
by the bicinchonic acid method using bovine albumin as the standard
(Smith et al., 1985). Prior to electrophoresis, appropriate quantities of
extract were diluted with 3× SDS-sample buffer (150 mM Tris-HCl,
pH 6.8, 10% β-mercaptoethanol, 20% glycerol, 3% SDS) and boiled
for 2 minutes.
For immunoblot analysis, SDS-PAGE was carried out according to
the protocol of Laemmli (1970). Gels were electrically transferred
(Towbin et al., 1979) to Immobilon-P sheets for 1.5 hours at 4°C and
500 mA. Sheets were blocked for 1 hour in a solution of 5% non-fat
dry milk in TBS containing 0.1% Tween-20, followed by overnight
incubation (room temperature) in an appropriate dilution of the
indicated primary antibody. CENP-B antiserum was a generous gift
from Drs Ann Pluta and William Earnshaw, Johns Hopkins University School of Medicine. Immunoreactive bands were visualized
following incubation (2 hours, room temperature) with 125I-conjugated Protein A (ICN, final dilution 0.5 µCi/ml) by autoradiography
Cell culture
Frozen stocks of skin cells (cell line CCL 157) from a male Muntiacus
muntjac (Indian muntjac) were obtained from the ATCC (Rockville,
MD). These cells were cultured in Ham’s F10 medium supplemented
with 20% fetal calf serum (FCS). HeLa cells were cultured in DMEM
supplemented with 10% FCS. All cells were grown in a humidified
5% CO2 atmosphere. Where indicated, cells were mitotically arrested
by incubating in the presence of 0.1 µg/ml colcemid (Aldrich) for 18
hours.
Expression and purification of CENP-E fusion protein
The full length CENP-E cDNA clone pBS-CENP-E 1-2663 (Brown
et al., 1994) was digested with HpaI and XhoI and a ~1.8 kb fragment
corresponding to CENP-E nucleotides 2,958-4,804 (Yen et al., 1992)
was isolated and subsequently subcloned into the bacterial
expression vector pRSET (Invitrogen), which places a 6-histidine
‘tag’ at the amino-terminal terminus of the encoded CENP-E
fragment. The resultant plasmid (pRSET CE 2958-4804) was transformed into the E. coli host strain BL21(DE3)pLysE (Studier et al.,
1990) and the encoded fusion protein was induced by adding isopropylthio-β-D-galactoside (IPTG, final concentration 1 mM) to a
culture of logarithmically growing cells. Following a 2-4 hour incubation at 37°C with vigorous shaking, the cells were harvested by
Polyclonal antibody preparation and purification
A female New Zealand White rabbit was immunized subcutaneously
with 100-250 µg of His6-CE 955-1571 suspended by sonication in
Freund’s complete adjuvant. Subsequent boosts were done at 3 week
intervals for a total of 5 injections. Anti-CENP-E immunoreactivity
was monitored by immunoblotting and immunofluorescence
microscopy.
For affinity purification, purified His6-CE 955-1571 was solubilized by boiling for 5 minutes in 25 mM Tris-HCl, pH 7.5, 5 mM
EDTA, 1% SDS followed by sonication and the addition of 10
volumes of coupling buffer (100 mM NaHCO3NaOH, pH 8.8, 500
mM NaCl). The solubilized protein was then coupled to CNBractivated Sepharose 4B (Sigma) at a ratio of 2 mg protein/ml of
column resin. Subsequently, affinity purification was carried out as
outlined by Harlow and Lane (1988). The Ig fraction was further
purified by chromatography on a column of Protein A-Sepharose
(Pharmacia Fine Chemicals) and subsequently dialyzed into TBS,
glycerol added to 25% (v/v), and stored at −80°C.
CENP-E is kinetochore associated throughout anaphase-A
on Kodak XAR film for 6-20 hours at −80°C with intensifying
screens. (Blots incubated with the tubulin monoclonal antibody 5H1
were incubated with rabbit anti-mouse antisera (Sigma) prior to incubation with 125I-conjugated Protein A.) Quantification of
immunoblots was carried out using a phosphorimager (Molecular
Dynamics).
Immunofluorescence microscopy
For immunofluorescence, Indian muntjac cells were cultured on presterilized glass coverslips to ~75% confluency prior to processing.
Alternatively, to facilitate the viewing and measurement of many
mitotic cells, cells were grown in large tissue culture flasks as
indicated above and mitotic cells were harvested by mitotic shakeoff. The cells were then washed twice with PBS and pelleted onto
coverslips using a Cytospin III cytocentrifuge (Shandon) at 1,000
rpm for 3 minutes. The cells were then fixed with cold methanol
(−20°C, 5 minutes), rinsed extensively in PBS and blocked for 5
minutes in PBS plus 5% bovine serum albumin (BSA), and then
incubated (30 minutes, 37°C) with the indicated primary antibodies
diluted in PBS + 5% BSA. The tubulin monoclonal antibody 5H1
(subclass IgM) was a gift from Dr L. I. Binder, Northwestern University School of Medicine, the anti-centromere/kinetochore autoimmune CREST serum (from patient SH; Zinkowski et al., 1991) was
a gift from Dr B. R. Brinkley, Baylor College of Medicine, and anticentrosome autoimmune serum (from patient SPJ; Balczon et al.,
1994) was a gift from Dr R. B. Balczon, University of South Alabama
School of Medicine. Following this incubation, the cells were rinsed
extensively in PBS, then incubated (37°C, 30 minutes) with a mixture
of Texas Red labeled goat anti-rabbit and FITC-labeled goat antihuman, or Texas Red labeled goat anti-rabbit and FITC-labeled goat
anti-mouse µ-chain secondary antibodies diluted to 10 µg/ml each in
PBS + 5% BSA. All secondary antibodies were purchased from
Kirkegaard and Perry Labs (Gaithersburg, MD) except Texas Red
conjugated goat anti-rabbit which was purchased from Vector Labs
(Burlingame, CA). Prior to mounting the coverslips, cells were counterstained with Hoescht 33258 (final concentration 0.02 µg/ml) to
visualize DNA.
To visualize metaphase chromosomes, mitotically arrested Indian
muntjac cells were obtained by culturing cells in the presence of the
microtubule disrupting agent colcemid (Aldrich) at a concentration of
0.1 µg/ml for 18 hours. The mitotic cells were then harvested by
mitotic shake-off, pelleted by centrifugation and then swelled by incubation in a hypotonic buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl,
5 mM MgCl2) for 10 minutes at room temperature. Following this,
the cells were pelleted onto coverslips using the cytocentrifuge (2,000
rpm, 5 minutes) and fixed and processed for immunofluorescence
microscopy as outlined above.
To clearly view individual kinetochores in cells which had
undergone anaphase-A, mitotic cells collected via shake-off were
hypotonically swollen using the above outlined swelling procedure.
Following this, the cells were cytocentrifuged onto coverslips and
subsequently fixed and processed for immunofluorescence
microscopy.
Morphometry and computer-assisted video microscopy
Cells were viewed using a Zeiss Axiovert 35 epifluorescence microscope equipped with a Hamamatsu SIT camera coupled to an image
analysis system (Image-1, Universal Imaging). Fluorescence bleedthrough from adjacent channels was minimal. Images were recorded
by summing 16 frames and were subsequently stored on floppy discs
as TIFF graphics files. Following this, images were imported into
Adobe Photoshop (Adobe Systems Inc.), image contrast adjusted,
pseudo-colored, merged to form three-color (RGB) images, and
printed using a Tectronix color printer.
To obtain the higher resolution micrographs required to document
the co-localization of CENP-E with kinetochores in anaphase cells,
an Olympus BH2 microscope equipped with a ×100 (NA 1.4) oil-
963
emersion lens was employed. Images were recorded using Kodak TMAX 400 ASA 35 mm film. Subsequently, the resultant negatives
were digitized using a Nikon Coolscan slide scanner and the micrographs digitally processed as indicated above.
Measurement of pole-to-pole distance was conducted on Indian
muntjac cells which were mitotically selected by shake-off. Cells were
scored to be in anaphase if they contained chromatids which were
obviously in the process of poleward migration and, since Indian
muntjac cells possess low chromosome numbers and large kinetochores of heterogenous size (Brinkley et al., 1984), anaphase onset
was scored based on the criterion that no obvious paired kinetochores
were observed. Measurements were made using the Image-1 ‘measure
with caliper’ function. Calibration was achieved using a mounted
diatom (Pleurosigma angulatum) with an inter-frustule distance of
0.625 µm.
Chromosome isolation
Logarithmically growing cultures of HeLa cells were cultured in
the presence of colcemid (0.1 µg/ml) for 3 hours. Following this,
the mitotically arrested cells (5×107) were harvested by mitotic
shake-off, collected by centrifugation and washed extensively in
cold (4°C) PBS. Subsequently, chromosomes were isolated from
these cells using the technique outlined by Mitchison and Kirschner
(1985a). Briefly, cells were hypotonically swollen for 10 minutes
on ice in 100 ml of 5 mM Pipes-KOH, pH 7.2, 5 mM NaCl, 5 mM
MgCl2, 0.5 mM EDTA. Cells were then pelleted by centrifugation,
and resuspended in 5 ml of cold (4°C) chromosome lysis buffer (10
mM Pipes-KOH, pH 7.2, 2 mM EDTA, 0.1% β-mercaptoethanol,
1 mM spermidine, 0.5 mM spermine, 1 mM PMSF, 2 µg/ml
aprotinin, 0.1% digitonin) and subsequently homogenized with 20
strokes in a 15 ml dounce homogenizer (tight pestle). Following
this, the lysate was cleared by centrifugation (250 g, 1 minute), the
supernatant applied to a 9 ml 20-60% linear sucrose gradient
prepared in 10 mM Pipes-KOH, pH 7.2, 1 mM EDTA, 0.1% β-mercaptoethanol, 0.5 mM spermidine, 0.25 mM spermine, 1 mM
PMSF, 2 µg/ml aprotinin, and the gradient centrifuged (2,500 g, 15
minutes, 4°C).
Following centrifugation, the supernatant above the sucrose
gradient was removed and concentrated 10-fold in an Amicon microconcentrator (Centricon-10) and stored at −80°C. The chromosome
containing fraction banded as a visible, flocculent mass at approximately 50% sucrose, as previously reported (Mitchison and Kirschner,
1985a). The top third of the sucrose gradient was removed, 10 ml of
chromosome lysis buffer was added, the tube vigorously vortexed and
the chromosomes pelleted by centrifugation (12,000 g, 4°C, 30
minutes). The chromosomes were then suspended in 200 µl of chromosome lysis buffer and stored at −80°C. Subsequently, equal proportions of the chromosome and supernatant fractions were analyzed
as outlined.
RESULTS
CENP-E localization during mitosis in Indian
muntjac cells
Since the chromosomes of the Indian muntjac possess
unusually large kinetochores (Brinkley et al., 1984), we
reasoned that this feature would facilitate assessment of CENPE binding to kinetochores at various points during mitotic progression. To generate an antibody to follow CENP-E localization in Indian muntjac cells, a ~ 67 kDa segment of the human
CENP-E was utilized to raise a polyclonal antiserum in rabbits.
Computer searches revealed that no other known proteins
possess significant homology to this domain, which lies within
the predicted α-helical, stalk domain of CENP-E (Fig. 1A).
964
K. D. Brown, K. W. Wood and D. W. Cleveland
Following affinity purification, this antibody (pAb-HpX)
reacted on immunoblots of extracts of Indian muntjac cells
with a single high molecular mass protein (Fig. 1D). As this
protein is of similar size to human CENP-E and increases 3.5-
A
pAb-HpX
Antigen
NH
COOH
2
stalk
head
B
Colcemid:
-
tail
C
+
-
+
121
205
-
50
33
-
27
121
86
dye
front
immunoblot
Coomassie
HeLa
D
Colcemid:
-
E
+
-
+
121205
-
50
33
121
86
-
27
dye
front
immunoblot
fold in abundance in mitotically enriched cells (Fig. 1D,E),
similar to the 6-fold increase in CENP-E seen in mitotically
arrested HeLa cells (Fig. 1B), we conclude that this antibody
reacts monospecifically with muntjac CENP-E.
As seen earlier for human chromosomes (Yen et al., 1991;
Fig. 2C), CENP-E binds to the kinetochores of chromosomes
isolated from Indian muntjac cells (Fig. 2F). Moreover, in
both human (Fig. 2B,C) and muntjac cells (Fig. 2E,F)
CENP-E localizes more peripherally than do the centromere
antigens recognized by a CREST autoantiserum that binds
to the DNA binding proteins CENP-A and CENP-B and the
inner kinetochore protein CENP-C. Also, as in human cells,
CENP-E is not detectable in most interphase cells (Fig. 3A,
lower right), but is found in the cytoplasm of cells in
prophase when the chromatin is beginning to condense, but
the nuclear envelope remains intact (Fig. 3A, arrowhead).
Immediately after nuclear envelope disassembly, CENP-E
targets to kinetochores (Fig. 3B), as indicated by the pairs of
dots (Fig. 3B). CENP-E remains kinetochore-associated
throughout chromosome congression to the metaphase plate
(Fig. 3C).
Concerning localization of CENP-E during anaphase, an
initial assay using lack of obvious kinetochore pairs and
chromatid separation to monitor progression into anaphase
revealed that CENP-E was detectable near the spindle poles
(Fig. 3D), indicating that some CENP-E remained kinetochorebound even at the completion of anaphase-A. In cells which
displayed more pronounced chromatid separation, CENP-E
was present at both kinetochores and interzonal microtubules
of the spindle (Fig. 3E) or at interzonal microtubules but not
kinetochores (Fig. 3F). Finally, in cells which had completed
mitosis and reassembled an interphase microtubule array,
CENP-E was limited to the midbody (Fig. 3G).
To verify that CENP-E adjacent to the poles of cells which
had completed anaphase-A was due to co-localization with
kinetochores, cells were hypotonically swollen to increase
resolution between kinetochores and antibodies to CENP-E
and to the CREST autoantigens were used simultaneously.
In cells which contained chromatids that had completed
poleward anaphase-A migration (Fig. 4A), CENP-E (Fig.
4B) and the CREST centromere antigens (Fig. 4C) colocalized to discrete, chromatid-bound dots adjacent to the
poles (Fig. 4B).
Coomassie
I. muntjac
Fig. 1. Characterization of the affinity purified CENP-E antisera
pAb-HpX. (A) Schematic of the CENP-E polypeptide denoting a
67 kDa domain (labeled pAb-HpX antigen) in the central helical
segment that was used as an immunogen after expression in
bacteria. (B) Immunoblot analysis with pAb-HpX of SDS-extracts
(50 µg/lane) from HeLa cells grown for 18 hours in the presence
(+) or the absence (−) of the microtubule-disrupting drug
colcemid. Arrowhead denotes the position of CENP-E. (C) The
same extracts from (B) (50 µg/lane), but stained with Coomassie
Blue following SDS-PAGE. (D) Immunoblot analysis with pAbHpX on extracts from Indian muntjac cells (100 µg/lane) grown in
the presence or absence of colcemid. The arrowhead denotes
position of CENP-E. (E) Indian muntjac cell extracts (50 µg/lane)
stained with Coomassie Blue following SDS-PAGE. Migration
positions (in kDa) of molecular mass standards are marked at the
left.
Quantitative assessment of kinetochore-associated
CENP-E during anaphase-A
To investigate more precisely when CENP-E dissociates from
kinetochores, cell cycle position was determined from the
extent of spindle elongation. Measurement of pole-to-pole
distance in 32 metaphase cells showed a mean pole-to-pole
distance of 3.85 µm (±0.85 µm s.d.). In cells which had
undergone poleward chromosome migration, but with relatively short pole-to-pole distances, CENP-E distribution was
limited to the kinetochores (Fig. 5E-H). Furthermore, kinetochores were found clustered around the spindle poles prior to
significant pole-to-pole lengthening clearly suggesting that
anaphase-A movement goes to completion prior to significant
anaphase-B advance in these cells. Cells with longer pole-topole distances (indicative of advance into anaphase-B)
displayed diminished kinetochore staining and marked staining
CENP-E is kinetochore associated throughout anaphase-A
965
Fig. 2. pAb-HpX reactivity with isolated HeLa
and Indian muntjac metaphase chromosomes.
Chromosome spreads from colcemid-arrested
HeLa cells (A-C) and Indian muntjac cells (DF) were subjected to immunofluorescence with
Hoescht 33258 to visualize DNA (A,D),
human autoimmune CREST sera (B,E), and
pAb-HpX (C,F). Note the co-localization of
both CREST sera and pAb-HpX at the
centromere of the chromosomes. Bar, 10 µm.
of the interzonal microtubules (Fig. 5I-L and M-P). With yet
longer pole-to-pole distances, CENP-E was limited to the
spindle microtubules (Fig. 5Q-T).
Measurement of pole-to-pole distance in ~300 anaphases
(Fig. 6) revealed that the majority (75%) of cells in early
anaphase (pole-to-pole distances of <7.0 µm) displayed
CENP-E staining limited to the kinetochores, while the
remaining 25% showed staining of both kinetochores and
interzonal microtubules (n=32). Cells with pole-to-pole
distances of 7.0-9.9 µm (n=60) and 10.0-12.9 µm (n=52)
displayed successively lower numbers of cells with CENP-E
located exclusively to the kinetochores (23% and 10%, respectively) and an increase in cells displaying staining at both
locations (73% and 86%, respectively). In cells with pole-topole distances of 13.0-15.9 µm (n=57), no cells were found
with CENP-E limited to the kinetochores, but a significant
population (27%) had CENP-E limited to the interzonal
microtubules (73% of the cells at this stage were positive for
CENP-E at both locations). At yet longer inter-pole distances
of 16.0-18.9 µm (n=49) and >18.9 µm (n=50), there were
decreasing percentages of cells with both kinetochore and
microtubule staining (28% and 22%, respectively) and, conversely, an increasing population of cells with staining
restricted to the interzonal microtubules (72% and 78%,
respectively). Cells in telophase, identified by the longest
spindles, segregated chromosomes and observable decondensation of chromatin, never displayed any CENP-E staining at
the kinetochores and possessed inter-polar distances ranging
from 25-32 µm (not shown).
Our observations on CENP-E localization during anaphase
suggest that CENP-E gradually dissociates from the kinetochore following the completion of anaphase-A and reassociates with the interzonal microtubules of the spindle in a gradual
manner during anaphase-B. However, another possibility is
that there are two populations of CENP-E: one becomes kinetochore-bound following nuclear envelope breakdown, while
the other never associates with kinetochores but is recruited
from a free pool to the interzonal microtubules following the
onset of anaphase. In this latter view, the ability of a non-kinetochore associated pool of CENP-E to bind microtubules
would be suppressed prior to anaphase, possibly due to
nucleotide-independent
microtubule
p34cdc2-regulated,
binding site present within the carboxy terminus of CENP-E
(Liao et al., 1994).
To distinguish between these two possibilities, chromosomes from mitotically arrested HeLa cells were fractionated
by sucrose density centrifugation (see Materials and
Methods) and equal proportions of the chromosome-enriched
fraction and the soluble supernatant were analyzed by
immunoblotting (Fig. 7). CENP-B, a well characterized centromeric component of metaphase chromosomes (Cooke et
al., 1990), was detected in the chromosome containing
fraction but not in the soluble component fraction (Fig. 7B),
confirming that the chromosome fraction was enriched in, and
the soluble component fraction was depleted of, chromosomes. Immunoblot analysis of equal proportions of these
fractions with a tubulin antibody revealed that this protein
was detected only in the soluble fraction (Fig. 7C), indicating no detectable contamination of the soluble component
fraction in the chromosome-enriched fraction. When both
fractions were assayed for CENP-E, it was found only in the
chromosome-enriched fraction (Fig. 7D). That post-translational modifications could account for masking the antigens
on a potential pool of soluble CENP-E seems unlikely since
the antibody to CENP-E is polyclonal and was generated
against a large (74 kDa) protion of CENP-E. Hence, the
evidence in Fig. 7 demonstrates that most, possibly all,
CENP-E in prometaphase cells fractionates with chromosomes. Further, since anaphase is very short, translation efficiency falls during mitosis (Fan and Penman, 1970), and
synthesis of the 2,663 amino acid CENP-E protein requires
about 20 minutes, it seems highly unlikely that new synthesis
of CENP-E can account for the interzonal CENP-E appearing
late in anaphase.
966
K. D. Brown, K. W. Wood and D. W. Cleveland
A
D
B
C
E
F
G
Fig. 3. CENP-E distribution during mitosis in Indian muntjac cells. Indian muntjac cells were fixed and stained with Hoescht 33258 (blue),
tubulin monoclonal 5H1 (green) and affinity purified CENP-E antisera pAb-HpX (red). (A) An image of an interphase cell (arrow) and a
prophase cell (arrowhead) is displayed. (B) Prometaphase cell. (C) Metaphase cell. (D) Early anaphase cell. (E) Mid-anaphase cell. (F) Late
anaphase cell. (G) Late telophase cell.
DISCUSSION
The dynamics of CENP-E associations during
mitosis: a clearer view
Initial studies on the localization of CENP-E during mitosis
in human cells determined that this protein is first detected as
a kinetochore-associated component following the breakdown
of the nuclear envelope at prometaphase and remains kinetochore-associated throughout chromosome congression and at
the onset of anaphase (Yen et al., 1991). It was also shown
that some CENP-E remains kinetochore-associated during
early anaphase (Yen et al., 1991; Lombillo et al., 1995), but
relocalizes exclusively to the midzone region of the mitotic
spindle at an undefined point in anaphase or telophase. Our
observations in Indian muntjac cells extend these findings to
prove that, following the onset of anaphase, CENP-E remains
kinetochore bound until poleward chromosome migration
(anaphase-A) has been completed. Following this, CENP-E
accumulates on the interzonal microtubules of the mitotic
spindle during spindle elongation (anaphase-B). The failure to
CENP-E is kinetochore associated throughout anaphase-A
A
DNA
gradual manner during the course of spindle elongation. As
anaphase-B nears completion, no CENP-E is detected at the
kinetochore, suggesting that by this point in mitotic progression CENP-E has quantitatively dissociated from the kinetochore and is now fully associated with the interzonal microtubules.
B
C
CENP-E
CREST
Fig. 4. Co-localization of CENP-E and CREST antigens during
anaphase. Mitotic Indian muntjac cells were hypotonically swollen
prior to fixation and immunofluorescent processing. (A) Hoescht
33258. (B) pAb-HpX. (C) CREST sera. The boxed region of the cell
in A is shown at higher magnification in B and C. Note colocalization of the pAb-HpX and CREST sera staining (arrowheads).
detect a soluble pool of CENP-E in prometaphase cells
indicates that, following completion of anaphase-A, CENP-E
dissociates from the kinetochore and then associates with the
interzonal microtubules. Furthermore, the relocation of
CENP-E from kinetochore to mitotic spindle occurs in a
CENP-E and the mechanics of anaphase-A
While the exact nature of anaphase-A mechanics awaits full
elucidation, several key aspects of the forces which guide
poleward chromosome movement have been uncovered. For
instance, it is well established that microtubules which extend
from the spindle poles to the kinetochores, referred to as kinetochore fibers, shorten during anaphase-A (see McIntosh,
1985). Furthermore, the shortening of kinetochore fibers is due,
primarily, to microtubule subunit loss at the kinetochore
(microtubule plus-end) (Gorbsky et al., 1987), although some
subunit loss has also been found at the spindle pole (microtubule minus-end) (Mitchison and Salmon, 1992). When
combined with the observations that the kinetochore possesses
an intrinsic ability to translocate along microtubules (Hyman
and Mitchison, 1991) and plays a role in poleward chromo-
Q
M
I
E
A
967
Centrosomes
pole-to-pole
distance (µm)
3.9
6.8
B
F
C
G
D
H
10.5
J
14.5
19.9
N
R
O
S
P
T
DNA
K
CENP-E
L
Merged
Color
Image
Fig. 5. CENP-E localization during anaphase. Mitotically selected Indian muntjac cells were processed for immunofluorescence with the anticentrosome autoimmune sera SPJ (A,E,I,M,Q), Hoescht 33258 to visualize DNA (B,F,J,N,R), and pAb-HpX (C,G,K,O,S). The measured pole-topole distance for each cell is shown. Additionally, these images were merged and pseudo-colored (D,H,L,P,T). Blue, DNA; green, centrosomes; red,
CENP-E.
968
K. D. Brown, K. W. Wood and D. W. Cleveland
P er cen t a ge of Cells in Ea ch Gr ou p
100
Fig. 6. Distribution of CENP-E during anaphase.
Mitotically selected Indian muntjac cells were stained
for centrosomes, DNA and CENP-E as described in Fig.
5. The pole-to-pole distance of metaphase and anaphase
cells was measured and the cells scored for CENP-E
staining limited to the kinetochores (open bars), limited
to the interzonal microtubules (filled bars), or in both
locations (shaded bars).
kinetochore only
kinetochore and
microtubule
microtubule only
75
50
25
0
x = 3.85
n = 32
Metaphase
e
m
A
ble
om
lu
r
So Ch
B
86
e
m
o
os
le m
lub hro
o
S
C
121
86
205
CENP-B
50
50
33
33
19
1
C
2
dye front
e
om
s
o
ble
om
lu
r
So Ch
1
D
2
86
Tubulin
121
1
2
dye front
10.0 - 12.9
13.0-15.9
16.0-18.9
> 18.9
n = 60
n = 52
n = 57
n = 49
n = 50
Early Anaphase
Mid-Anaphase
: Pole-to-Pole
Pole-to-Pole
distance distance
(µ m)
: Mitotic
Stage
shortly after the poleward translocation of chromosomes along
them.
The finding that cytoplasmic dynein, a minus-end directed
microtubule motor, was present at the kinetochore (Steuer et
al., 1990; Pfarr et al., 1990) fueled speculation that this
protein was, in part, responsible for powering chromosome
movement during anaphase-A. However, antibody microinjection experiments have not supported involvement of
dynein in this process (Vaisberg et al., 1993) and genetic
approaches in yeast have failed to reveal a role in chromosome movement per se (Li et al., 1993; Saunders et al., 1995).
On the other hand, we have shown here that the kinesin-like
protein CENP-E is kinetochore associated throughout all
mitotic chromosome movements including anaphase-A.
Combined with the findings that: (1) some kinesin-like
proteins support minus-end directed motility (McDonald et
al., 1990; Walker et al., 1990; Endow et al., 1994); (2) a
minus-end motor activity has been shown to be associated
with CENP-E (Thrower et al., 1995); and (3) antibodies to
CENP-E block minus end-directed chromosome movement
powered by microtubule disassembly (Lombillo et al., 1995),
these observations strengthen the case that CENP-E is the
e
ble om
u
l
r
So Ch
205
50
33
7.0 - 9.9
m
o
os
CENP-E
121
n = 32
Late Anaphase
some movement (Nicklas, 1989; Rieder and Alexander, 1990),
these findings imply that the kinetochore actively participates
in poleward chromosome movement along microtubules which
either disassemble at the kinetochore/microtubule interface or
o
os
< 7.0
86
1
2
Fig. 7. Biochemical analysis of CENP-E distribution in fractionated
mitotic cells. Chromosomes from mitotically arrested HeLa cells
were isolated by sucrose density centrifugation as outlined in
Materials and Methods. Subsequently, equal proportions of the
soluble component fraction (lane 1) and the chromosome enriched
fraction (lane 2) were analyzed. (A) 10% of each fraction was
electrophoresed on a 5-15% gradient gel followed by Coomassie
staining. (B) 10% of each fraction was electrophoresed on a 10%
polyacrylamide gel and immunoblotted with CENP-B antisera.
(C) 2.5% of each fraction was electrophoresed on a 7.5%
polyacrylamide gel and immunoblotted with the tubulin monoclonal
antibody 5H1. (D) 10% of each fraction was electrophoresed on a
5% polyacrylamide gel and immunoblotted with the CENP-E
antisera pAb-HpX.
CENP-E is kinetochore associated throughout anaphase-A
motor (or one of the motors) for anaphase-A chromosome
movement.
We thank Dr Trina Schroer, Department of Biology, Johns Hopkins
University for allowing us use of her video microscope system and
her comments on the manuscript prior to submission. We also thank
Drs Ron Balczon, Bill Brinkley, Skip Binder, Ann Pluta and Bill
Earnshaw for their generous gifts of antibodies. K.D.B. was supported
by a postdoctoral fellowship from the American Cancer Society.
K.W.W. was supported by a postdoctoral fellowship from the Damon
Runyan/Walter Winchell Cancer Fund (DRG-1258). This work has
been supported by a grant (GM 29513) to D.W.C. from the National
Institutes of Health and funds from the Ludwig Institute for Cancer
Research.
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(Received 12 November 1995 - Accepted 31 January 1996)