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Antagonistic microtubule-sliding
motors position mitotic centrosomes in
Drosophila early embryos
David J. Sharp*, Kristina R. Yu†, John C. Sisson†, William Sullivan† and Jonathan M. Scholey*‡
*University of California at Davis, Section of Molecular and Cellular Biology, 1 Shields Avenue, Davis, California 95616, USA
†University of California at Santa Cruz, Department of Biology, 317 Sinsheimer Labs, Santa Cruz, California 95064, USA
‡e-mail: [email protected]
The positioning of centrosomes, or microtubule-organizing centres, within cells plays a critical part in
animal development. Here we show that, in Drosophila embryos undergoing mitosis, the positioning of
centrosomes within bipolar spindles and between daughter nuclei is determined by a balance of opposing
forces generated by a bipolar kinesin motor, KLP61F, that is directed to microtubule plus ends, and a
carboxy-terminal kinesin motor, Ncd, that is directed towards microtubule minus ends. This activity
maintains the spacing between separated centrosomes during prometaphase and metaphase, and
repositions centrosomes and daughter nuclei during late anaphase and telophase. Surprisingly, we do not
observe a function for KLP61F in the initial separation of centrosomes during prophase. Our data indicate
that KLP61F and Ncd may function by crosslinking and sliding antiparallel spindle microtubules in relation
to one another, allowing KLP61F to push centrosomes apart and Ncd to pull them together.
entrosomes are the primary microtubule-organizing centres
within animal cells and thus they play a fundamental part in
numerous cellular and developmental events. For example,
during mitosis, duplicated centrosomes form the bipolar mitotic
spindle poles that determine the position and orientation of cleavage planes which, in turn, determine the sizes of daughter cells and
spatial relationships that exist between them1. Moreover, in multinucleated cells, such as the Drosophila syncytial blastoderm, the relative positioning of centrosomes also contributes to the spacing of
multiple adjacent nuclei2. Work in fungal and animal systems3 indicates that the microtubule-associated motors termed bipolar and
carboxy-terminal kinesins4 may be important in determining the
relative position of adjacent centrosomes, particularly during
mitosis3–25. For example, severe loss-of-function mutations in the
gene encoding the Drosophila bipolar kinesin, KLP61F, result in the
formation of mono-astral mitotic spindles with duplicated but
closely spaced centrosomes, leading to the proposal that this bipolar
kinesin participates in the initial separation of centrosomes during
mitotic spindle assembly13–17. This suggestion, however, is difficult
to reconcile with our recent observation that KLP61F motors are
sequestered in the nucleus during prophase when the centrosomes
initially separate17. A mitotic function has also been proposed for
the Drosophila C-terminal kinesin, Ncd, on the basis of the abnormal morphology of the mitotic spindles that form in its absence22,
but the precise mitotic function of this motor remains unclear.
Here we test the hypothesis that KLP61F and Ncd position mitotic
centrosomes by generating opposing microtubule-sliding forces
within interpolar microtubule bundles17 and telophase midbodies2.
C
Results
Spindles assemble but collapse after loss of KLP61F function. To
investigate the function of KLP61F in positioning centrosomes, we
used the Drosophila early embryo system to study the effects of
injecting antibodies against functionally essential epitopes found in
the tail of KLP61F (ref. 17) into embryos containing fluorescently
labelled tubulin and chromatin (Fig. 1). This allowed us to assess
the influence of KLP61F on centrosome positioning in real-time by
simultaneously visualizing nuclei (data not shown) and the microtubules emanating from centrosomes (Fig. 1).
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During mitosis in control injected embryos, sets of duplicated
centrosomes separated by about 120° to nearly opposite sides of the
nucleus, at a rate of roughly 5 µm min-1. As the nuclear envelope
broke down at the onset of prometaphase, microtubules from
opposite centrosomes began to fill the nuclear region and intercalate to form interpolar bundles. Over the next minute, the metaphase spindle was established and then maintained for 2–3
minutes before the onset of anaphase. During this time the relative
position of centrosomes within mitotic spindles did not change
substantially.
In embryos injected with anti-KLP61F antibodies, the movement of centrosomes in relation to one another during early spindle
assembly appeared unaffected and duplicated centrosomes separated at a rate nearly identical to that of controls (~5.5 µm min-1).
The onset of prometaphase, as marked by the appearance of microtubules within nuclei and the formation of interpolar microtubule
bundles, was also relatively normal. However, shortly after this the
effects of KLP61F inhibition become apparent. Instead of forming
stabilized bipolar metaphase spindles as in controls, separated centrosomes quickly slid back together, at a rate of 5–7 µm min-1, in the
absence of KLP61F activity. Within 3 minutes after nuclear-envelope breakdown, nearly all the centrosomes that previously constituted the two spindle poles had collapsed together and appeared as
small ‘mono-asters’ of microtubules. Analysis of multiple focal
planes revealed that chromosomes remained attached at the distal
ends of the centrosomal microtubules, which extend down into the
interior of the embryo (data not shown). These data indicate that
KLP61F may not function during the initial separation of centrosomes during prophase, as previously thought (although a redundant role in this process or the inaccessibility of specific antigenic
sites on KLP61F molecules during prophase cannot be ruled out),
and show, unequivocally, that KLP61F is essential for maintaining
the spacing between centrosomes within mitotic spindles after they
have first separated.
The inhibition of KLP61F also affects the positioning of centrosomes between mitotic nuclei, because once the collapse of individual spindles was complete, adjacent collapsed spindles began to
slide together ( Fig. 2). This sliding was preceded by the formation
of interspindle microtubule bundles (Fig. 2b, small arrowheads),
which were quite similar to the microtubule bundles that form
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articles
Figure 2 Mitotic centrosomes associated with adjacent spindles interact
through microtubule bundles to slide together and form large arrays of
collapsed mono-asters in the absence of KLP61F function. Time series of
tubulin fluorescence, showing the interactions between centrosomes from adjacent
spindles following the spindle collapse that results from injection of anti-KLP61F
antibodies. a–c, Centrosomes from adjacent collapsed spindles (a) become bridged
by bundles of microtubules (b, small arrows) and slide together (c). Sets of
centrosomes that are not bridged by microtubule bundles remain separate (a–c,
large arrows). d, Over time, large numbers of spindles coalesce to form large
microtubule monoasters. Tubulin is shown in yellow and chromosomes in blue. Note
how the chromosomes remain attached to the distal ends of the microtubules
emanating from these asters. The times shown at the bottom right of each panel are
measured as in Fig. 1. Similar results were obtained with at least 15 different
embryos. Scale bar represents 7 µm in a–c and 15 µm in d.
Figure 1 Centrosomes within bipolar spindles separate but then collapse
together following the inhibition of the bipolar kinesin KLP61F. Time series
of images showing tubulin fluorescence during mitotic spindle formation, in
Drosophila embryos injected with anti-KLP61F antibodies (right panels) or with
nonspecific rabbit IgG (left panels) at the same concentration as a control. Arrows
follow the centrosomes in two individual spindles. Times shown at the bottom right
of each panel are the times from the onset of prophase. Centrosome separation
during prophase occurs similarly in both conditions, but as metaphase spindles are
established in controls the centrosomes slide back together in embryos injected
with anti-KLP61F antibodies. Similar results were obtained in more than 20 different
embryos in 5 experiments. Drosophila melanogaster is an ideal system for real-time
functional analyses of mitosis31. Most important, Drosophila early embryos are
large, multinucleated cells that undergo rapid and synchronous mitoses; thus the
factors that position adjacent centrosomes both within and between mitotic
spindles can be studied easily2,32.
between the spindle poles during prometaphase. As collapsed spindle mono-asters that were not bridged by microtubule bundles did
not slide together (Fig. 2, large arrowheads), the forces driving the
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centrosomes together appear to require microtubule–microtubule
interactions. The rate at which this occurs, ~6 µm min-1, is similar to
the rate of spindle collapse, indicating that a similar mechanism
may drive both.
Over time, the coalescence of multiple adjacent centrosomes led
to the formation of extremely large microtubule asters, with
numerous centrosomes at their centres and condensed chromosomes attached to the distal ends of the microtubules (Fig. 2d).
Although this phenotype indicates a probable metaphase arrest,
chromosomes associated with these asters showed a pattern of
phosphorylation of histone H3 that is characteristic of anaphase in
wild-type cells (data not shown and ref. 26), a result that is being
studied at present.
The collapse of centrosomes within and between spindles
resulted in the production of superficially similar mono-astral
microtubule arrays and associated circular mitotic figures. Similar
structures resulting from the inhibition of bipolar kinesins in
mitotic extracts12, antibody-injected cells18 and mutants5–10,13 have
been widely interpreted as resulting from defects in the initial separation of centrosomes, yet our data show that they could also arise
from the collapse of preassembled bipolar spindles. Several other
mitotic proteins have been assigned functions in the initial separation of centrosomes on the basis of similar phenotypes; our results
indicate that the functions of these proteins should be re-evaluated.
The effects of Ncd counterbalance those of KLP61F. The rate at
which adjacent centrosomes slide together in the absence of
KLP61F activity and the apparent requirement for interdigitating
microtubules indicate that, in wild-type cells, a driving force may
be generated by another microtubule-crosslinking and-sliding
motor with the opposite transport properties to those of KLP61F.
The C-terminal kinesin Ncd is a microtubule crosslinker that is
directed to microtubule minus (more slowly growing) ends; it
moves at a rate of about 8–15 µm min-1 in vitro19,20 and is therefore
an attractive candidate for counterbalancing KLP61F activity. In
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articles
a
Isometric tension
b
Collapse
Centriole
Ncd
Microtubule
KLP61F
Figure 4 The relative positioning of mitotic centrosomes is determined by a
balance of microtubule–microtubule sliding motors. The motors KLP61F and
Ncd crosslink antiparallel microtubules in interpolar microtubule bundles. The force
generated by KLP61F, a plus-end-directed motor, pushes centrosomes apart while
Ncd, a minus-end-directed motor, pulls them together. a, Normally, during
prometaphase and metaphase, these forces are counterbalanced, maintaining the
spindle under isometric tension. b, If this balance is tipped, following the inhibition of
KLP61F by antibody injections, the repulsive force between centrosomes is
dissipated, the inward force generated by Ncd predominates, and centrosomes slide
together. Small arrows show the direction of the force generated by individual motors
on microtubules and large arrows show the direction of the resulting forces on
centrosomes.
Figure 3 Positioning of mitotic centrosomes and daughter nuclei involves an
interaction between bipolar kinesins and C-terminal kinesins. Mitotic-spindle
formation in embryos carrying a null allele (cand) for the C-terminal kinesin Ncd and
injected with anti-KLP61F antibodies. a, Metaphase spindles assemble but are
abnormally positioned in relation to one another. ‘Spurs’ or bundles of microtubules
can also be seen running between adjacent spindles. b, The chromosomes
segregate and early anaphase B occurs. Arrows indicate the spindle midbodies,
consisting of interdigitating microtubules that span between the separating
chromosomal masses. c, During later phases of anaphase B, these microtubule
bundles shorten significantly and fray as centrosomes and daughter nuclei move
back together. The inset shows the midbodies in wild-type embryos during the same
stage of mitosis. These are longer and the microtubules within them are more
organized. Daughter nuclei were never observed to slide together in either wild-type
or cand controls. Similar results were obtained from >10 embryos and spindle
collapse was never observed as either a direct or an indirect consequence of
injection of anti-KLP61F antibodies into cand embryos. Scale bar represents 12.5 µm
in a–c and 13.5 µm in the inset.
support of this proposal are the findings that Ncd becomes concentrated on interpolar microtubule bundles within spindles in vivo22,
as does KLP61F (ref. 17), and that null mutations in the gene
encoding Ncd result in abnormally long and unstable spindles21,22
that could arise from defects in spindle-pole positioning. Genetic
interactions between bipolar and C-terminal kinesins have been
reported in fungi23–25.
To determine whether KLP61F and Ncd do, in fact, generate
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opposing forces between mitotic centrosomes, we injected embryos
carrying a null allele for Ncd (termed cand; ref. 27) with the antiKLP61F antibodies (Fig. 3). In striking contrast to results obtained
in wild-type embryos, the inhibition of KLP61F in an Ncd-null
background did not inhibit the formation of bipolar metaphase
spindles (Fig. 3a). Although the spindles that formed under these
conditions appeared to be randomly positioned, and abnormal
bundles of microtubules extended between them, they did not collapse but instead successfully completed anaphase A and appeared
to be relatively normal during early anaphase B (Fig. 3b). During
the later stages of anaphase B and telophase, however, marked
defects in the organization of microtubules within the spindle midbody became visible, and the spacing between the centrosomes
associated with daughter nuclei decreased substantially (Fig. 3c).
These observations lead us to draw two conclusions. First,
because genetic deletions of Ncd suppress spindle collapse and the
subsequent formation of microtubule mono-asters that otherwise
occur in the absence of KLP61F function, we conclude that KLP61F
and Ncd generate opposing forces to determine the relative positioning of centrosomes within spindles during prometaphase and
metaphase. Second, as the inhibition of both KLP61F and Ncd
results in spindle defects during late anaphase B and telophase that
affect the relative positioning of daughter nuclei, we conclude that
the activity of these motors also helps to determine internuclear
spacing within the early embryo. Thus, the forces generated by
KLP61F and Ncd are not important solely for mitosis; they may also
influence other facets of development, such as nuclear positioning.
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articles
Discussion
RECEIVED 10 MARCH 1999; REVISED 31 MARCH 1999; ACCEPTED 1 APRIL 1999; PUBLISHED MAY 1999.
Our results provide, to our knowledge, the first evidence that bipolar and C-terminal microtubule-sliding motors interact to generate
antagonistic forces that position centrosomes during animal development. However, it is likely that other motors are also important
in this process. For example, studies of several systems implicate the
minus-end-directed motor cytoplasmic dynein in the initial separation of centrosomes during prophase28 and in spindle elongation
during anaphase B29. Our findings also clarify the mitotic function
of Ncd and reveal that this motor functions together with the bipolar kinesin KLP61F to position centrosomes during stages of mitosis
after prophase (that is, following the initial separation of centrosomes, which appears to occur by a KLP61F- and Ncd-independent
mechanism).
These observations, together with the previous findings that
KLP61F and Ncd are both able to crosslink microtubules, are similarly localized to interpolar microtubule bundles, and generate
motility in opposite directions along the surface lattice of the
microtubule, lead us to propose that these motors position centrosomes by using a ‘sliding-filament mechanism’30 to generate force
between antiparallel microtubules (Fig. 4). When these antagonistic
forces balance one another, centrosome spacing is maintained
under isometric tension. However, tipping this balance results in
movements of centrosomes in the direction of the prevailing motor.h
Methods
Drosophila stocks.
We used Drosophila mutant stocks. Flies carrying the histone–green fluorescent protein (GFP) construct
were provided and maintained by the Sullivan laboratory. Claret non-disjunctional (Ncd) flies (denoted
cand) that are a genetic null for Ncd were provided by R. Scott Hawley.
Embryo collections.
Adult flies were maintained at 25 °C in small plastic containers inverted over agar dishes. To obtain
embryos at the correct stage of development (during the formation of the syncytial blastoderm), flies
were allowed to lay on fresh agar trays for 1 h. These trays were then removed and allowed to age for
another hour at room temperature. Embryos were mechanically dechorionated on double-sided sticky
tape and immobilized on heptane glue. Embryos were then dessicated for 5–9 min and injected under
halocarbon oil.
Microinjections.
Microinjections of rhodamine-conjugated tubulin (Molecular Probes) were performed as described31.
Embryos were then allowed to sit for 5 min and were then re-injected at the same site with either
anti-KLP61F antibodies or non-specific rabbit IgG. The anti-KLP61F antibody injected was an affinitypurified peptide antibody against specific epitopes in the tail domain of KLP61F (ref. 17). Nonspecific
rabbit IgG was obtained from the flow-through off the KLP61F affinity column. Both were concentrated
to an identical optical density (OD 280) by spin filtration in all side-by-side experiments. Optimal
effects of injections of anti-KLP61F antibody were observed at antibody concentrations between 7 and
12 mg ml-1. Injections of antibody at concentrations of <7 mg ml-1 produced only a marginal effect.
Successive freeze-thawing of the KLP61F antibody diminished its effects. Because of this, antibodies
were aliquotted, flash-frozen in liquid nitrogen, and stored at −80 °C shortly after their purification and
concentration.
Embryo preparations for anti-phospho-histone-H3 immunofluorescence.
Following injections with anti-KLP61F antibody, embryos were aged at 25 °C for 15–40 min, washed free
of halocarbon oil with 100% heptane, fixed in 100% heptane:37.5% formalin (1:1) for 5 min, handdevitellinized on double-sided sticky tape, and then incubated in 10 mg ml-1 RNase at 37 °C for 3 h.
Standard antibody incubations and washes were performed. The antibodies used were: rabbit antiphospho-histone-H3 (1:500 dilution; a gift from T.-T. Su) and anti-rabbit-Cy5 (1:250 dilution, Chemicon). DNA was detected with propidium iodide.
Laser-scanning confocal microscopy.
All images shown were obtained on a Leica TCS NT confocal microscopes. Time series were
generated using pre-set programs in Leica TCS software (xyt mode). In these, images were acquired
either from the same optical section or from serial Z sections every 30–60 s over variable periods
of time.
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ACKNOWLEDGEMENTS
This work was supported by grants from the NIH (to J.M.S. and W.S.) and NIH postdoctoral fellowships
(to D.J.S. and J.C.S.). We thank S. Hawley and members of his laboratory for help with this study and for
providing the Cand fly stocks; R. Saint for providing the histone–GFP line; G. Rogers for Fig. 4 and his
intellectual contribution to this work; and the other members of the Scholey and Sullivan laboratories
for assistance. The anti-KLP61F antibody used here was made in collaboration with T. Mitchison and we
thank him for his continuing interest and for discussions about mitosis.
Correspondence and requests for materials should be addressed to J.M.S.
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