Pole-to-chromosome movements induced at metaphase: sites of
microtubule disassembly
VICTORIA E. CENTONZE* and GAEY G. BORISY
University of Wisconsin-Madison, Laboratory of Molecular Biology, 1525 Linden Drive, Madison, WI53706, USA
•Author for correspondenceat: present address: Integrated Microscopy Resource, University of Wisconsin, 1675 Observatory Dr.,
Madison, WI 53706, USA
Summary
Metaphase spindles can be induced to shrink by
treating cells with microtubule-depolymerizing
agents. During treatment, the paired sister chromatids remain at the metaphase plate and the poles
move toward them. The question we asked is
whether this pole-to-chromosome movement was
accompanied by a loss of subunits from the kinetochore ends of the microtubules, the polar ends, or
both ends. LLC-PK cells were injected at late
prometaphase with Xrhodamine tubulin and at
metaphase the fluorescent spindles were marked by
photobleaching a bar between one pole and the
chromosomes. Nocodazole at low concentrations was
briefly applied to the cells to induce the shortening of
the spindle and movement of the poles inward
toward the chromosomes. In the induced shortening,
the distance between the photobleached bar and the
chromosomes decreased substantially while the distance between the bar and the pole showed a smaller
change. Upon reversal from nocodazole, new
polymer was added to the spindle as determined by
recovery of fluorescence, and the cells progressed
through mitosis and cytokinesis. We conclude that
the movement of the poles to the chromosomes
induced by nocodazole treatment during metaphase
is similar to the chromosome-to-pole movement
occurring during anaphase in that under both
conditions the primary site for kinetochore microtubule disassembly is at the kinetochore.
Introduction
al. 1987, 1988; Nicklas, 1989). However, recent experiments by Mitchison (1989) indicate that metaphase
microtubules exhibit a slow poleward flux of subunits. The
flux is too slow to account entirely for anaphase chromosome movement but it may provide a redundant mechanism for segregation. It may also have a role in congression.
An open question is whether the forces responsible for
poleward movement of a kinetochore at anaphase are
operational in both prometaphase and metaphase (Wise,
1978; Pickett-Heaps etal. 1982). The prometaphase stretch
of chromosomes indicates a poleward force acting early in
mitosis on kinetochores of sister chromatids (HughsSchrader, 1943; Hughs-Schrader, 1947). The poleward
force acting on one kinetochore is counterbalanced by the
force pulling its sister kinetochore toward the opposite
pole (Nicklas and Koch, 1969). This results in a net force of
zero until anaphase when the sister chromatids separate.
When one kinetochore of a prometaphase or metaphase
chromosome is ablated with a laser beam, the whole
chromosome moves toward the pole to which the unirradiated kinetochore is attached (McNeill and Berns, 1981;
Hays and Salmon, 1990). By damaging one kinetochore
the bipolar tension is disrupted and the force acting on the
undamaged kinetochore can be observed.
We wanted to investigate whether the poleward force
operating in prometaphase and metaphase is related to
the poleward force of anaphase. Anaphase A is characterized by the decrease in distance between the chromosomes
and the pole to which they are attached. Movement of
Anaphase is defined as the disjunction of sister chromatids
followed by their physical separation to opposite poles of
the mitotic spindle. In contrast, congression refers to the
prometaphase movement of chromosomes toward the
equator, including frequently occurring oscillations about
the equatorial plane (Nicklas, 1971; Salmon, 1989). The
prometaphase movement of chromosomes toward a pole
shares with anaphase the property that the microtubules
connecting the chromosome to the pole shorten. However,
the molecular basis of poleward movement and whether
anaphase and prometaphase employ the same or different
mechanisms has yet to be established.
Chromosome movement may be the result of a forcegenerating translocator (Rieder and Alexander, 1990;
Hyman and Mitchison, 1991), such as a dynein or a
kinesin-like molecule, located at the kinetochore (Steuer
et al. 1990; Pfarr et al. 1990). Alternatively, chromosome
movement may be driven by the disassembly of the
microtubules to which the chromosomes are attached, and
models invoking conformational changes in the tubulin
lattice or biased diffusion with multiple binding sites have
been proposed (Hill, 1985; Garel et al. 1987; Koshland et al.
1988).
Previous experiments have shown that as a chromosome
moves poleward in anaphase, its kinetochore microtubules
remain essentially stationary and disassemble primarily
from the kinetochore end (Mitchison et al. 1986; Gorbsky et
Journal of Cell Science 100, 205-211 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
Key words: mitosis, metaphase, microtubule dynamics,
nocodazole.
205
chromosomes to the pole can be induced at metaphase by
placing mitotic cells under microtubule-destabilizing
conditions. Cold, hydrostatic pressure and drugs such as
colchicine and nocodazole induce the spindle to shorten
(Inoue and Ritter, 1975; Salmon, 1975; DeBrabander et al.
1986). As the spindle shortens, the poles are drawn to the
chromosomes, which remain at the metaphase plate
(Cassimeris et al. 1990). This movement can be viewed as
an anaphase-like movement, since the kinetochore microtubules shorten as the distance between the pole and
chromosomes decreases. Using this treatment in combination with fluorescence imaging and photobleaching, we
evaluated whether the kinetochore microtubules disassemble at the kinetochore as they do at anaphase (Gorbsky
et al. 1987, 1988), at the pole as would be predicted by
microtubule flux (Mitchison, 1989), or at both ends due to a
general destabilization of the microtubule polymer.
Materials and methods
Cell culture and microinjection
LLC-PK cells were cultured in DME medium (Gibco, Grand
Island, NY) supplemented with 10 % fetal bovine serum (Hyclone
Lab., Logan, UT), 20 mM Hepes and antibiotics. Two days prior to
an experiment, the cells were cultured onto coverslips marked
with a carbon locator pattern and mounted in plastic 35 mm Petri
dishes modified for microinjection (Gorbsky et al. 1987). Prometaphase cells were injected with Xrhodamine-derivatized tubulin
(Gorbsky et al. 1988; Sammak and Borisy, 1988) with a dye to
protein ration of 0.5:1.0. After microinjection the cells were
returned to the incubator until they reached metaphase.
Photobleaching
As previously described (Sammak et al. 1987) an argon ion laser
beam (Spectra-Physics, Inc., Mountain View, CA) was focused
through a 300 mm focal length lens and a 200 mm focal length
cylindrical lens to produce a bar-shaped beam. This beam was
directed through the epi-illumination path of a Zeiss IM-35
microscope (Carl Zeiss, Inc., Thornwood, NY) and focused through
a Zeiss 100 X planapo objective, NA 1.3 to produce a Gaussian bar
of width=1.3/nn (measured at one-half peak intensity). Cells
were positioned on the microscope stage so that the long axis of
the spindle was perpendicular to the bar-shaped beam. Irradiation time was set to 100 ms with an electronically controlled
shutter (Vincent Associates, Uniblitz, Rochester, NY).
Nocodazole treatment, lysis and fixation
After photobleaching, a fluorescence image of the marked spindle
was recorded with a CCD image sensor as described below. The
DME medium was then aspirated from the injection dish and
replaced with medium containing 0.04, 0.08 or 0.12/igml" 1 of
nocodazole. The cells were incubated with nocodazole for 3-5 min
after which an image of the treated spindle was recorded. The
nocodazole-containing medium was aspirated from the dish and
replaced with fresh medium. The cells released from nocodazole
were monitored for recovery of fluorescence in the spindle and for
completion of mitosis. While on the microscope stage, the cells
were maintained at 33 °C to 37 °C by an air curtain incubator
(Nicholson Precision Instruments, Inc., Gaithersburg, MD). To
minimize evaporation, the dish was kept covered whenever
possible. The cells were not kept out of a 5 % CO2 atmosphere
longer than 30 min. For long-term monitoring, the cells were kept
in a tissue culture incubator and placed back on the warmed stage
for observation.
Observation and analysis
Efforts were made to minimize the total amount of irradiation
delivered to each cell. For scanning and focussing, the mercury
arc source was attenuated with neutral density filters and images
were collected with a SIT camera (SIT-66, Dage MTI Inc.,
206
V. E. Centonze and G. G. Borisy
Michigan City, IN) and Quantex (QX-9200, Quantex Corp.,
Sunnyvale, CA) image processor. In general, three frames (0.1s
exposure) were grabbed, digitized and averaged. These images
were noisy but adequate for determining focal level. For higher
quality and for quantitation, fluorescence images were recorded
with the cooled CCD image sensor (model 200, Photometric Inc.,
Tucson, AZ) and the mercury arc source was not attenuated. In
total, each cell received less than 5 s equivalent of irradiation
with an unattenuated mercury arc source.
The images were digitized to 14 bit depth and stored on a
WORM drive optical disk (type 3363, IBM Corp., Armonk, NY).
The stored images were later analyzed using the Photometries
imaging software. The fluorescent spindle was positioned in each
image with its long axis aligned on the horizontal axis. The
intensity values for each vertical row of 25 pixels above and below
the spindle axis were averaged using the image processor and
displayed as a plot of the averaged intensities uersus their
horizontal pixel position. Hard copies of the graphs were obtained
by means of a video printer (model P-61U, Mitsubishi Electric,
Rancho Dominguez, CA). The poles were defined as the pixel
positions along the outermost descending limbs of the plot where
the intensity value was 80% of the peak value. The spindle
equator was denned as the pixel equidistant between the poles.
The position of the bleached bar was defined as the pixel in the
center of the trough of reduced fluorescence. Pixel distances were
later calibrated to micrometers using the image of a stage
micrometer.
Results
Excessive irradiation of fluorescently derivatized tubulin
could perturb the progression of the cell through mitosis as
well as cause structural damage to the microtubules
containing derivatized subunits (Vigers et al. 1988), thus
creating artifactual results. From previous experiments
we have determined the doses of light from both the
mercury arc source and the laser, which cause ultrastructural damage to microtubules (Centonze and Borisy, 1989;
Centonze and Borisy, unpublished data). In the work
presented here, each cell was subjected to an imaging and
photobleaching protocol such that the total dose of light
delivered was below the level required to cause detectable
structural damage.
To verify lack of damage to kinetochore microtubules in
the spindle, photobleached cells were lysed, fixed and
processed for anti-tubulin immunofluorescence. At the
light-microscopic level, the indirect immunofluorescence
showed that kinetochore fibers were continuous through
the photobleached zone (data not shown). Similar results
were obtained by Gorbsky and Borisy (1989) and microtubule integrity was confirmed by electron microscopy of
irradiated cells. Thus, our protocol of photobleaching and
imaging did not appear to affect the microtubules of the
spindle or the progression of the cell through mitosis.
Fig. 1 shows a series of micrographs of the direct
fluorescence image of a spindle from a cell previously
injected with Xrhodamine tubulin. Prior to photobleaching (Fig. 1A), the spindle appears symmetric with respect
to the metaphase plate and the corresponding fluorescence
intensity profile (Fig. 1A') along the length of the spindle
demonstrates an equal distribution of fluorescent material
in each half of the spindle, indicating an equivalent
amount of incorporation of derivatized tubulin. After
photobleaching (Fig. IB), a distinct bar of reduced fluorescence is seen in the irradiated half-spindle, corresponding to the trough in the intensity profile (Fig. IB')- The
laser produced a bleached zone approximately 1.5 jan
wide, as would be expected from the width of the Gaussian
profile of the beam (see Materials and methods; and
Fig. 1. Nocodazole-induced pole-to-chromosome movement. An
LLC-PK cell was microinjected with Xrhodamine tubulin and
analyzed byfluorescencedigital imaging. Panels show the
directfluorescenceimages and thefluorescenceintensity
distribution along the spindle axis as determined with a CCD
image sensor. (A,A') A metaphase cell prior to laser
irradiation. The distance between the poles (P-P) is 20 /an.
(B,B') A bleached bar, b, was placed 3.5//m from the pole, P,
and 7.0/im from the spindle1 equator, Eq. (C,C) After a 3min
treatment with 0.08/igml" nocodazole, the poles, P', and the
bleached bar, b', have moved closer to the chromosomes at the
spmdle equator, Eq, resulting in a P' to P' distance of 16 /an.
The P' to b' distance remained 3.5/an. The b' to Eq distance
decreased by 2 /an, which accounts for the observed decrease in
length of the half-spindle.
ently corresponded to the center of the centrosomes seen in
phase-contrast micrographs of the same spindles (see
Gorbsky et al. 1988).
Cells were treated briefly with a low dose of nocodazole
to induce depolymerization of microtubules (Fig. 1C). The
intensity profile (Fig. 1C) shows a decrease in fluorescence throughout the entire spindle; however, the
kinetochore bundles remained distinct. This suggests, as
other workers have reported (DeBrabander et al. 1986;
Cassimeris, et al. 1990), that the drug treatment preferentially depolymerizes non-kinetochore microtubules emanating from the centrosome, leaving the previously
established kinetochore bundles relatively intact. The
poles of the mitotic spindle were drawn toward the
chromosomes with both poles moving an equal distance.
The rate of pole-to-chromosome movement in this cell was
0.7/nnmin" 1 , which approximates the rate of chromosome-to-pole movement observed at anaphase (Gorbsky et
al. 1988). Cells treated with nocodazole concentrations of
0.04jUgml-1 showed less movement while those treated
with 0.12//gml" 1 showed more movement (data not
shown). As reported previously (Mullins and Snyder, 1981;
Vandre and Borisy, 1989), higher concentrations of
nocodazole disrupted the metaphase configuration; kinetochore fibers dissociated from the poles and disassembled,
and the chromosomes at the metaphase plate became
disorganized. Careful comparison of the fluorescence
intensity profiles of the untreated (Fig. IB') and the
treated cell (Fig. 1C) revealed that the distance between
the pole and the bleached bar did not change substantially
even though there was a pronounced decrease in the
distance between the pole and the chromosomes, which
remained at the spindle equator. This result indicates that
as the distance between the poles and chromosomes
decreases, tubulin is lost from the kinetochore fibers that
are equatorial to the bleached bar.
I enuth I mil)
Centonze and Borisy, 1989). The position of the bleached
bar was taken as the center of the trough. The positions of
the poles were determined from the fluorescent images.
The poles were assigned to a point just beyond the peak of
microtubule fluorescence since this position most consist-
Fig. 2 shows that the nocodazole-induced shortening is
reversible. A bleached bar was placed on one half-spindle
and the original position of the bar and the poles were
noted (Fig. 2A,A'). After treatment with O.OS^gml"1
nocodazole for 3min, the poles moved 1.5/nn toward the
spindle equator at a rate of 0.5/nnmin" 1 while the
distance between the pole and the bleached bar remained
unchanged (Fig. 2B,B'). After lOmin in medium without
nocodazole, the spindle length and fluorescence intensity
increased up to initial levels, interpolar microtubules
reappeared, and the bleached bar was no longer visible
(Fig. 2C,C). The fluorescence recovery suggests both that
non-kinetochore microtubules have nucleated and grown
from the centrosomes and that the kinetochore microtubules have turned over their polymer and elongated.
Pole-to-chromosome movements induced at metaphase
207
Fig. 2. Recovery from nocodazole treatment. (A,A') A
metaphase spindle with a pole-to-pole distance of 12.5 /on was
marked with a bleached bar, b, 2.1 /an from a pole and 4.4//m
from the spindle equator, Eq. (B,B') After a 3min treatment
with 0.08/(gml" 1 nocodazole the poles, P', and the bleached
bar, b', have moved closer to the equator, Eq, resulting in a P'
to P' distance of 9.5/an. The P' to b' distance remained 2.1/on.
The b' to Eq distance decreased by 1.5/an, equal to the
decrease in the length of the half-spindle. (C,C) The
nocodazole-containing medium was washed from the cell and
replaced with fresh medium. Within 5min new polymer was
added to the spindle as evidenced by the disappearance of the
bleached zone and the increase in fluorescence intensity of the
spindle. The spindle has also increased in length.
E
3-
i
2-
XI
u 1-
2
'•3
a7 1
S 0
o
-1
0
1
1
-+2
3
4
Decrease in P-b distance (/mi)
5
Fig. 3. Quantitative analysis of pole-to-chromosome
movement. (A) Decrease in b-Eq distance versus decrease in
P-Eq distance. The equation of this line is Y=0.89±0.12
X+0.14±0.25 [fl2=0.9]. This plot indicates a direct correlation
between the movement of the bleached bar toward the
chromosomes with the decrease in spindle length. (B) Decrease
in P-b distance versus decrease in P-Eq distance. The
equation of this line is Y=0.18±0.12 X-0.14±0.25 [R2=0.3].
This plot indicates that the movement of the pole to the
bleached bar does not correlate with the decrease in spindle
length. (Errors were determined within a 95 % confidence
interval.) Number of cells plotted=27.
Length (/mi)
The cell subsequently divided, indicating that the drug
treatment and photobleaching produced no irreversible
damage to mitotic function.
The graphs in Fig. 3 summarize the results from 27
cells. If the induced shortening occurred exclusively by the
loss of subunits at the kinetochore, the kinetochore
microtubules (and the bleached bar marking them) would
208
V. E. Centonze and G. G. Borisy
be expected to move at the same rate and to the same
extent as the poles move towards the chromosomes. The
plot of the decrease in bleached bar-to-equator distance
versus the decrease in pole-to-equator distance in Fig. 3A
gives a least-squares regression line with a slope that is
not significantly different from unity. This indicates that
the movement of the pole to the chromosome can be
accounted for primarily by shortening of the kinetochore
bundle at the kinetochore end. On the same assumption,
one would expect no relative motion of the pole with
respect to the bleached bar. Fig. 3B shows the analysis of
the change in the pole-to-bleached bar distance with
respect to the decrease in the pole-to-chromosome distance. The low correlation coefficient indicates that the
distance between the pole and the bleached bar was only
weakly related to the position of the poles and was
certainly not sufficient to account for the decrease in the
pole-to-chromosome distance. However, analysis of the
data did reveal that the change in distance between
the pole and the bleached bar was statistically significant,
raising the possibility that tubulin subunits could be lost
from the pole as well as from the kinetochore, albeit at a
lower rate.
There are several possible explanations for an apparent
loss of subunits from the pole: poleward micro tubule flux
(Mitchison, 1989), which implies that the connection of the
microtubules to the pole is dynamic; destabilization of the
pole end of the kinetochore microtubules by the nocodazole
treatment; or, a systematic error in the designation of pole
position in the nocodazole-treated cells leading to an
apparent but not real movement. Measurements of poleto-pole distance and pole-to-bleached bar distance were
made from graphs of fluorescence intensity profiles of the
spindle (see Materials and methods). The position of the
pole in the profiles of fluorescence intensity was assigned
to the point that best corresponded to the position of the
centrosome by comparison with phase-contrast micrographs (see Gorbsky et al. 1988). However, in the
nocodazole-treated cells the peak of fluorescence narrowed
as the astral microtubules depolymerized. As a result, the
80 % peak fluorescence position was closer to the equator
even when there was no movement of the peak itself.
Therefore, the assignment of the position of the poles at
80% of the peak may have introduced a small but
systematic error biasing the results. Consequently, the R2
of the regression line plotted in Fig. 3B is probably an
overestimate of the correlation between the change in
pole-to-bleached bar distance and the decrease in pole-toequator distance. Our experiments have shown that the
kinetochore is the primary site of subunit loss as
microtubules shorten during drug induced pole-to-chromosome movement. The uncertainties in the analysis of the
pole-to-bleached bar distance do not permit us to exclude
the possibility that the pole is a secondary site for tubulin
loss, but neither do the results demand it.
Discussion
Treatment of mitotic cells with low concentrations of
nocodazole induces the spindle poles to be drawn in toward
the chromosomes at the spindle equator. The relative
motion is similar to anaphase, since the distance between
the chromosomes and the poles decreases. Also, the rate at
which the poles are drawn to the chromosomes is similar to
the rate of chromosome movement during anaphase.
Using fluorescent analogue cytochemistry and photobleaching we have visualized the spindle during nocodazole treatment and monitored the dynamics of spindle
microtubules. Our data show that as the poles move
toward the chromosomes the bleached bar marking them
also moves, indicating that the kinetochore microtubules
shorten by losing subunits primarily from the kinetochore
end. The kinetochore end is also the primary site of
subunit loss as the chromosomes move to the poles during
anaphase. Therefore, with regard to the relative motion,
its rate and magnitude, and the site of subunit loss, the
two motions are similar.
Poleward flux of microtubules, observed by Mitchison
(1989), does not satisfy the requirements to be the motive
force for either the nocodazole-induced movement or the
movement of anaphase chromosomes. First, microtubule
flux is characterized by subunit loss from the pole end of
the microtubule, opposite to the primary site from which
subunits are lost during nocodazole-induced movement
and during anaphase. Second, the rate reported for flux in
LLC-PK cells (0.3-0.7 jrni min" 1 ), although greater than
the upper bound for pole end disassembly (<0.3jummin~1)
seen in our experiments, is not sufficient to account for the
rate of chromosome movement observed at anaphase
(Gorbsky et al. 1987, 1988).
An inference from our results is that the chromosomes
at metaphase are subjected to a poleward force and that
the poles are subjected to an equatorial force. Sister
chromosomes remain at the equator because their poleward forces are equal and opposite. However, the poleward
force, by Newton's third law, must also exert an equal and
opposite force on the poles that would tend to cause the
poles to move to the equator. The fact that this normally
does not occur implies the existence of some resistive or
opposing force operating on the poles, preventing their
equatorial movement. Our results provide experimental
evidence that the opposing force is nocodazole-sensitive,
since the predicted equatorial movement of poles does
occur in drug-treated cells. Since non-kinetochore microtubules are preferentially depolymerized under the conditions of nocodazole treatment employed, the results
suggest that the opposing force is dependent upon the
maintenance of the astral or non-kinetochore microtubules.
Since little attention has been given in the literature to
the existence or mechanism of the 'opposing force,' it is
worth briefly exploring potential explanations. Four
possible mechanisms for the opposing force may be
considered, all of which share the property that the force is
dependent on the continued presence of non-kinetochore
microtubules. The opposing force may be the result of the
polar ejection field generated by the dynamic instability of
astral microtubules (Eieder et al. 1986). At anaphase the
ejection field would normally decrease as the centrosome
changes to an interphase state with a diminished capacity
to nucleate microtubules (Kuriyama and Borisy, 1981;
Snyder et al. 1982; Kuriyama, 1984). Nocodazole treatment would cause a similar decrease in the magnitude of
the ejection field, since microtubule assembly would be
blocked.
A second model for the opposing force is based on
interzonal microtubule crossbridges. Molecules that crosslink interpolar microtubules may be mechanochemical or
structural. A motor molecule could exert an opposing force
by causing antiparallel microtubules to slide against one
another, generating an outward pushing force that would
counterbalance the tendency of the poles to move inward
(LaFountain, 1972; Mclntosh et al. 1989). Alternatively,
the microtubule crossbridges could be purely structural
links generating a rigid interpolar structure that would be
strong under compression and therefore resist inward pole
movement. Nocodazole treatment would destabilize the
interpolar microtubules and, under either the mechanochemical or structural variation of this model, without
these microtubules the poles would be free to be drawn
toward the chromosomes at the equator.
Pole-to-chromosome movements induced at metaphase
209
A third model for the opposing force results from the
interaction of astral microtubules with translocator
molecules. Minus-end-directed translocators could be
bound by their non-ATP-sensitive site to an immovable
structure in the cytoplasm or to the membrane cytoskeleton. Not being able to move toward the minus (pole) end
of astral microtubules, the action of these translocators
would be expressed as a pull on the poles away from the
spindle equator. Alternatively, the translocators need not
be attached to any permanent cytoplasmic structure. The
active stroke of the translocators alone may exert enough
force to act as 'oars' against the cytoplasm causing the
poles to 'swim' away from the spindle equator. A
requirement of these models is that the distribution of
astral microtubules about the mitotic pole be asymmetric,
being more highly developed on the cytoplasmic side, as is
observed. Nocodazole treatment would depolymerize the
astral microtubules and thus remove the structures on
which the translocator molecules operate.
A fourth model for a force opposing the movement of
chromosomes to the pole invokes the presence of forcegenerating molecules at or near each sister kinetochore
that are switched on and off. By in vitro motility assays
and immunocytochemistry, it has been shown that
kinetochores have associated with them both a plus-enddirected motor (Mitchison and Kirschner, 1985; Hyman
and Mitchison, 1990) and a minus-end or pole-directed
motor (Steuer et al. 1990; Pfarr et al. 1990; Hyman and
Mitchison, 1991). Throughout prometaphase and metaphase, chromosomes oscillate about the metaphase plate
(Ostergren, 1949, 1950; Nicklas, 1971; Centonze etal.,
unpublished data). As a chromosome moves poleward, the
leading kinetochore could be pulling by means of a minusend-directed motor while the trailing kinetochore pushes
by means of a plus-end-directed motor. Were this to be
true, the Newtonian reaction forces on the poles would be
opposite in character. The pulling of the leading kinetochore would tend to pull its pole inward whereas the
pushing of the trailing kinetochore would tend to push its
pole outward. After a reversal in the cycle of chromosome
oscillation, the leading and trailing kinetochores would
reverse roles and the signs of their reactive forces on the
poles would also reverse, resulting in a net, time-averaged
polar force of zero. Thus, the 'opposing force' would result
from the activity and switching of motors of opposite force
polarity operating at sister kinetochores. If nocodazole, by
promoting depolymerization, diminished the activity of
the plus-end motor or pushing force, the balance of forces
would be upset and the poles would tend to be drawn
inward.
Finally, we note that motions of poles also occur at other
stages of mitosis. In prophase, the asters move apart to
establish the poles of the mitotic spindle and, in anaphase
B the poles move apart, augmenting the separation
between the sister chromosomes. Whether the forces
implied by these motions are related to the opposing force
revealed in our experiments is a question warranting
further investigation.
While this work was in preparation, Cassimeris and
Salmon (1991) in a study of the effects of nocodazole on
newt lung cells reported essentially the same result that
prometaphase or metaphase kinetochore microtubules
shorten by loss of subunits at the kinetochores. Taken
together, our results support the generality of the
conclusion that the mechanism of poleward motion of
chromosomes in prometaphase, metaphase or anaphase is
the same.
210
V. E. Centonze and G. G. Borisy
We thank Mr Steven Limbach for his excellent technical
assistance in preparing thefigures.This work was supported by
NIH grants GM 30385 and GM 25062 (G.G.B.).
References
CASSIMERIS, L., RIEDBR, C. L., Rirpp, G. AND SALMON, E D (1990).
Stability of microtubule attachment to metaphase kinetochorea in
PtK, cells J Cell Sci. 96, 9-15.
CASSIMERIS, L. AND SALMON, E. D. (1991). Kinetochore microtubulea
shorten by loss of subunits at the kinetochores of prometaphase
chromosomes. J. Cell Sci. 98, 151-158.
CENTONZE, V. E AND BORISY, G. G. (1989). Applications and limitations
of photobleaching and fluorescence imaging. In Optical Microscopy for
Biology (ed B Herman and K. Jacobson), pp 184-194. New York:
John Wiley & Sons, Inc.
DE BRABANDER, M., GEUEN8, G., NUYDENS, R , WILLEBRORDS, R., AERTS,
F., DE MEY, J. AND MCINTOSH, J. R. (1986). Microtubule dynamics
during the cell cycle The effects of taxol and nocodazole on the
microtubule system of PtK2 cells at different stages of the mitotic
cycle. In International Review of Cytology (ed. G. H. Bourne), pp.
215-274. Orlando- Academic Press, Inc.
GAREL, J. R., JOB, D AND MARGOLIS, R. L. (1987) Model of anaphase
chromosome movement based on polymer-guided diffusion. Proc. natn
Acad. Sci. U.S.A. 84, 3599-3603.
GORBSKY, G. J. AND BORISY, G. G. (1989). Microtubules of the
kinetochore fiber turn over in metaphase but not in anaphase. J. Cell
Biol. 109, 653-662.
GORBSKY, G. J., SAMMAK, P. J. AND BORISY, G. G. (1987). Chromosomes
move poleward in anaphase along stationary microtubules that
coordinately disassemble from their kinetochore ends. J. Cell Biol.
104, 9-18.
GORBSKY, G. J., SAMMAK, P. J. AND BORISY, G. G. (1988). Microtubule
dynamics and chromosome motion visualized in living anaphase cells.
J. Cell Biol. 106, 1185-1192.
HAYS, T S. AND SALMON, E D (1990). Poleward force at the kinetochore
in metaphase depends on the number of kinetochore microtubules. J.
Cell Biol. 110, 391-404.
HILL, T. L. (1985). Theoretical problems related to the attachment of
microtubules to kinetochores. Proc. natn. Acad. Sci. U.SA. 82,
4404-4408.
HUGHES-SCHRADER, S. (1943) Polarization, Kinetochore movements, and
Bivalent Structure in the Meiosis of Male Mantids. Biol. Bull mar.
Biol. Labs Woods Hole 85, 265-300.
HUGHES-SCHRADBR, S (1947). The "pre-metaphase stretch" and
kinetochore orientation in plasmids. Chromosome 3 1—21.
HYMAN, A. A AND MITCHISON, T. J. (1990) Modulation of microtubule
stability by kinetochores in vitro. J. Cell Biol. 110, 1607-1616
HYMAN, A. A. AND MITCHISON, T J. (1991). Kinetochores contain two
different microtubule based motor-activities with opposite polarities.
Nature (in press).
INOUE, S. AND RITTER, H. JR (1975). Dynamics of mitotic spindle
organization and function. Soc. gen. Physiol. Ser. 30, 3-30.
KOSHLAND, D. E , MITCHISON, T. J. AND KIRSCHNER, M W (1988)
Polewards chromosome movement driven by microtubule
depolymerization in vitro. Nature 331, 499-504.
KURIYAMA, R. (1984). Activity and stability of centrosomes in Chinese
hamster ovary cells in nucleation of microtubules in vitro J. Cell Sci
66, 277-295.
KURIYAMA, R. AND BORISY, G. G. (1981). Centriole cycle in Chinese
hamster ovary cells as determined by whole-mount electron
microscopy. J. Cell Biol. 91, 814-821
LAFOUNTAIN, J. R. JR (1972). Spindle shape changes as an indicator of
force production in crane-fly spermatocytes. J. Cell Biol. 10, 79-93.
MCINTOSH, J. R., VIGERS, G P A. AND HAYS, T. S. (1989). Dynamic
Behavior of Mitotic Microtubules. In Cell Movement, vol. 2: Kinesin,
Dynein, and Microtubule Dynamics (ed. F. D. Warner and J. R.
McIntosh), pp. 371-382. New York: Alan R. Liss, Inc.
MCNEILL, P. A. AND BERNS, M W (1981). Chromosome behavior after
laser microirradiation of a single kinetochore in mitotic PtK2 cells. J.
Cell Biol 88, 643-553.
MITCHISON, T. J. (1989). Polewards microtubule flux in the mitotic
spindle: Evidence from photoactivation offluorescence.J. Cell Biol.
109, 637-652.
MITCHISON, T., EVANS, L., SCHULZE, E. AND KIRSCHNER, M. (1986). Sites
of microtubule assembly and disassembly in the mitotic spindle. Cell
45, 515-527.
MITCHISON, T. J. AND KIRSCHNER, M. W. (1985). Properties of the
kinetochore in vitro. II. Microtubule capture and ATP-dependent
translocation J. Cell Biol. 101, 766-777.
MULLINS, J. M AND SNYDER, J. A. (1981). Anaphase progression and
furrow establishment in nocodazole-arrested PtKl cells. Chromosoma
83, 493-505.
NICKLAS, R. B. (1971). Mitosis. Adv. Cell Biol. 2, 225-297.
NICKLAS, R. B. (1989). The motor for poleward chromosome movement
in anaphase is in or near the kinetochore. J. Cell Bwl. 109,
2245-2255.
NICKLAS, R. B. AND KOCH, C. A. (1969). Chromosome
micromanipulation. 3. Spindle fiber tension and the reorientation of
mal-oriented chromosomes. J. Cell Bwl. 43, 40-50.
OSTEROREN, G. (1949). Luzula and the mechanism of chromosome
movements. HeredUas 35, 445-468.
OSTERGREN, G. (1950). Considerations on some elementary features of
mitosis. Hereditas 36, 1-18.
PFARR, C. M., COUE, M., GRISSOM, P. M., HAYS, T. S., PORTER, M. E. AND
MCINTOSH, J. R. (1990). Cytoplasmic dynein is localized to
kinetochores during mitosis [see comments]. Nature 345, 263-265.
PICKETT-HEAPS, J. D., TIPPIT, D. H. AND PORTKR, K. R. (1982).
Rethinking mitosis. Cell 29, 729-744.
RIEDER, C. L. AND ALBXANDER, S. P. (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.
RIEDER, C. L., DAVISON, E. A., JENSEN, L. C, CASSIMERIS, L. AND
SALMON, E. D. (1986). Oscillatory movements of monooriented
chromosomes and their position relative to the spindle pole result
from the ejection properties of the aster and half-spindle. J. Cell Biol.
103, 581-591.
SALMON, E. D. (1975). Pressure-induced depolymerization of spindle
microtubules. I. Changes in birefringence and spindle length. J. Cell
Bwl 65, 603-614.
SALMON, E. D (1989). Microtubule Dynamics and Chromosome
Movement. In Mitosis: Molecules and Mechanisms (ed. J. S. Hyams
and B. R. Brinkley), pp. 119-181. London: Academic Press.
SAMMAK, P. J AND BORISY, G. G. (1988). Direct observation of
microtubule dynamics in living cells. Nature 332, 724-726.
SAMMAK, P. J., GORBSKY, G. J. AND BORISY, G. G. (1987). Microtubule
dynamics in vivo' a test of mechanisms of turnover. J. Cell Biol. 104,
395-405.
SNYDER, J A., HAMILTON, B. T. AND MULLJNS, J. M. (1982). Loss of
mitotic centrosomal microtubule initiation capacity at the metaphaseanaphase transition. Eur. J. Cell Bwl. 27, 191-199.
STBUEH, E. R., WORDEMAN, L. SCHROER, T A AND SHEETZ, M. P. (1990)
Localization of cytoplasmic dynein to mitotic spindles and
kinetochores. Nature 345, 266-268
VANDRE, D. D AND BORISY, G. G. (1989). Anaphase onset and
dephosphorylation of mitotic phosphoproteins occur concomitantly. J.
Cell Biol. 94, 245-268.
VIGERS, G. P., COUE, M. AND MCINTOSH, J. R. (1988). Fluorescent
microtubules break up under illumination. J. Cell Biol. 107,
1011-1024.
WISH, D. (1978). On the mechanism of prometaphase congression.
chromosome velocity as a function of position on the spindle
Chromosoma 69, 231-241.
(Received 14 May 1991 - Accepted 21 May 1991)
Pole-to-chromosome movements induced at metaphase
211