* Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania 19103; Department of
Biology, University of North Carolina, Chapel Hill, North Carolina 27599; and § Fox Chase Cancer Center, Philadelphia,
Pennsylvania 19111
CENP-E is a kinesin-like protein that binds to kinetochores and may provide functions that are critical for normal chromosome motility during mitosis. To directly test the in vivo function of CENP-E, we microinjected affinity-purified antibodies to block the assembly of CENP-E onto kinetochores and then examined the behavior of these chromosomes. Chromosomes lacking CENP-E at their kinetochores consistently exhibited two types of defects that blocked their alignment at the spindle equator. Chromosomes positioned near a pole remained mono-oriented as they were unable to establish bipolar microtubule connections with the opposite pole. Chromosomes within the spindle established bipolar connections that supported oscillations and normal velocities of kinetochore movement between the poles, but these bipolar connections were defective because they failed to align the chromosomes into a metaphase plate.
Overexpression of a mutant that lacked the amino-terminal 803 amino acids of CENP-E was found to saturate limiting binding sites on kinetochores and competitively blocked endogenous CENP-E from assembling onto kinetochores. Chromosomes saturated with the truncated CENP-E mutant were never found to be aligned but accumulated at the poles or were strewn within the spindle as was the case when cells were microinjected with CENP-E antibodies. As the motor domain was contained within the portion of CENP-E that was deleted, the chromosomal defect is likely attributed to the loss of motor function.
The combined data show that CENP-E provides kinetochore functions that are essential for monopolar chromosomes to establish bipolar connections and for chromosomes with connections to both spindle poles to align at the spindle equator. Both of these events rely on activities that are provided by CENP-E's motor domain.
THE kinetochore establishes and maintains the connection between the chromosome and the dynamic
plus ends of microtubules to generate force for accurately segregating chromosomes. After the nuclear envelope disassembles at the onset of mitosis, chromosomes establish connections to microtubules from each spindle
pole, as each member of a kinetochore pair that resides on
opposite sides of the centromere captures microtubules
that are nucleated from the pole that they face. The bipolar attached chromosome congresses toward the spindle
equator through the coordinated actions of the kinetochore pair. Net displacement of the chromosome occurs
when one kinetochore moves towards its pole, while its sister kinetochore moves away from its pole. Concomitant
with these motions, microtubules attached to the kinetochore that is moving poleward must shorten, and those
that are attached to its sister elongate. Shrinkage and elongation of microtubules occur primarily at the kinetochore
because this attachment site is where tubulin subunits are
incorporated into or mostly lost from the microtubule
(Mitchison et al., 1986 The discovery of various microtubule-based motors that
reside at kinetochores suggest that these molecules generate force to move chromosomes and also maintain the mechanical link between kinetochores and the tips of shrinking and growing microtubules (Desai and Mitchison, 1995 CENP-E is a 312-kD protein (Yen et al., 1992 A role for CENP-E in chromosome motility has come
from recent in vitro studies on the mechanism that is responsible for microtubule depolymerization-dependent
movement of chromosomes (Lombillo et al., 1995 The discrepancy between the in vitro data and the early
microinjection studies necessitated a re-evaluation of the
in vivo function of CENP-E. This renewed effort required
that we develop strategies that directly test the contribution of CENP-E to kinetochore function. Optimally, this
would require examining the behavior of chromosomes
whose kinetochores lacked CENP-E. Our first strategy was
to microinject highly specific polyclonal CENP-E antibodies to sterically block CENP-E from binding to kinetochores
at mitosis. The second approach was to overexpress a motorless CENP-E mutant in transiently transfected cells to
saturate limiting binding sites for CENP-E at kinetochores
and thus competitively block endogenous CENP-E from
binding. In both strategies, depletion of endogenous
CENP-E from kinetochores to levels below our limits of
detection blocked chromosome alignment but not bipolar
spindle formation. Chromosome alignment was reproducibly blocked at very specific stages as they were found to
be trapped very close to a pole (most likely with a monopolar connection), or chromosomes established defective
bipolar connections that sustained normal velocities of oscillations but often failed to attain a stable position at the
spindle equator. Thus, CENP-E and its motor domain provide functions that are essential for two events during
prometaphase: monopolar chromosomes to establish bipolar connections and for bipolar chromosomes to align and
form a stable metaphase plate at the spindle equator.
Production of Antibodies and Immunological Methods
A 1.3-kb Dra I CENP-E cDNA fragment was subcloned into pMAL expression vector (New England Biolabs, Beverly, MA) and purified maltose-binding (MBP)1 fusion protein was used to raise rabbit antibodies
(Dra B) against the carboxy-terminal 360 amino acids. For HX-1 antibodies, an Xho/HindIII fragment that encodes amino acids 1,571-1,859 of
CENP-E was subcloned into pGEX (Pharmacia Biotech, Piscataway, NJ),
and the purified glutathione-S-transferase (GST) fusion protein was used
to immunize rabbits. Antibodies were affinity purified by binding whole
IgG to an Affigel-10 (Bio Rad, Richmond, CA) column that was covalently coupled with either the MBP:DraB or GST:HX-1 fusion proteins.
Antibodies were eluted with 100 mM glycine, pH 2.5, neutralized, concentrated, and stored in PBS. Preimmune rabbit IgG was purified using
protein A sepharose (Pierce, Rockland, IL). Mouse monoclonal antibody
177 was described previously (Yen et al., 1991 Microinjections and Video Microscopy of Injected Cells
Microinjections were performed with a Nikon Diaphot that is equipped
with an Eppendorf semi-automatic microinjector and micromanipulator. Antibodies were first filtered through a 0.2-µm membrane (Millipore, Bedford, MA), and injections were performed with Eppendorf femtotips. HeLa or U2OS cells that were grown to 30% confluence in Hepes buffered DME plus 10% FBS were injected in the cytoplasm with an antibody
concentration of 0.5 mg/ml for the anti-carboxy terminus (DraB) antibody, 10 mg/ml for anti-rod domain (HX-1) antibody, and 20 mg/ml for
preimmune antibodies. After injection, cells were returned to 37°C for
~12 h before observation. For quantitative studies, HeLa cells were synchronized by double thymidine block as described (Yen et al.,1992). After
release from the block, the tissue culture dish was placed into a TC-102
(Medical Systems Corp., Greenville, NY) microscope stage incubator that
maintained the medium at 37°C. Cells were injected with antibody, the
culture medium was overlaid with mineral oil, and the entire field of injected and uninjected cells were recorded using a CCD camera (Ultrachip;
Javelin Electronics, Torrance, CA) and a time-lapse S-VHS video recorder (Panasonic, Secaucus, NJ). The duration of mitosis for each cell
was determined by recording when each cell rounded up (entry into mitosis) and when they separated (cytokinesis).
Interphase U2OS cells were microinjected with DraB antibodies and
incubated for ~12 h at 37°C. When the cells were confirmed to be blocked
in mitosis, the coverslip was removed from the culture dish and inverted
onto a clean glass slide that had two strips of double stick tape. Hepes-buffered DME plus 10% FBS was added, and the observation chamber
was sealed with a 1:1:1 mixture of Vaseline/lanolin/beeswax. The slide
chamber was mounted on a microscope (Diaphot; Nikon, Inc.) and cells
were observed under a 63× 1.4 NA differential interference contrast
(DIC) objective and a 1.4 NA condenser. Images were captured with a
Newvicon video camera (Hamamatsu Phototonics, Bridgewater, NJ),
real-time background subtraction was performed with a DSP 2000 image
processor (DAGE-MTI, Michigan City, IN), and the subtracted images
were recorded onto time-lapse S-VHS VCR (Panasonic). Individual video
frames were photographed directly from the monitor. To measure the velocities of the kinetochores, the videotape was analyzed as described
(Skibbens et al., 1993 Constructs and Transient Transfections
Construction of the motorless CENP-E was accomplished by fusing PCR
fragments that contain either GFP (Chalfie et al., 1993 Microinjection of CENP-E Antibodies Depletes
CENP-E from Kinetochores
Our first strategy to deplete CENP-E from kinetochores
took advantage of an earlier observation that CENP-E is a
cytoplasmic protein during interphase and assembles onto
kinetochores after nuclear envelope breakdown in mitosis
(Yen et al., 1991
Initial experiments showed that when interphase (asynchronous) HeLa or U2OS cells were microinjected with
either of the two CENP-E antibodies, they were found to
accumulate in mitosis when they were examined after an
overnight incubation. These cells were probably blocked
in mitosis as their mitotic index of injected cells was much
higher than cells injected with nonimmune IgG. To examine the distribution of CENP-E in the mitotically blocked cells, the injected cells were stained with a murine monoclonal CENP-E antibody (mAb177) whose epitope did not
overlap those recognized by the injected rabbit polyclonal
antibodies. mAb177 staining did not reveal the presence of
CENP-E at the kinetochores of these mitotic cells (Fig. 1,
B and D) even though prominent levels of kinetochore
staining were observed in adjacent uninjected prometaphase cells (Fig. 1, F and G). Direct visualization of the injected CENP-E antibodies also did not reveal specific localization at kinetochores (data not shown). The fate of
CENP-E/antibody complexes in the injected cells is not
known but most likely was released from the cytoplasm
during extraction.
Inhibition of CENP-E binding to kinetochores was specific for the affinity-purified antibodies as injection of 40-fold more nonimmune rabbit IgG did not disrupt mitotic
progression (Fig. 2, A, D, and G; and see below) or the
normal distribution of CENP-E at kinetochores (Fig. 2, B,
E, and H) at metaphase or at the interzonal microtubules
at anaphase. As mAb177 was able to identify the normal subcellular distribution of CENP-E in the cells injected
with control IgG, these results also show that the inability
of mAb177 to stain kinetochores of cells injected with
CENP-E polyclonal antibodies was not simply due to an
overwhelming level of rabbit IgG in the cell. Microinjection of CENP-E antibodies was found to be a reliable
method to deplete CENP-E from kinetochores and was
therefore used to examine the in vivo function of CENP-E.
Loss of CENP-E at Kinetochores Block Cells in Mitosis
To monitor the fates of the injected cells in a more quantitative way, 27 HeLa cells that were synchronized at the G1/S
boundary were injected with the affinity-purified carboxy-terminal CENP-E antibodies shortly after they were released from the cell cycle block (Table I). Time-lapse
videomicroscopy at low magnification (10× phase contrast) showed that the injection did not interfere with progression of the cells into mitosis as they took the same amount of time to enter mitosis as did adjacent uninjected
cells. However, cells injected with the affinity-purified antibodies to CENP-E were arrested in mitosis in a period
that varied between 4 to 17 h while mock-injected cells or
cells injected with nonimmune IgG completed mitosis in
~1 to 2 h. All of the cells arrested in mitosis by CENP-E
antibodies eventually died by apoptosis. These results show
that CENP-E is not required for progression of cells into
mitosis but is essential for completion of mitosis.
Table I.
Time Course of Microinjected Cells
; Gorbsky et al., 1987
; for review see
Inoue and Salmon, 1995
; Yen and Schaar, 1996
; Nicklas, 1997
).
;
Lombillo et al., 1995
). Thus far, the motor composition of
mammalian kinetochores includes cytoplasmic dynein and
its associated dynactin complex (Pfarr et al., 1990
; Steuer
et al., 1990
) and two kinesin-related proteins, CENP-E (Yen et al., 1992
) and MCAK (XKCM1 is its frog homolog; Wordeman and Mitchison, 1995
; Walczak et al.,
1996
). Video analysis and correlative electron microscopy
of chromosome movements in newt lung cells show that if
a kinetochore makes a lateral connection with a microtubule that glances past it, this interaction was sufficient to
pull the chromosome polewards at a velocity that is consistent with the in vitro polarity and velocity of cytoplasmic dynein (Rieder et al., 1989
; Hayden et al., 1990
). In addition to a potential role in pulling chromosomes into the
spindle proper, dynein and dynactin contribute to spindle
morphogenesis, as disruption of their functions interferes
with bipolar spindle formation (Vaisberg et al., 1993
; Echeverri et al., 1996
). XKCM1 (and perhaps MCAK) may also
play a role in spindle morphogenesis as depletion of this
protein from frog egg extracts disrupts spindle assembly (Walczak et al., 1996
). However, the ability of XKCM1 to
induce catastrophic depolymerization of microtubules in
vitro suggests that this activity at the kinetochore may
stimulate kinetochore microtubule depolymerization and
facilitate poleward movement of chromosomes (Walczak
et al., 1996
).
) that assumes a highly elongated shape and was found to be associated with a minus end microtubule motor activity (Thrower
et al., 1995
). Recent immunogold EM data show that it is
concentrated at the fibrous corona that occupies the surface of the outer kinetochore plate (Cooke et al., 1998
).
Early functional studies had shown that microinjection of
an anti-CENP-E monoclonal antibody (mAb177) arrested
cells in mitosis with chromosomes aligned in a metaphase plate at the spindle equator (Yen et al., 1991
). The nature
of this arrest was never clear, as the antibody was not directed towards obvious functional domains such as the
motor or the carboxy-terminal microtubule binding domains (Liao et al., 1994
), but rather, it recognized epitopes
along the extended rod domain. Although the presence of
this monoclonal antibody at kinetochores did not prevent chromosomes from aligning at the spindle equator, it interfered with critical events that were necessary to initiate
chromosome separation and anaphase onset.
). Chromosome movement in this in vitro system does not require
ATP but relies simply on the ability of the kinetochore to
remain attached to the shrinking end of a single microtubule induced to depolymerize by the dilution of free tubulin subunits. CENP-E was found to be important for coupling the kinetochore to the microtubule as antibodies
directed against its neck region, which connects the motor
domain of CENP-E to its stalk domain, disrupted chromosome movement by dissociating the kinetochore from a
shrinking microtubule against a flow of buffer. This finding suggested that the kinetochore-microtubule interactions mediated by CENP-E in vitro might be important to
chromosome attachment and alignment on the spindle in
vivo.
Materials and Methods
). To detect the green fluorescent protein (GFP):CENP-E fusion protein by immunoblot, rat polyclonal antibodies were generated against bacterially expressed GFP. Affinity-purified GFP antibodies were used to probe filters at a final
concentration of 1 µg/ml. Filters were washed, incubated with alkaline
phosphatase-conjugated anti-rat secondary antibodies (Sigma Chemical
Co., St. Louis, MO), and bound antibodies were visualized by chemiluminescent detection. Western blots were performed according to manufacturer's protocol (Tropix, Bedford, MA). HX-1 and DraB antibodies were used at 1 µg/ml final concentration. Immunofluorescence staining was performed as described (Yen et al., 1991
) and visualized with a 100× 1.3 NA
objective attached to a microscope (Microphot SA; Nikon, Inc., Melville,
NY) equipped with epifluorescence optics. Photographs were taken with
Kodak TMax 400 film.
).
) or the IgG-binding region of protein A (pRIT2T; Pharmacia Biotech) in the correct reading frame with a 5.6-kb cDNA fragment that encoded amino acids 880 to
the carboxy terminus of CENP-E. The hybrid gene was subcloned into the
expression vector pWS that uses a CMV promoter and a segment of the
adenovirus 5
tripartite leader sequence to achieve high level expression
in mammalian cells (Sheay et al., 1993
). CsCl purified plasmid DNA was
used to transfect subconfluent HeLa cells according to Chen and Okayama (1987)
. Cells were harvested 30-40 h after transfection and lysates were prepared for immunoblot analysis. Immunofluorescence staining was performed as described above.
Results
). If microinjection of CENP-E-specific antibodies into interphase cells can block cytoplasmic CENP-E
from assembling onto kinetochores after nuclear envelope
breakdown, it would then be possible to examine the behavior of chromosomes that lacked CENP-E. Polyclonal
antibodies were raised against either a segment of the stalk
(amino acids 1,571-1,859) or the carboxy-terminal 360 amino
acids that included a large portion of the kinetochore targeting domain (Chan, G.K.T., and T.J. Yen, unpublished
observations). Both sets of antibodies were expected to
form mutivalent complexes with CENP-E and thus sterically interfere with its ability to bind to kinetochores. In
addition, the carboxy-terminal antibodies were expected
to directly block CENP-E from binding to kinetochores by
obscuring the kinetochore-targeting domain from its binding site. Both of the affinity-purified antibodies were
highly specific as CENP-E was the only protein that was
identified when they were used to probe filters that contained total protein from mitotic HeLa cells (Fig. 1 A).
Fig. 1.
Depletion of CENP-E from kinetochores by microinjection of rabbit polyclonal antibodies directed against CENP-E.
(A) Western blots of mitotic HeLa lysates probed with stalk HX-1
(lane 1) and carboxy-terminal DraB (lane 2) affinity-purified antibodies. (B, D, F, and I) Monoclonal antibody mAb177 (7) staining of Hela cells injected with 0.5 mg/ml of DraB antibodies (B
and D), an uninjected prometaphase cell (F). mAb177 was visualized with FITC-conjugated anti-mouse antibodies. (C, E, and G)
DAPI staining to visualize chromosomes. Images from B, D, and
F were exposed for identical times. Bar, 10 µm.
[View Larger Version of this Image (109K GIF file)]
Fig. 2.
Preimmune antibodies injected at a 40-fold higher concentration have no effect on mitotic progression or CENP-E localization. Preimmune HX-1 antibodies were injected in an identical manner to immune antibodies. DNA was detected with
DAPI (A, D, and G) CENP-E was detected with mAb 177 (B, E,
and H) and injected antibodies were detected with anti-rabbit Ig
secondary antibodies (C, F, and I). No aberrations were seen in
the organization of the chromosomes during alignment (A).
CENP-E localized normally in these cells during anaphase (E and
H). Anaphase cells as shown in D through F and G through I
were never observed in immune HX-1 nor DraB antibody-
injected cells. Bar, 10 µM.
[View Larger Version of this Image (163K GIF file)]
Chromosomes Lacking CENP-E at Kinetochores Fail to Align
The nature of the mitotic defect that resulted from the depletion of CENP-E from kinetochores was next examined.
Tubulin staining revealed that the mitotically blocked cells
had established a bipolar spindle (Fig. 3 C). The overall
morphology of the spindle in many cells took on a ragged
appearance that is likely due to the prolonged mitotic
block. Nevertheless, this result showed that CENP-E is
not essential for bipolar spindle formation. Examination of the distribution of chromosomes in >100 blocked cells
showed that they were either positioned very close to the
poles or scattered between the poles (Fig. 3, D and F). The
accumulation of large numbers of chromosomes at the poles
in the mitotically blocked cells contrasts with a normal prometaphase cell where chromosomes are rarely seen so
close to the poles but lie mostly within the spindle (Fig. 3,
A and B). The chromosomes at the poles did not result
from premature separation of the chromatids because immunofluorescence staining with anti-centromere autoimmune serum (ACA) produced the characteristic double dot
staining pattern that is indicative of paired chromosomes
(Fig. 3 E, open arrows). In addition to the accumulation of
monopolar chromosomes in cells where CENP-E function
was inhibited, there were also chromosomes that were strewn throughout the spindle. Normally, the chromosomes that are positioned in between the two poles at the
time of nuclear envelope breakdown rapidly establish bipolar connections and immediately begin to congress towards the spindle equator (Fig. 3 B) (Alexander and Rieder,
1991; Rieder and Salmon, 1994
) The loss of CENP-E affected their ability to align, as we never observed an injected cell with aligned chromosomes at the metaphase
plate.
To further characterize the behavior of chromosomes whose kinetochores were depleted of CENP-E, the motions of chromosomes in normal and antibody-injected U2OS cells were monitored by time-lapse video enhanced (VE)-DIC microscopy. In a normal cell, chromosomes were found to be completely aligned into a metaphase plate at the spindle equator ~30 min after nuclear envelope breakdown (Fig. 3, G and I). In an injected cell that was already blocked in mitosis for 2 h before viewing, the mono-oriented chromosomes never moved away from their pole to establish bipolar connections during a 2-h period of continuous observation. In contrast, chromosomes within the spindle were connected to both poles as they oscillated back and forth between the poles at velocities typical of chromosomes in uninjected cells (1.7 vs. 1.6 µm/ min). However, these bipolar connections are defective as the chromosomes failed to align into a metaphase plate during the 2-h period of observation (Fig. 3, H and J). As these chromosomes retained the ability to oscillate between the poles during the extended period of the block, the loss of CENP-E from the kinetochores does not appear to affect their ability to move with normal velocities and maintain a bipolar connection. However, the inability of these chromosomes to align into a metaphase plate demonstrates that kinetochores that lack CENP-E are defective in the mechanisms that govern their movements.
Bipolar Attached Centromeres Lacking CENP-E Separate
In ~25% of the mitotically blocked cells that were injected with CENP-E antibodies, some of the chromosomes within the spindle appeared to have separated at their centromeres because ACA produced single dots of staining instead of the typical double dot pattern that was indicative of paired centromeres (Fig. 3 E, solid arrows). The failure to detect a double dot pattern was not because one of the centromeres was obscured as a second dot of ACA staining was not found within close proximity in any focal plane. In all cases, the single foci of ACA staining were located at the extreme ends of what appeared to be centromeres that had separated from each other and were pulled towards opposite poles. However the chromosomes located at the poles remain paired based on the double dot ACA staining at their centromeres (Fig. 3 E, open arrows).
An example of a cell that was examined by time-lapse
VE-DIC microscopy (Fig. 4, A-N) showed that the chromosomes within the spindle were indeed separated at their
centromeres (Fig. 4, B and I, solid arrows) and along the
length of their arms but remained connected near the telomeres (Fig. 4, B and I, open arrows). It is noteworthy
that all separated metacentric chromosomes remained connected at both telomeres. However, acrocentric chromosomes were invariably found to be connected only by
the telomere region of the long arm as the connections at
the telomeres of the short arm were never observed.
To search for clues that would help us understand how
depletion of CENP-E might cause the bipolar chromosomes
to prematurely separate, several parameters of kinetochore movement were quantitated. We were able to track
the movements of sister kinetochores of partially separated chromatids in cells like that shown in Fig. 4 for
nearly 50 min before the cell went into anaphase or the chromosomes went out of focus. First, we determined that
the separated chromosomes were still connected to both
poles as they were found to oscillate between the poles at
rates that were not significantly different than for a normal
bipolar chromosome. The kinetochores on partially separated sister chromatids (Fig. 5, Ks1P and Ks2P) oscillated
back and forth between the poles with average velocities (0.65 ± 0.2 µm/min, n = 12) similar to those exhibited by
kinetochores on bipolar-oriented chromosomes in uninjected cells (0.8 ± 0.2 µm/min, n = 5). In addition, the
plots (Fig. 5, Ks1P and Ks2P) showed that sister kinetochores on the partially separated sister chromatids oscillated at frequencies that were similar to normal paired kinetochores; the duration of kinetochore movement in one direction of oscillation was 1.7 ± 0.7 min for both injected
and uninjected cells. We also quantitated the distance that
separated the two kinetochores from one of the poles and
plotted these measurements over time. While both kinetochores were separated from each other by the partially
separated arms, they were able to maintain a relatively
constant distance of 3 µm between them (Fig. 5, Ks1P-Ks2P). This indicated that for the majority of time (63%),
the two kinetochores exhibit coordinated motions where
one sister is able to sense the direction of motion of the
other sister (Skibbens et al., 1993). There were discrete
points in time when the sister kinetochores moved away
from each other, stretching their chromatid arms (Fig. 5,
peaks in Ks1P-Ks2P plot) and also towards each other so
that the chromosome appeared compressed (Fig. 5, valleys
in Ks1P-Ks2P plot; and Fig. 4, B-E, and I-L). The overall
similarity in velocity of movement, frequency of oscillation
and frequency of coordinated kinetochore movements between the prematurely separated chromosomes and a normal bipolar chromosome (Skibbens et al., 1993
) indicates that substantial depletion of CENP-E does not impair
these kinetochore motility parameters.
Unlike the bipolar chromosomes that oscillated for extended periods of time, the video images also showed that
chromosomes that were positioned near the poles never
exhibited any motion (data not shown). Separation of the
bipolar chromosomes in these cells was not due to premature anaphase, because in rare instances, a cell overcame
its checkpoint arrest and entered anaphase (Figs. 5 and 6).
The telomeric connections that once held the separated chromosomes together were synchronously dissolved and
the completely separated chromatids moved towards opposite poles at rates that were typical of that seen in normal anaphase (Figs. 5 and 6, A-E).
Amino-Terminal Truncated CENP-E Behaves as a Dominant-Negative Mutant
The disruption of kinetochore motility when CENP-E was
depleted from kinetochores led us to adopt a second approach to directly test the contribution of its motor domain
to kinetochore function. This method relied on the ability
of an overexpressed CENP-E mutant to saturate the binding sites on kinetochores and competitively block endogenous CENP-E from assembling onto kinetochores. We deleted the amino-terminal 803 amino acids from CENP-E
that included the kinesin-like motor domain and replaced
the deleted portion with green fluorescent protein (GFP;
Chalfie et al., 1993). Western blots of lysates prepared from
transiently transfected HeLa cells showed that the GFP:
CENP-E N
803 mutant expressed a protein of the expected size (Fig. 7 A). Examination of the distribution of
the GFP:CENP-E N
803 (Fig. 7, D and G) in transfected
mitotic cells showed that it accumulated at kinetochores as
confirmed by colocalization with ACA staining (Fig. 7 H).
Because the mutant CENP-E lacked its amino-terminal
803 amino acids, an antibody that specifically recognized
this domain was used to detect the distribution of endogenous CENP-E. This antibody was able to detect endogenous CENP-E at the kinetochores of an untransfected
metaphase cell (Fig. 7, B and C). In contrast, overexpression of the CENP-E mutant saturated the kinetochore
binding sites (Fig. 7 D) as endogenous CENP-E was not
detected at kinetochores (Fig. 7 E).
The reproducible increase in the mitotic index of cells
that expressed the CENP-E N 803 mutant relative to untransfected or mock-transfected cells suggested that the
accumulation of the mutant protein blocked cells in mitosis. In all cases (>100 cells), the occupation of the kinetochore by the mutant did not disrupt bipolar spindle formation but selectively disrupted chromosome alignment.
Inspection of the chromosomes in fixed preparations of
cells that were saturated with the CENP-E mutant at their
kinetochores showed that chromosomes were found at the
poles as well as in between the poles (Fig. 7, F and I). This
data provides independent confirmation of the microinjection data. As the region that was deleted contains the motor domain that was shown to exhibit ATP-sensitive microtubule binding in vitro (Liao et al., 1994
), the results strongly suggest that the motor domain of CENP-E is critical for proper kinetochore function. It is formally possible
that the ~350 amino acids that extend beyond the motor
domain might contribute to some non-motor function of
CENP-E that is also important for kinetochore function.
The ability of the CENP-E mutant to compete effectively
with the endogenous CENP-E for kinetochore-binding sites
suggests that the mutant protein retains the same interactions with other proteins at the kinetochore as with the
endogenous CENP-E. Thus, the chromosome defects observed when CENP-E is depleted from kinetochores by
microinjecting antibodies is unlikely the result of compromising the overall structural integrity of the kinetochore.
The collective data obtained by antibody injection and overexpressing the CENP-E N
803 mutant show that we have
specifically disrupted CENP-E function at kinetochores
and demonstrated that this molecule is critical for monopolar chromosomes to establish bipolar connections and
for bipolar chromosomes to align into a metaphase plate.
CENP-E Functions to Align Chromosomes
In this study, we have directly examined the kinetochore
function of CENP-E in vivo by characterizing the behavior
of chromosomes whose kinetochores were depleted of
CENP-E or were saturated with a CENP-E mutant that
lacked its motor domain. In both approaches, cells arrested in mitosis as they failed to align their chromosomes at the spindle equator. Our current findings differ from
early experiments, which showed that cells microinjected
with a CENP-E monoclonal antibody became blocked at
metaphase with aligned chromosomes. The disparity between the data can be accounted for by differences in experimental approaches. In the earlier study (Yen et al., 1991),
microinjection of the monoclonal antibody did not block assembly of CENP-E at kinetochores, as was the case here.
Instead, the monoclonal antibody was bound to the kinetochore-associated CENP-E at what we now know to be a
segment of the central rod domain. When the rod domain
of CENP-E was bound by this antibody, it did not interfere with its normal function, which is to align chromosomes
at the spindle equator. However, the presence of this antibody at the kinetochores was sufficient to prevent the aligned chromosomes from separating. Similar outcomes have been
reported for other antibodies, which when bound to kinetochores or centromeres arrested cells in mitosis with aligned
chromosomes (Bernat et al., 1989; Tomkiel et al., 1994
).
CENP-E Is Essential for Two Steps during Chromosome Alignment
Our observation that chromosomes that lacked CENP-E
at their kinetochores were reproducibly trapped at the
poles or within the spindle reveal two discrete steps during
chromosome alignment, when CENP-E function is critical.
In normal cells, chromosomes that are positioned near a
pole at the onset of nuclear envelope breakdown establish
a transient monopolar connection that is converted to a bipolar connection when its unoccupied kinetochore captures a microtubule from the opposite pole (Nicklas, 1988;
Nicklas and Ward, 1994
). As the frequency that the unoccupied kinetochore encounters a microtubule that traverses across the entire spindle is low, it is critical that a
stable connection is made when the kinetochore comes in
contact with a microtubule (Nicklas and Kubai, 1985
). One
likely kinetochore function of CENP-E is to use its motor
domain to capture and stabilize kinetochore-microtubule interactions. This possibility is supported by in vitro data
that show the motor domain of CENP-E directly binds to
microtubules (Liao et al., 1994
). When CENP-E is depleted
from kinetochores, the ability of the unoccupied kinetochore
of a monopolar chromosome to capture microtubules and
establish a bipolar connection is reduced (Fig. 8, A and B).
In normal cells, chromosomes that are positioned within
the spindle after nuclear envelope breakdown encounter
microtubules at higher frequencies than the unoccupied
kinetochore of a monopolar chromosome and therefore
establish rapid bipolar connections (Cassimeris et al., 1994;
Rieder and Salmon, 1994
). Even when CENP-E is depleted from these kinetochores, the higher frequency of
microtubule encounters within this region of the spindle
appears to be sufficient to establish and maintain bipolar
connections. These connections may be mediated by other
kinetochore proteins or by a residual amount of endogenous CENP-E that is below the limits of our detection. Although these bipolar connections are sufficient to sustain
relatively normal rates of oscillations, the CENP-E depleted kinetochores are unable to align at the spindle equator. The reason for this very specific defect is unclear, but
it may be due to the inability to establish critical kinetochore microtubule connections that are essential for alignment. A normal mammalian kinetochore can be attached to
as many as 20 to 30 microtubules (McIntosh, 1991
; McEwen, 1997). These microtubule connections are not static,
as they undergo cycles of release and capture at the kinetochores even after the chromosomes have aligned. At
metaphase, a microtubule is estimated to have a half-life of
~5 min so that it spends ~80% of its time attached to the
kinetochore (Zhai et al., 1995
). Whether this represents
the critical number of kinetochore microtubules that are
required for chromosome alignment is unknown. If the role of CENP-E is to maintain stable kinetochore microtubule connections, kinetochores that lack CENP-E may be
unable to establish the connections that are essential for
alignment at the equator. This explanation is consistent
with our interpretation of why monopolar chromosomes that lack CENP-E fail to establish bipolar connections as
well as in vitro data that suggest that CENP-E is important
for maintaining stable kinetochore connections (Lombillo et
al., 1995
). On the other hand, the failure to align is also
consistent with the argument that kinetochores that lack
CENP-E exhibit an unregulated poleward force that sustains prolonged oscillations but cannot maintain chromosomes at a steady position at the equator.
Bipolar Chromosomes Can Partially Separate in the Absence of CENP-E
The separation of centromeres and chromatid arms when
CENP-E was depleted from the kinetochores were only
observed for chromosomes that established bipolar attachments as monopolar chromosomes that accumulated in
the same cell remained paired. This aberration is not due
to a general consequence of a prolonged mitotic block for
two reasons. First, chromosomes that are arrested at the
metaphase plate by microinjection of centromere-specific
antibodies remain paired even after several hours of the
block. It is perhaps noteworthy that in the two cases that
were previously examined, CENP-E was still present at
the kinetochores of the metaphase-arrested chromosomes (Yen et al., 1991; Tomkiel et al., 1994
). Second, when cells
are blocked in mitosis for extended periods with microtubule-destabilizing drugs, the connections between the
chromsome arms separate but the centromeres remain
paired (Rieder and Palazzo, 1992
).
As we have not observed chromosomes that lack CENP-E
in the process of separating, we can only speculate about
their origin. We favor an explanation where we consider
CENP-E's role in kinetochore motility although it could also
contribute in an unknown way to the structural glue that
holds the centromeres together. The glue hypothesis alone
is inconsistent with our observation that only some chromosomes in a cell become separated while others, mostly at the poles, remain paired. Separation is likely due to secondary effects that resulted from the prolonged periods of
oscillations exhibited by the chromosomes that achieved
bipolar connections. This explanation is consistent with
the lower frequency at which cells with separated chromosomes are seen. Our observations are consistent with experiments in which cells blocked in metaphase by a low
dose of taxol show a transient increase in the distance between sister kinetochores (Waters et al., 1996a,b). Separation of these sister kinetochores is thought to occur as a
consequence of the poleward force that is simultaneously
exerted on both kinetochores as a result of microtubule
flux. If kinetochores lacking CENP-E tended to move towards opposite poles at higher frequency, the extended
periods of oscillations due to a mitotic block might provide
sufficient time for the centromeres to come apart. Another possibility is that CENP-E-deficient kinetochores produce
an unchecked poleward force that increases the frequency
or magnitude of force at which both kinetochores are simultaneously pulling towards their respective poles. The
inappropriate application of poleward force would initially
pull apart the connections holding together the centromeres, which would then propagate along the length of the
chromosome arms.
Proposed Mechanism of CENP-E Function
We propose that CENP-E plays a critical role in maintaining stable kinetochore-microtubule connections that allows chromosomes to establish bipolar connections and to
align at the spindle equator. This possibility is supported
by in vitro experiments that showed the importance of
CENP-E in microtubule depolymerization-dependent motility of kinetochores (Lombillo et al., 1995). In this assay, depolymerization of a single microtubule that was attached
at the kinetochore of an isolated chromosome was sufficient to induce movement of the chromosome in the direction of the shrinking microtubule. Although this assay was
performed in the absence of detectable ATP and low tubulin concentrations, there are interesting parallels that
can be drawn between the in vitro and in vivo data. Both the in vitro and in vivo data revealed the importance of
CENP-E's motor domain in kinetochore function. The ability of an isolated chromosome to move while remaining attached to a depolymerizing microtubule was completely
inhibited if chromosomes were preincubated with CENP-E
antibodies that were directed against the neck region that
connected the motor domain to the stalk. The antibodies
disrupted motility most likely by interfering with CENP-E's motor domain. Likewise, kinetochores saturated with
a motorless CENP-E mutant were defective in their ability to align chromosomes. Both studies also showed that
CENP-E was not critical for kinetochores to bind microtubules. Inhibition of CENP-E function with antibodies in vitro did not prevent microtubule capture by kinetochores.
Similarly, kinetochores depleted of CENP-E in vivo were
able to bind microtubules, although the connections were
defective.
The basis of the in vivo defects observed for kinetochores that lack CENP-E may be explained by the in vitro
finding that CENP-E is essential for coupling the kinetochore to a shrinking microtubule. Specifically, the in vitro
studies showed that CENP-E was essential for kinetochores
to remain attached to a shrinking microtubule or against a
counterflow of buffer (i.e., under tension). This suggests
that the connection between the kinetochore that was defective in CENP-E function and its microtubule was disrupted when tension was exerted on it. Perhaps, the critical function of CENP-E in vivo is to maintain and stabilize
the connections between the kinetochore and microtubules. As tension is known to stabilize kinetochore microtubule connections in vivo (Nicklas and Ward, 1994),
CENP-E could fulfill its role as a kinesin-like protein generating motive force at the kinetochore.
Received for publication 24 September 1997 and in revised form 10 October 1997.
Address all correspondence to Tim J. Yen, Fox Chase Cancer Center, 770 Burholme Avenue, Philadelphia, PA 19111. Tel.: (215) 728-2590. Fax: (215) 728-3616.The authors are grateful to S. Jablonski, D. Gately, E. Golemis, and T. Coleman for critical reading of the manuscript.
This work was supported by National Institutes of Health grant GM24364 to E.D. Salmon. T.J. Yen was supported by grants GM44762, Leukemia Society Scholar's Award, core grant CA06927, Council for Tobacco Research, and an Appropriation from the Commonwealth of Pennsylvania.
ACA, anti-centromere autoimmune serum; DIC, differential interference contrast; GFP, green fluorescent protein; GST, glutathione-S-transferase; MBP, maltose-binding protein; VE, video enhanced.
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