Journal of Cell Science 104, 961-973 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
961
Odd chromosome movement and inaccurate chromosome distribution in
mitosis and meiosis after treatment with protein kinase inhibitors
R. Bruce Nicklas*, Lawrence E. Krawitz and Suzanne C. Ward
Department of Zoology, Duke University, Durham, NC 27706, USA
*Author for correspondence
SUMMARY
Errors in chromosome orientation in mitosis and meiosis are inevitable, but normally they are quickly corrected. We find that such errors usually are not corrected in cells treated with protein kinase inhibitors.
Highly inaccurate chromosome distribution is the result.
When grasshopper spermatocytes were treated with the
kinase inhibitor 6-dimethylaminopurine (DMAP), 84%
of maloriented chromosomes failed to reorient; in
anaphase, both partner chromosomes were distributed
to the same daughter cell. These chromosomes were
observed for a total of over 60 h, and not a single reorientation was seen. In contrast, in untreated cells, maloriented chromosomes invariably reoriented, and
quickly: in 10 min, on average. A second protein kinase
inhibitor, genistein, had exactly the same effect as
DMAP. DMAP affected PtK1 cells in mitosis as it did
spermatocytes in meiosis: improper chromosome orientations persisted, leading to frequent errors in distribution. We micromanipulated chromosomes in spermatocytes treated with DMAP to learn why maloriented
chromosomes often fail to reorient. Reorientation
requires the loss of improper microtubule attachments
and the acquisition of new, properly directed kinetochore microtubules. Micromanipulation experiments
disclose that neither the loss of old nor the acquisition
of new microtubules is sufficiently affected by DMAP to
account for the indefinite persistence of malorientations.
Drug treatment causes a novel form of chromosome
movement in which one kinetochore moves toward
another kinetochore. Two kinetochores in the same
chromosome or in different chromosomes can participate, producing varied, dance-like movements executed
by one or two chromosomes. These kinetochore-kinetochore interactions evidently are at the expense of kinetochore-spindle interactions. We propose that malorientations persist in treated cells because the
kinetochores have numerous, short microtubules with a
free end that can be captured by a second kinetochore.
Kinetochores capture each other’s kinetochore microtubules, leaving too few sites available for the efficient
capture of spindle microtubules. Since the efficient capture of spindle microtubules is essential for the correction of errors, failure of capture allows malorientations
to persist. Whether the effects of DMAP actually are
due to protein kinase inhibition remains to be seen. In
any case, DMAP reveals interactions of one kinetochore
with another, which, though ordinarily suppressed, have
implications for normal mitosis.
INTRODUCTION
(reviewed by Nicklas, 1988). By metaphase, malorientations are extremely rare. We have discovered that malorientations are extremely common in cells treated with
either of two very different protein kinase inhibitors, DMAP
(6-dimethylaminopurine; Néant and Guerrier, 1988) and
genistein (Akiyama et al., 1987). These malorientations persist in both mitosis and meiosis and the result is wholesale
nondisjunction. One of the processes required for reorientation might be affected by the drugs, such as the acquisition of new, properly directed kinetochore microtubules or
the loss or redirection of misdirected ones. In fact, neither
process is much affected by the drugs. The most significant
effect of DMAP is a novel kinetochore-kinetochore interaction that it induces. Kinetochores move toward other
Mitosis is naturally prone to error, but normally the errors
are flawlessly corrected. Early in mitosis, improper arrangements of kinetochore microtubules are common. For example, the microtubules associated with the two kinetochores
of a pair of chromosomes may extend to the same pole
rather than to opposite poles. Such a malorientation, if it
persisted, would lead to nondisjunction: chromosomes
would be distributed to the same daughter cell. Normally,
however, malorientations are unstable and are soon corrected by reorientation. One kinetochore or the other gains
microtubules extending toward the opposite spindle pole
and the old, misdirected microtubules are lost or redirected
Key words: mitosis, chromosome distribution, nondisjunction,
protein kinase, DMAP (6-dimethylaminopurine)
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R. B. Nicklas, L. E. Krawitz and S. C. Ward
kinetochores, rather than toward the spindle. Evidently the
kinetochores are so bound up in interacting with each
other’s microtubules that they often fail to interact properly
with spindle microtubules.
MATERIALS AND METHODS
Grasshopper (Melanoplus sanguinipes, Fabricius) spermatocytes
were cultured at 25-26˚C, observed by phase contrast microscopy,
and micromanipulated by standard procedures (Nicklas et al.,
1982, and references therein). The cells were exposed to drugs by
soaking the testes in medium containing the drug for 20 min before
the cell cultures were prepared. Mammalian cells (line PtK1 rat
kangaroo kidney cells from American Type Culture Collection,
Rockville, MD) were cultured in Ham’s F-12 medium supplemented with 2% LPSR-1 (low protein serum replacement-type 1;
Sigma, St. Louis, MO) and were observed at 34˚C in chambers
as described by Lee (1989). The cells were exposed to inhibitors
by replacing the standard medium with one containing the
inhibitor. Chromosome behavior in spermatocytes and PtK1 cells
was recorded on an optical disk recorder (model TQ-2021FCB,
Panasonic Video Systems, Secaucus, NJ).
The inhibitor 6-dimethylaminopurine (DMAP; Sigma, St.
Louis, MO) was added to the medium just before use to give a
concentration of 0.35-0.65 mM for spermatocytes and 0.25-0.3
mM for PtK1 cells. Genistein (4′,5,7-trihydroxy-isoflavone; Indofine, Somerville, NJ) was used only on spermatocytes. It was dissolved in DMSO (dimethylsulfoxide; Mallinkrodt, Paris, KY) and
added to the spermatocyte culture medium to give a final concentration of 40-50 µg/ml genistein and 2% DMSO; 2% DMSO
by itself has no effect on spermatocyte division. For both
inhibitors, the range of useful concentrations is narrow: half the
lowest concentration used has little or no effect, while twice the
highest concentration has general cytotoxic effects.
Some living cells were observed by high extinction/high resolution polarization microscopy, as described by Inoué (1986,
1988). Nikon (Melville, NY) optics selected for freedom from
strain were used: a rectified achromatic-aplanatic condenser with
a numerical aperture (NA) of 1.4 and a 1.4NA/60× plan apochromatic objective. The images were acquired and processed
using an Image1 system (Universal Imaging Corp., West Chester,
PA); noise was reduced by averaging, haze was removed by
unsharp masking, and contrast was optimized.
Immunofluorescence staining of microtubules was carried out
as described earlier (Nicklas et al., 1989), except for the use of a
monoclonal anti-tubulin antibody (TU-27, generously provided by
Lester Binder, University of Alabama, Birmingham, AL) followed
by a Cy3-labeled secondary antibody (Jackson ImmunoResearch
Laboratories, West Grove, PA); Cy3 has outstandingly bright
fluorescence. Several antibodies in addition to anti-tubulin were
also tried, as noted in Results; all were used with a Cy3-labeled
secondary antibody. The stained cells were viewed on a confocal
microscope (model MRC-600, Bio-Rad, Cambridge, MA); images
were averaged to reduce noise, sharpened by a high-pass filter,
and contrast was optimized.
RESULTS
DMAP effects on spermatocytes
Cells treated with DMAP are like no others ever described.
Maloriented chromosomes, normally rare, are common in
cells at all stages of division. In the cell shown in Fig. 1,
four bivalents out of eleven were maloriented. One of these
eventually reoriented (arrow, 162- and 202-min prints), and
the partner chromosomes segregated normally in anaphase.
This malorientation was stable for over 2.5 h before reorientation occurred. The other three maloriented chromosomes persisted in their misguided orientation, and the partner chromosomes segregated to the same spindle pole in
anaphase (425.8- and 426-min prints). These malorientations were stable for 421 min, or 7 h, measured from the
time they were first observed until the beginning of
anaphase; the time spent without reorientation in anaphase
was not counted because reorientation never occurs in
anaphase even in untreated cells. We studied a total of 25
maloriented chromosomes in 16 cells treated with DMAP;
4 (16%) of them reoriented while 21 (84%) persisted, leading to nondisjunction in anaphase. The chromosomes that
did not reorient were observed for a total of 3,691 min,
over 60 h, in totally stable malorientations. The average
time before reorientation of the four chromosomes that did
reorient was 87 min (range: 30-152 min).
Untreated control cells are very different. Reorientation
always follows soon after a chromosome is induced to malorient (Nicklas and Koch, 1969). In the present experiments, the average time before reorientation in a sample of
10 chromosomes was 9.9 min (range: 2 to 19.5 min).
Maloriented chromosomes in cells treated with DMAP
are seen as soon as the preparations are made, after 20 min
in DMAP. Some of these are due to the usual errors in early
prometaphase, but others arise in cells at later stages, presumably because of temporary spindle disorganization
caused by mechanical abuse of the cells during culture
preparation. We never saw a properly oriented chromosome
become maloriented in the presence of DMAP. Hence,
DMAP does not induce malorientations, but it makes those
that arise from other causes abnormally stable. At midprometaphase, the majority of DMAP-treated cells have one
or more maloriented chromosomes. In contrast, in untreated
cells at the same stage, the frequency is so low as to discourage counting; certainly not more than one cell in a thousand has a maloriented chromosome.
We used micromanipulation to characterize the malorientations induced by DMAP. When a maloriented chromosome is pulled toward the opposite pole with a microneedle, both partner chromosomes are stretched (Fig. 2,
0- and 26-min prints). Experiments on an additional 15 maloriented chromosomes gave the same result. Thus, both partner chromosomes of maloriented chromosomes are attached
to one spindle pole. Electron microscopy of untreated cells
disclosed that in such malorientations the kinetochore microtubules of both partners extend to the pole to which they
are attached (Ault and Nicklas, 1989). Micromanipulation
permits malorientations to be induced (Nicklas and Koch,
1969). In Fig. 2, 29-min print, a properly oriented chromosome was detached from the spindle and bent to face the
same pole as its partner. Orientation to that pole promptly
followed, as shown by stretching the chromosome with the
micromanipulation needle - both partners were attached to
the lower pole (Fig. 2, 33 min). After the new orientation
was stabilized by applying tension for 3 min, the chromosome was released from the needle (Fig. 2, 34 min). It
moved to the lower spindle pole (Fig. 2, 51 min) where it
remained in a perfectly stable malorientation. Both partner
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Fig. 1. Persistent malorientations and nondisjunction in a spermatocyte treated with 0.55 mM DMAP. The time in min is given on each
print. Four maloriented chromosomes were present initially (arrows, 0-min print). One of these subsequently reoriented (arrow, 162 and
202 min), but the other three did not, and their partner chromosomes were distributed to the same pole in anaphase rather than to opposite
poles (arrows, 425.8- and 426-min prints; arrowhead: the unpaired X-chromosome, segregating to one pole, which is normal).
Chromosome movement was generally normal in this cell, but one chromosome (lower left, 162-425.8 min) failed to congress to a
position midway between the poles. Bar, 10 µm.
chromosomes segregated to the same pole in anaphase, like
the naturally occurring, maloriented chromosome at the
other pole (Fig. 2, 275 min). The naturally occurring malorientation was stable for over 271 min and the induced malorientation was stable for 237 min (from the time of its
release from the needle to the beginning of anaphase).
In all, 9 malorientations induced by micromanipulation
were studied in 9 cells treated with DMAP. Not one of the
maloriented chromosomes reoriented, and collectively they
were stable for a total of 1,184 min: almost 20 h without
a single reorientation.
DMAP at the concentration used affects mitotic
processes in addition to chromosome distribution, though
not so dramatically. Chromosome congression to a
metaphase position at the spindle equator sometimes fails
in treated cells (Fig. 1), and such failure is rare in untreated
cells. However, most chromosomes congress normally in
the presence of DMAP (Figs 1 and 2). Chromosome movement in anaphase looks normal (Figs 1 and 2), but the rate
is greatly decreased - from an average of 1.04 µm/min in
untreated cells to 0.38 µm/min in treated cells (the rates are
averages for 3-4 chromosomes in each of 4 untreated and
Fig. 2. Micromanipulation experiments in a spermatocyte treated with 0.55 mM
DMAP. The time in min is given on each print. A maloriented chromosome
(arrow, 0-min print) was pulled downward with a microneedle (26 min). Both
partner chromosomes were stretched (arrows at kinetochores, 26 min), revealing
that both were attached to the same (upper) spindle pole. In a second experiment,
a properly oriented chromosome was induced to malorient (arrow, 29 min). One
partner chromosome was detached from the spindle and bent, so that the
kinetochores of both partners faced the same (lower) pole. Soon, both partners
had attached to the lower pole, as shown by stretching the chromosome (arrows
at kinetochores, 33 min) with the microneedle. After release from the needle, the
maloriented chromosome moved to the lower pole (34-51 min), and both
partners were distributed to that pole in anaphase (arrows, 275 min). The original
malorientation (upper pole, 0-275 min) also persisted, and nondisjunction
followed in anaphase (275 min). Bar, 10 µm.
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R. B. Nicklas, L. E. Krawitz and S. C. Ward
3 treated cells). DMAP has more drastic effects on the less
healthy cells that inevitably occur in spermatocyte cultures
(Nicklas et al., 1979). In particular, hypotonicity causes
short, ill-organized spindles in untreated cells, and DMAP
exacerbates this effect so that the spindles quickly collapse.
Prometaphase-metaphase lasts a long and variable time
(4-8 h) in both untreated and treated cells. The onset of
anaphase may be somewhat delayed in treated cells when
several maloriented chromosomes are present (Fig. 1), but
certainly the cells are not blocked in metaphase. Nor is the
entry into mitosis blocked: spindle formation and
prometaphase are initiated in cells after 5 h or more of exposure to DMAP.
Genistein effects on spermatocytes
In cells treated with genistein, maloriented chromosomes
are indefinitely stable and nondisjunction often is the consequence (Fig. 3), just as with DMAP. Experiments with
genistein were limited to confirming in 8 cells the more
extensive observations on cells treated with DMAP. Six
maloriented chromosomes reoriented but only after an average of 40.7 min (range: 25 to 60 min). Three chromosomes
did not reorient; they spent a total of 255 min in completely
stable malorientations and showed nondisjunction in
anaphase. Genistein has a less specific effect on chromosome distribution than does DMAP. Even at concentrations
that cause fewer persistent malorientations than DMAP,
genistein inhibits chromosome movement in anaphase (Fig.
3), and the spindles at all stages are shorter and subject to
collapse.
DMAP effects on PtK1 cells
DMAP stabilizes improper orientations in PtK1 cells, and
thereby causes nondisjunction in anaphase (Fig. 4). Eight
improperly oriented chromosomes in 5 cells spent a total
time of 580 min, over 9 h, without a single correction nondisjunction of the sister chromatids invariably occurred.
Metaphase is prolonged in these cells, with an average duration of 91 min, as compared to 25-40 min in controls. Chromosome movement to the spindle equator in prometaphase
and to the poles in anaphase is not noticeably affected by
0.25 mM DMAP, but spindle elongation in anaphase is
often inhibited, as in the cell in Fig. 4.
The effects of DMAP on PtK 1 cells are swiftly reversible
(Fig. 5). Soon after the DMAP-medium was replaced with
normal medium, the maloriented chromosomes oriented
properly, as signaled by movement toward the equator (32and 35-min prints), and a normal anaphase followed (73
min). Two additional experiments gave the same result; on
average the maloriented chromosome oriented properly 9.2
min after the DMAP was removed, and anaphase followed
20.0 min later.
Why do malorientations persist in treated cells?
The cause of persistent malorientations was investigated in
DMAP-treated spermatocytes. The correction of malorientation by reorientation requires the formation of a new,
proper kinetochore attachment and the loss of an old,
improper one.
Attachment tests
We tested the possibility that the formation of new attachments is defective in treated spermatocytes by detaching
chromosomes from the spindle by micromanipulation. In
untreated cells, detached chromosomes initially lack kinetochore microtubules, but invariably acquire new ones and
begin to move again (Nicklas and Kubai, 1985). Therefore,
the lag time between detachment and the beginning of
movement is a measure of the time required for reattachment to the spindle by the acquisition of new kinetochore
microtubules. We detached relatively small chromosomes
and placed them at one pole of the spindle. One kinetochore faced the farther pole, nearly as far from that pole as
a kinetochore in a maloriented chromosome. The average
time required for the reattachment of that kinetochore, signaled by movement toward the farther pole, was 1.6 min
in untreated cells (range: 0.2-4.7 min; 13 experiments) and
4.4 min in cells treated with DMAP (range: 1.8-11.8 min;
13 experiments). The delay in reattachment in cells treated
with DMAP is statistically significant (t-test, P = 0.01), but
a delay of a few minutes cannot be the reason that malorientations often persist for several hours.
Tether tests
Micromanipulation was also used to test the possibility that
the old, improper attachment persists in treated cells, and
Fig. 3. A second inhibitor, genistein, also causes persistent malorientation and nondisjunction. This spermatocyte was treated with 40
µg/ml genistein. The time in min is given on each print. A maloriented chromosome (arrows) was stable for 60 min, and both partners
were distributed to the same pole in anaphase (61 and 65 min prints). Bar, 10 µm.
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965
Fig. 4. DMAP causes nondisjunction in a mammalian cell in mitosis. This PtK1 cell was treated with 0.25 mM DMAP. The time in min is
given on each print. Two chromosomes (arrows, 0-min print) lying near the upper spindle pole remained there, and their sister chromatids
were distributed to that pole in anaphase (41 and 54 min) rather than to opposite poles. Bar, 10 µm.
therefore keeps the kinetochores tethered to the same pole.
A maloriented chromosome was produced by micromanipulation, as described above (Fig. 2). After some time, a
needle was inserted near a kinetochore and gently moved
toward the opposite pole, to mimic the movement following the capture of a microtubule extending toward that pole.
Sometimes the tug of the needle was resisted, and the kinetochore region was pulled out (Fig. 6, upper row). Obviously the kinetochore was still tethered to the spindle, and
therefore if a microtubule from the opposite pole had been
captured by the kinetochore, the associated motors would
not have been able to shift the kinetochore toward the opposite pole. Hence, capture at that time probably would not
have led to reorientation. In contrast, sometimes the kinetochore could be moved by the microneedle without resistance (Fig. 6, lower row). At such a time, a properly
directed microtubule, if captured by the kinetochore, could
have led to reorientation.
The experiment must be done somewhat differently in
control and treated cells. The time before reorientation is
highly variable in control cells, so we obtained a random
sample by waiting until one of the two kinetochores of a
maloriented bivalent began to reorient and then probed the
attachment of the other kinetochore. This would not work
with cells treated with DMAP, simply because waiting for
reorientation often would be futile. We reasoned that if
DMAP prevents reorientation by stabilizing old spindle
attachments, it must do so when reorientation normally
occurs in untreated cells, at an average of 10 min after malorientation. Hence, we probed for attachment in DMAPtreated cells at 10 min after malorientation. We found that
improper attachments commonly persist in both treated and
untreated cells, but there is not much difference between
them. About half of the kinetochores were tethered to the
pole, 53% in untreated cells (15 experiments) and 63% in
cells treated with DMAP (24 experiments).
Kinetochore-kinetochore interactions
More by accident than design, we discovered that DMAP
causes a unique form of chromosome movement that is
based on kinetochore-kinetochore interactions. A maloriented chromosome (arrows, Fig. 7, 0-min print) was
detached from the spindle and moved to the cytoplasm (13
min). It remained at rest for several min, and then was suddenly jerked toward the spindle (21 min). The movement
did not continue, but instead, the two kinetochoric ends of
the chromosome curled toward each other (22 min): the end
nearer the spindle at 21 min actually moved away from the
spindle, toward the other kinetochore. This movement, too,
did not persist; the chromosome uncurled (29 min) only to
curl again (31 min). In this curled condition, the chromosome finally began to move back toward the spindle (34
Fig. 5. The effects of DMAP are reversible. The time in min is given on each print. Two chromosomes (arrows, 30-min print) in this PtK1
cell remained maloriented for 30 min while in 0.28 mM DMAP (one of the two chromosomes is obscured by the other in the 0-min print).
At 30 min, the DMAP was washed out, and first one chromosome (lower arrow, 32 min) and then the other (35 min) oriented properly
and moved to the metaphase plate. A normal anaphase followed (73 min). Bar, 10 µm.
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R. B. Nicklas, L. E. Krawitz and S. C. Ward
Fig. 6. Tether tests on a
spermatocyte treated with 0.6
mM DMAP. The time in min
is given on each print. A
chromosome was induced by
micromanipulation to
malorient to the upper pole
and then released from the
needle. 10 min later, one
partner chromosome was
pulled gently toward the lower
pole with a microneedle (too
small to be visible) inserted
near its kinetochore (arrows,
0-0.5-min prints). The
kinetochore region was pulled
out (0.3 and 0.5 min),
revealing that the kinetochore
was firmly tethered to the
upper pole. In contrast, the
kinetochore of the other
partner chromosome was
freely movable (arrow, 1.6-2.5
min): it was not tethered and
could have reoriented and
moved to the lower pole if it
had captured a microtubule
from that pole. Bar, 10 min.
min) and reached the spindle with its kinetochores together,
maloriented. The malorientation (70-min print) persisted for
42 min, but eventually the chromosome reoriented and
moved toward the equator (105 min).
These kinetochore-kinetochore interactions are not limited to maloriented chromosomes, and more than one chromosome can join in the dance. In the cell in Fig. 8, two
chromosomes, one maloriented chromosome and one normally oriented, were detached from the spindle and placed
near one another in the cytoplasm, far from the spindle (Fig.
8, 0 min). A kinetochore of one chromosome approached
a kinetochore of the other chromosome (1- and 7-min
prints) until the two kinetochores touched. The duo became
attached to the spindle and moved to it, still kinetochoreto-kinetochore (12- and 20-min prints). Eventually, both
oriented properly on the spindle (30 min).
The average velocity of the kinetochore-to-kinetochore
movements in these two cells was 2.1 µm/min (n = 5). This
value is indistinguishable from the velocity of the chromosome-to-spindle movements that follow chromosome
detachment from the spindle and for ordinary chromosomespindle movement early in prometaphase (0.7-2.3 µm/min:
Nicklas, 1967).
The kinetochores that move toward one another are
mechanically connected, very likely by microtubules. The
mechanical linkage was revealed by micromanipulation.
For instance, the chromosomes in Fig. 8 were both moved
closer to the spindle (7- and 12-min prints) by pulling on
just one of the chromosomes. Occasionally, presumptive
microtubules between interacting kinetochores can be seen
by polarization microscopy (Fig. 9A-C); they look just like
microtubules in spindles (Fig. 9D). Attempts to demonstrate
kinetochore-to-kinetochore microtubules by immunostain-
ing yielded no really satisfactory evidence. This is probably because the interactions are so transitory (Fig. 7) and/
or because the microtubules are hard to preserve.
Kinetochore-kinetochore interactions have never been
seen in hundreds of detachment experiments in untreated
cells. In contrast, one or more kinetochore-kinetochore
interactions were seen in 56% (15 of 27) of experiments
on cells treated with DMAP. Interactions were not seen
when the detached chromosome or chromosomes soon
moved back to the spindle, and hence there was little time
for kinetochore-kinetochore interactions. The comparison
between normal and DMAP-treated cells is imperfect, of
course, because kinetochores in untreated cells usually have
relatively little time to interact with each other before they
become attached to the spindle. We did a set of detachment
experiments in untreated cells in which detached chromosomes were placed very far from the spindle, as in the
experiments on DMAP-treated cells such as those in Figs
7 and 8. In 16 experiments, the chromosome began a sustained movement back toward the spindle in an average of
1.3 min (range, 0.2-6 min). Thus, in these cells, there is
very little time for kinetochore-kinetochore interaction
before interactions with the spindle begin. In 15 comparable experiments in DMAP-treated cells, the first movement
toward the spindle occurred after an average of 5.7 min
(range, 0.2-18 min). This is not very different from the
value for controls, but often the movement was not sustained. The time from detachment until sustained movement
(to be compared with the average of 1.3 min in untreated
cells) was 28 min, with a range of 0.2-180 min.
Labile initial attachments
The difference between the time for initial movement
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Fig. 7. A solo dance. Kinetochore-kinetochore movements within a single chromosome in a spermatocyte treated with 0.35 mM DMAP.
The time in min is given on each print. A maloriented chromosome (arrows at kinetochores, 0-min print) was detached from the spindle
with a microneedle and placed far out in the cytoplasm (arrows, 13 min). After darting a short distance toward the spindle (21 min), the
chromosome stopped, and one kinetochore moved toward the other so that the chromosome curled on itself (arrows, 22 min). The
chromosome relaxed (29 min) but then curled again (31 min). With kinetochore pressed to kinetochore, the chromosome moved back
toward the spindle (34 min), and both kinetochores oriented to the upper pole (70 min). Eventually, the chromosome oriented properly
and moved toward the equator (105 min). Bar, 10 µm.
versus sustained movement in the above experiments is due
to the lability of the initial attachment of chromosomes in
DMAP-treated cells. A good example is the movement of
one kinetochore in Fig. 7. Between 13 and 21 min, the kinetochore moved several µm toward the spindle, and then,
1 min later, it moved in the opposite direction. When
detached chromosomes are placed far from the spindle,
twitches of one kinetochore or the other toward the spindle occur and recur but movement is rarely sustained. In
consequence, chromosomes often remain for many minutes
without stable interactions with the spindle, allowing time
for kinetochore-kinetochore interactions to occur.
Immunostaining observations
Spindle microtubule organization in DMAP-treated cells
cannot be distinguished from that in untreated spermato-
cytes (Fig. 10) or PtK1 cells (not illustrated). Note that there
are numerous microtubules in the cytoplasm in both treated
and untreated cells that detached chromosomes might capture (Fig. 10). Consistent with a plentiful supply of cytoplasmic microtubules is the short time required for the
initial reattachment of detached chromosomes in both
untreated and treated cells.
We tried several antibodies specific for various phosphoproteins and other proteins that might be affected by the
inhibitors: (1) monoclonal anti-phosphotyrosine antibodies:
number 5-321 (Upstate Biotechnology, Inc., Lake Placid,
NY), PY-20 (ICN Biomedicals, Inc., Costa Mesa, CA) and
PY-54 (Zymed Laboratories, South San Francisco, CA); (2)
polyclonal anti-phosphorylation antibodies: #702 (Jean
Wang, University of California, San Diego, CA); (3) antisarc (David Morgan, University of California, San Fran-
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R. B. Nicklas, L. E. Krawitz and S. C. Ward
Fig. 8. A pas de deux. Kinetochore-kinetochore movements of two chromosomes in a spermatocyte treated with 0.35 mM DMAP. The
time in min is given on each print. Two chromosomes were detached and placed in the cytoplasm (arrows at two kinetochores, 0-min
print). One kinetochore of the upper chromosome approached and touched a kinetochore of the other chromosome (1-7 min). The two
chromosomes were mechanically connected, as shown by pulling one of them toward the spindle with a micromanipulation needle - the
other chromosome was towed along (12 min). The two chromosomes moved together to the spindle, but later they oriented properly and
separated (20-30 min). Bar, 10 µm.
cisco, CA); (4) anti-γ-tubulin (Berl Oakley, Ohio State University, Columbus, OH) and (5) MPM-2, an antibody that
reacts with several phosphoproteins prominent in mitotic
cells (Potu Rao, M. D. Anderson Cancer Center, Houston,
TX). We found no significant difference between treated
and untreated cells with any of these antibodies. This negative result is disappointing but not meaningful. General
phosphoprotein antibodies such as MPM-2 may or may not
react with particular proteins, e.g. those phosphorylated on
tyrosine, and the staining with anti-phosphotyrosine antibodies is so low in intensity and so diffuse in control cells
that important differences between controls and treated cells
would be undetectable even if present.
DISCUSSION
Two protein kinase inhibitors, DMAP and genistein, cause
inaccurate chromosome segregation at meiosis in grasshopper spermatocytes. Reorientation, the normal process of
error correction, often fails to occur, and improper chromosome orientations persist for hours. In sharp contrast, in
untreated cells, on average only 10 min pass before maloriented chromosomes reorient. DMAP also causes mal-
orientations to persist in mammalian (PtK1) cells in mitosis and again, inaccurate chromosome distribution to the
daughter cells is the result. In PtK1 cells, the effects of
DMAP are completely reversible. Reversibility of drug
effects in grasshopper spermatocytes cannot be tested in our
preparations because the cells are not attached to the glass
and hence the medium around the cells cannot be replaced.
Many other treatments and conditions are known to produce abnormal chromosome complements (Dellarco et al.,
1985), including inhibitors of certain protein phosphatases
(Vandré and Wills, 1992) and kinases (Andreassen and
Margolis, 1991). DMAP and genistein, however, are the
only agents that are known to do so by stabilizing malorientations.
The inhibitors we studied affect other processes in addition to chromosome distribution. Chromosome congression
sometimes fails. Chromosome movement in anaphase is
reduced in extent and speed. Especially with genistein, the
spindles sometimes collapse, halting cell division. The
onset of anaphase may be delayed in spermatocytes and
certainly is delayed in PtK1 cells. Multiple effects are no
surprise in view of the numerous processes affected by
DMAP, including progression through the cell cycle (e.g.
see Rebhun et al., 1973; Néant and Guerrier, 1988; Luca
Mitosis and kinase inhibitors
969
Fig. 9. (A-C) Polarization microscopy of chromosome-chromosome movement in a spermatocyte treated with 0.5 mM DMAP. Fibers
running vertically appear in maximum dark contrast. (A) Two chromosomes (arrows at kinetochores) were detached from the spindle a
minute earlier and moved to a cytoplasmic region above one spindle pole. A dark fiber extends between the kinetochores. (B,C) The
chromosomes move together. The fiber seen in A is scarcely visible 5 s later (B), but this is commonly the case, because the fibers are
constantly in motion and the depth of focus is very shallow. The elapsed time between A and C is 20 s. (D) Very early prometaphase in
another living spermatocyte to show the appearance of microtubules when viewed by polarization optics. One chromosome (arrowheads
at kinetochores) is shown. The spindle poles lie out of view, above and just below the region shown. Several fibers, one of which
terminates in a kinetochore, are identified by arrows. At early prometaphase, electron microscopy discloses that microtubules are sparse,
so the fibers probably are composed of one or a very few microtubules. Bar, 5 µm.
and Ruderman, 1989; Jessus et al., 1991) and spindle organization (Dufresne et al., 1991). What is noteworthy is that
proper chromosome distribution is more sensitive to the
drugs than most other processes in the cells we studied.
DMAP and genistein are structurally dissimilar compounds yet both are potent and specific inhibitors of particular protein kinases, tyrosine kinases, in vitro (Néant
and Guerrier, 1988; Akiyama et al., 1987). In living cells,
specific inhibition of protein tyrosine phosphorylation by
these drugs has been directly demonstrated in a variety of
cells. For example, DMAP inhibits the phosphorylation of
particular proteins in echinoderm, Xenopus and mouse
oocytes (Néant et al., 1989; Jessus et al., 1991; Rime et
al., 1989). As for genistein, the list includes A431 human
carcinoma cells, mouse fibroblasts, human platelets and
lymphocytes (Akiyama et al., 1987; Hill et al., 1990;
Gaudette and Holub, 1990; Lane et al., 1991). Comparably direct evidence that the inhibitors affect protein phosphorylation in spermatocytes and PtK1 cells is lacking at
present. We note, however, that we used DMAP and
genistein at the same concentrations that specifically
inhibit tyrosine kinase in a great variety of other living
cells and that the two very different inhibitors have identical effects on chromosome distribution in spermatocytes.
Thus, reduced protein phosphorylation may be the cause
of inaccurate chromosome distribution in cells treated with
the inhibitors, but other possibilities certainly are not ruled
out. Our major focus in the present report is the unique
effects of the drugs on chromosome movement and distribution, leaving the molecular mechanism an open question.
Whatever the inhibitors do to molecules, how does this
result in abnormally stable malorientations and inaccurate
chromosome distribution? Two conditions are necessary for
reorientation: a new chromosome-spindle attachment must
form, and an old attachment must be lost. Either of these
processes might be affected by the drugs.
The first condition for reorientation is satisfied when a
kinetochore captures microtubules growing outward from a
pole (Mitchison and Kirschner, 1985b; Rieder and Alexander, 1990; Hayden et al., 1990). This requires a plentiful
supply of microtubules extending from each pole
970
R. B. Nicklas, L. E. Krawitz and S. C. Ward
Fig. 10. Microtubule immunostaining
of two untreated spermatocytes (A and
B) and two spermatocytes treated with
DMAP (C and D; 0.55 mM and 0.35
mM DMAP, respectively). Neither the
spindle nor cytoplasmic microtubules
of treated cells can be distinguished
from untreated cells; the spindle of the
cell in C is shorter than the others in
this sample but not outside the normal
range. Both the treated cells had
maloriented chromosomes though they
are not visible in these images (cell C
had one and cell D had two). Bar, 10
µm.
nearly to the opposite pole and kinetochores that are
competent to capture them.
A deficient supply of long microtubules probably
explains the persistence of maloriented chromosomes in
PtK1 cells treated with DMAP. “Centrophilic chromosomes” are common in untreated cells in mitosis. They lie
near a pole to which one sister kinetochore is oriented,
while the second kinetochore faces away from the pole
and usually is not oriented at all - it lacks kinetochore
microtubules (Rieder, 1990; Ault and Rieder 1992). After
a few minutes, the second kinetochore orients to the opposite pole and normal anaphase follows. When cells are
exposed to DMAP, however, orientation to the opposite
pole is indefinitely delayed and nondisjunction often
results. The kinetochores are competent to capture microtubules, since those facing a nearby pole do so. The unoriented kinetochore, however, faces a pole much farther
away and hence a shortage of long microtubules would
account for its failure to attach to the spindle. Certain protein kinases are known to affect microtubule length distribution (Verde et al., 1992 and references therein), and
DMAP affects those kinases, if indirectly (Luca and Ruderman, 1989). These studies provide no direct evidence
that DMAP treatment reduces the number of long micro-
Fig. 11. A diagram of one model for kinetochore-kinetochore
movement in the presence of DMAP. Kinetochores are
represented by black circles and microtubules by thin lines; in (B),
the polarity of the ends of two microtubules is indicated by plus or
minus signs and the direction of movement by the nearby arrow.
(A) Kinetochores have short microtubules attached to them, as a
result of either kinetochore nucleation of microtubules or of
capture of microtubule fragments nucleated elsewhere. (B) The
free end of a microtubule attached to one kinetochore can be
captured by another kinetochore, regardless of microtubule
polarity. Movement results from minus-end directed kinetochore
motors.
Mitosis and kinase inhibitors
tubules in mitosis, but at least they show that such an
effect is plausible.
The situation is different in grasshopper spermatocytes,
however, and the remainder of the discussion centers on
them. In spermatocytes, the partner kinetochores of a maloriented chromosome are both attached to the same pole
(Fig. 2), and their kinetochore microtubules extend toward
that spindle pole (Ault and Nicklas, 1989). Reorientation
requires the formation of a new spindle attachment via the
capture of microtubules from the opposite pole by one kinetochore or the other. We determined the time required for
the formation of new attachments. A longer time was
required in cells treated with DMAP, but still was only 4.4
min on average. Obviously, a delay of a few minutes in
making a new attachment cannot explain why malorientations generally persist for several hours.
Formation of a new attachment is not sufficient for reorientation. A second requirement is that the old, misdirected
kinetochore microtubules must be lost or must lose their
anchorage to the spindle. Otherwise, the reorienting kinetochore is not free to move toward the opposite pole (Nicklas and Kubai, 1985; Ault and Nicklas, 1989). We directly
tested for loss of anchorage in maloriented chromosomes by
using a micromanipulation needle to mimic a microtubule
and the motors associated with it, asking whether or not the
kinetochore was free to move at a given moment. Cells
treated with DMAP had a somewhat smaller fraction of
freely movable kinetochores, but in both treated and
untreated cells, only a minority of kinetochores was free to
move at a given time. Thus, anchorage to the pole appears
to be a significant impediment to reorientation even in
untreated cells. While the impediment may be a little greater
in cells treated with DMAP, it is an unlikely source of the
enormous difference in how long malorientations persist.
We conclude that in spermatocytes, the processes necessary for reorientation are not much affected by DMAP.
Hence the explanation for persistent malorientations must
lie elsewhere, probably in the novel kinetochore-kinetochore interactions that DMAP causes. Often, a kinetochore
moves toward another kinetochore, rather than toward the
spindle. The kinetochores of one chromosome (Fig. 7) or
of two chromosomes (Fig. 8) can move together. The velocity of kinetochore-kinetochore movement is typical of ordinary, microtubule-based, chromosome movement. The
interacting kinetochores are mechanically linked together
(Fig. 8) by connections that have the appearance of spindle microtubules as seen by polarization microscopy (Fig.
9). The possibility that the connections are chromatin fibers
can be dismissed: while chromosomes sometimes stick
together in hypertonic cells (whether or not DMAP is present), such connections once formed are stable and never
lead to dances such as those in Figs 7 and 8. A striking
example of the impact that kinetochore-kinetochore associations can have on connection to the spindle is seen in Fig.
7, from 13 to 22 min. A kinetochore achieves a connection
to the spindle, but that connection lapses. The kinetochore
then becomes connected to its partner’s kinetochore and
moves toward it, actually moving away from the spindle.
Kinetochore-kinetochore interactions via microtubules
provide a natural explanation for the failure of reorientation. Each kinetochore very likely has a definite, limited
971
number of sites for capturing and binding microtubules
(Nicklas, 1988; Zinkowski et al., 1991). We suggest that
the capture of microtubules from other kinetochores
reduces the number of spindle microtubules the kinetochore can capture: the maloriented kinetochores of DMAPtreated spermatocytes are literally too attached to one
another to attach properly to the spindle. Notice that any
reduction in the number of unoccupied capture sites by
kinetochore-kinetochore interactions is particularly apt to
affect maloriented chromosomes. Their kinetochores face
an abundance of easily captured microtubules from the
nearby pole (e.g. see Fig. 3) and do not directly face the
microtubules growing from the opposite pole. Even in
untreated cells, the initiation of reorientation by the capture of a microtubule from the opposite pole is an improbable event (Nicklas, 1988). Hence reorientation may well
be inhibited if the number of available capture sites for
spindle microtubules is reduced by the capture of kinetochore microtubules.
The impact of kinetochore-kinetochore interactions on
reorientation is probably enhanced by the lability of the
initial attachment of chromosomes to the spindle. In treated
cells, the initial movements of detached chromosomes
toward the spindle often do not continue and may even
reverse (Fig. 7, 13-22 min). Apparently, the first microtubules to be captured are unstable themselves or have
unstable connections at the kinetochore or pole. The frequent failure of the initial spindle attachments to persist
allows more time for kinetochore-kinetochore attachments
to form in treated cells. It should be noted that unstable
kinetochore-spindle interactions are not unique to cells
treated with DMAP. The initial movements of chromosomes in untreated cells are intermittent and jerky (Alexander and Rieder, 1991; Nicklas, 1967), though pauses or
reversals of the sort common in treated cells have not been
seen.
Assuming that microtubules are the basis of kinetochorekinetochore movements, where do those microtubules come
from and how does movement result? There are several possibilities, but the following is perhaps the most likely. Suppose that DMAP treatment leads to kinetochores having
short microtubules attached to them (Fig. 11A). Those
microtubules might arise because the drug increases the
number of short microtubules not associated with a pole
which then are captured by kinetochores (normally, such
microtubule fragments probably are rare, to judge from the
few microtubules with unexpected polarity: McIntosh and
Euteneuer, 1984). Alternatively, the drug might make kinetochores more efficient in nucleating microtubules, so that
they grow their own microtubules (kinetochores from
untreated cells are inefficient nucleators in vitro: Mitchison
and Kirschner, 1985a). Whatever the origin of short kinetochore microtubules might be, the free ends of these
microtubules could be captured by a second kinetochore
(Fig. 11B). Kinetochores capture microtubule ends of either
polarity, at least in vitro (Mitchison and Kirschner, 1985b).
Single microtubules would connect two kinetochores, with
the plus end at one kinetochore and the minus end at the
other (Fig. 11B). The two kinetochores could then move
toward each other by the minus-end-directed kinetochore
motors thought to produce normal chromosome movement
972
R. B. Nicklas, L. E. Krawitz and S. C. Ward
in anaphase (reviewed by McIntosh and Pfarr, 1991; Rieder,
1991).
There is some electron microscopic evidence that kinetochore-kinetochore microtubules can arise under conditions in which kinetochores nucleate microtubules (McGill
and Brinkley, 1975; Witt et al., 1980) and perhaps also, if
very rarely, in normal mitosis (Bajer and Molè-Bajer, 1969;
Luykx, 1970). As all of these authors imply, however, it is
not certain from the electron microscopic images that the
microtubules observed actually terminated in the kinetochores and were attached to them.
In conclusion, a protein kinase inhibitor, DMAP, produces a sort of chromosome movement that has never been
reported before, which is based on kinetochore-kinetochore
interactions. Evidently these interactions occur at the
expense of normal kinetochore-spindle interactions. The
outcome is persistent malorientation and inaccurate chromosome distribution.
The existence of frequent kinetochore-kinetochore interactions in treated cells raises the question of how they are
avoided in normal mitosis and meiosis. Microtubule dynamics and kinetochore-microtubule encounters must be precisely regulated to achieve a balance between unworkable
stability (no reorientation) and chaotic change (continuous
reorientation) so that errors in chromosome attachment to
the spindle can be corrected. The normal absence of interactions between kinetochores very likely is a consequence
of the essential regulation of mitotic encounters in general.
If so, the interaction of one kinetochore with another,
though aberrant, may reveal important features of regulation in normal cells.
We thank Robin Fulmer and Danette Miller for expert technical help, Dahong Zhang for assistance with confocal and polarization microscopy and Donna Maroni for unsparing editorial
review. For generous gifts of antibodies, we are grateful to Lester
Binder, David Morgan, Berl Oakley, Potu Rao and Jean Wang.
This investigation was supported in part by grant GM 13745 from
the Institute of General Medical Sciences, National Institutes of
Health.
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(Received 12 November 1992 - Accepted 5 January 1993)