ZM 447439

Kinetochore motors drive congression of peripheral
polar chromosomes by overcoming random
arm-ejection forces
Marin Barisic1
, Paulo Aguiar2
, Stephan Geley3
and Helder Maiato1,4,5
Accurate chromosome segregation during cell division in
metazoans relies on proper chromosome congression at the
equator. Chromosome congression is achieved after
bi-orientation to both spindle poles shortly after nuclear
envelope breakdown, or by the coordinated action of motor
proteins that slide misaligned chromosomes along pre-existing
spindle microtubules1
. These proteins include the
minus-end-directed kinetochore motor dynein2–5, and the
plus-end-directed motors CENP-E at kinetochores6,7 and
chromokinesins on chromosome arms8–11. However, how these
opposite and spatially distinct activities are coordinated to
drive chromosome congression remains unknown. Here we used
RNAi, chemical inhibition, kinetochore tracking and laser
microsurgery to uncover the functional hierarchy between
kinetochore and arm-associated motors, exclusively required for
congression of peripheral polar chromosomes in human cells.
We show that dynein poleward force counteracts
chromokinesins to prevent stabilization of immature/incorrect
end-on kinetochore–microtubule attachments and random
ejection of polar chromosomes. At the poles, CENP-E becomes
dominant over dynein and chromokinesins to bias chromosome
ejection towards the equator. Thus, dynein and CENP-E at
kinetochores drive congression of peripheral polar
chromosomes by preventing arm-ejection forces mediated by
chromokinesins from working in the wrong direction.
Chromosome congression to establish a metaphase plate is a well￾conserved feature of mitosis in metazoans that promotes the faithful
segregation of sister chromatids into daughter cells. Chromosome
congression is a robust process that relies on multiple strategies that
ultimately lead to bi-orientation relative to spindle poles1
. Owing
to favourable spatial distribution after nuclear envelope breakdown
(NEB) and direct microtubule capture from both centrosomes,
many chromosomes become rapidly bi-oriented. This may be
facilitated by the presence of surrounding membranes that promote
spindle assembly and microtubule interaction with chromosomes12,13
and/or chromosome prepositioning along the nascent spindle14
In addition, several microtubule-dependent motor proteins have
been implicated in chromosome congression before bi-orientation.
One of these motors is the microtubule minus-end-directed motor
dynein at kinetochores that brings chromosomes to the vicinity of the
spindle poles through lateral interaction with astral microtubules2–5
Subsequently, two microtubule plus-end-directed activities are
thought to counteract the poleward pulling force exerted by
kinetochore dynein and drive chromosomes towards the cell equator.
One is mediated by the chromokinesins Kid (kinesin-10) and Kif4A
(kinesin-4) on chromosome arms that, together with microtubule
polymerization against the chromosomes15, account for the so-called
polar ejection forces8–11,15,16 (PEFs). The other is mediated by CENP-E
(kinesin-7) at kinetochores, to slide chromosomes along pre-existing
spindle microtubules6,7. These activities have been elegantly integrated
into a comprehensive model of chromosome congression based on
motile kinetochores and PEFs (ref. 17). However, it is not understood
why some, and only some, chromosomes are first brought to the
pole when the goal is to reach the equator, and how the random
polar ejection of these chromosomes along astral microtubules is
prevented to bias chromosome movement towards the equator. Here
we addressed these questions by investigating how the opposite and
spatially distinct activities mediated by different motor proteins
are coordinated, and determined the respective correlation with
chromosome position soon after NEB in human cells.
To do so, we started by monitoring chromosome and spindle
microtubule behaviour after NEB at high spatial and temporal
resolution by live-cell spinning-disc confocal imaging of U2OS
1Chromosome Instability and Dynamics Laboratory, Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
Instituto de Engenharia Biomédica, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal. 3Biocenter, Division of Molecular Pathophysiology,
Innsbruck Medical University, Innrain 80, 6020 Innsbruck, Austria. 4Cell Division Unit, Department of Experimental Biology, Faculty of Medicine, University of Porto,
4200-319 Porto, Portugal.
5Correspondence should be addressed to H.M. (e-mail: [email protected])
Received 11 March 2014; accepted 9 October 2014; published online 10 November 2014; DOI: 10.1038/ncb3060
Figure 1 Only a subgroup of chromosomes rely on dynein and CENP-E
to congress to the metaphase plate. U2OS cells stably co-expressing
H2B–GFP and mCherry–α-tubulin were recorded by spinning-disc confocal
microscopy. Control cells (upper panel; n = 13 cells) were imaged at
high temporal resolution (10 s or 2 min). White arrows label the positions
of two peripheral chromosomes whereas red and green lines track their
motion during congression. Red arrowheads indicate poleward movement
of chromosomes in early prometaphase; red arrows indicate chromosomes
moving from the pole towards the metaphase plate. DHC was depleted
by RNAi (middle panel; n = 14 cells) and CENP-E was inhibited with
GSK923295 (lower panel; n=18 cells). Dashed lines represent the estimated
border between central (C) and peripheral (P) chromosomal regions. All
experimental conditions were reproduced by 3 independent experiments.
Scale bars, 5 µm. Time, h:min:s.
cells stably co-expressing GFP–histone H2B and mCherry–α-
tubulin. In control cells we distinguished two distinct modes of
chromosome alignment. One related to chromosomes apparently
within reach of both centrosomes that were able to bi-orient
within just a few minutes after NEB (Fig. 1 and Supplementary
Video 1). The other involved poleward movement of more peripheral
chromosomes and their accumulation at spindle poles, followed by
congression to the metaphase plate with a measured peak velocity
of 1.25±0.39 µm min−1
(mean ± s.d.; n = 15 chromosomes from 4
cells; Fig. 1 and Supplementary Video 1). RNA interference (RNAi)-
mediated depletion of dynein heavy chain (DHC) compromised
the extent of poleward motion of peripheral chromosomes, which
remained significantly expelled from spindle poles and delayed
congression to the metaphase plate (120±62 min versus 15±4 min
in controls; mean ± s.d.; Fig. 1 and Supplementary Fig. 1a and
Video 1). In contrast, inhibition of CENP-E with the small￾molecule inhibitor GSK923295, which locks CENP-E in a ‘rigor’
microtubule-bound state18, compromised the alignment of ∼15%
of the chromosomes (n = 5 cells), which first underwent poleward
motion and accumulated in close proximity to the spindle poles
(Fig. 1 and Supplementary Video 1). Importantly, chromosomes
that were apparently within reach of the two centrosomes at NEB
completed congression in a dynein- and CENP-E-independent
manner (Fig. 1 and Supplementary Video 1). These results suggest
that only a subset of peripheral chromosomes rely on dynein and
CENP-E at kinetochores for their poleward transport and subsequent
equatorial congression.
To determine the accurate position of the chromosomes that
depend on kinetochore motors for congression we started by
tracking kinetochores in three dimensions (x, y, t) using U2OS
cells stably expressing mCherry–α-tubulin and GFP–CENP-A. These
experiments confirmed that several peripheral chromosomes that
could not bi-orient shortly after NEB first initiated poleward
movement, followed by congression to the metaphase plate along
spindle microtubules (Fig. 2a,b and Supplementary Video 2). Then,
we back-tracked polar chromosomes that resulted from CENP-E
inhibition and found that they tend to occupy a peripheral position
near one of the spindle poles (Fig. 2a,b). Quantification of the
© 2014 Macmillan Publishers Limited. All rights reserved.
Figure 2 Kinetochore motors are exclusively required to align peripheral
chromosomes that are closer to one of the spindle poles. (a) U2OS cells
stably co-expressing GFP–CENP-A (green) and mCherry–α-tubulin (red) were
recorded by spinning-disc confocal microscopy with and without the CENP-E
inhibitor GSK923295 (n(control) = 6 cells, n(CENP-E inh.) = 11 cells). All
experimental conditions were reproduced by 4 independent experiments.
Scale bars, 5 µm. Time, h:min:s. (b) Corresponding three-dimensional (x, y,t)
tracking (Control) and back-tracking (CENP-E inhibition) of kinetochore
pairs from the cells in a. Different colours indicate distinct kinetochore
pairs tracked. Scale bars, 5 µm. Time, h:min:s. (c) Quantitative mapping
of kinetochore positions relative to the spindle pole, the metaphase plate
and the spindle (normalized as an ‘ellipsoid’) obtained by four-dimensional
(x, y, z, t) back-tracking of CENP-E-inhibited cells (see selected region in
a for reference). Note that most kinetochore pairs (green dots) occupy
a position at the spindle periphery and much closer to one of the
poles (red dot). Exceptional kinetochore pairs that fall inside the spindle
region are also indicated (black dots). n = 54 kinetochore pairs from
5 cells.
initial position of polar chromosomes that depended on CENP-E
for congression, now using four-dimensional (4D; x, y, z,t) tracking,
revealed that 96.3% of the kinetochores were excluded from the spindle
region and were closer to one of the poles than to the midpoint between
both poles (Fig. 2c and Supplementary Video 3). Thus, although most
chromosomes are able to align in a dynein- and CENP-E-independent
manner, these kinetochore motors are critical to align chromosomes
that might be positioned at the spindle periphery and much closer to
one of the spindle poles, where bi-orientation after NEB is less likely
to occur.
Next, we focused on understanding the distinct behaviour of polar
chromosomes, which were found to be ‘ejected’ away from the pole on
depletion of DHC or ‘locked’ very close to the pole after inhibition of
CENP-E (Fig. 1). For this purpose we simultaneously depleted DHC
by RNAi and inhibited CENP-E motor activity with GSK923295. As
for DHC depletion alone, these cells showed polar chromosomes that
were ejected towards the cortex, with chromosome arms often found
oriented away from the pole, consistent with the predicted action
of PEFs (refs 10,16; Fig. 3a and Supplementary Video 3). Quanti￾tative analysis in fixed material revealed that depletion of DHC in
CENP-E-inhibited cells caused the ejection of polar chromosomes
(3.3±1.4 µm away from the centrosome; mean ± s.d.; n=255 kine￾tochores, 10 cells) when compared with CENP-E inhibition alone
(1.6±0.6 µm; mean ± s.d.; n=283 kinetochores, 10 cells; P <0.001,
Mann–Whitney test). Surprisingly, nearly all ejected polar chromo￾somes in 92% of the cells in which CENP-E and dynein activities
were simultaneously abrogated revealed robust kinetochore fibres
(k-fibres; Fig. 3a and Supplementary Video 3), in striking contrast to
CENP-E-inhibited cells, which showed at best one such polar chromo￾some in only 18% of the cells. Similar findings were obtained on rescue
of Spindly RNAi with an RNAi-resistant point mutant (F258A) that
specifically prevents dynein recruitment to kinetochores19 (Supple￾mentary Fig. 2 and Video 4). Thus, the capacity of polar chromosomes
to tether on microtubule plus-ends does not require CENP-E motor
activity (in agreement with previous in vitro reconstitution experi￾ments, despite the requirement of the CENP-E motor domain20,21) or
kinetochore dynein. Moreover, kinetochore dynein seems to coun￾teract arm-mediated ejection forces on polar chromosomes that oth￾erwise would stabilize end-on kinetochore–microtubule attachments
leading to chromosome ejection towards the cortex.
© 2014 Macmillan Publishers Limited. All rights reserved.
Figure 3 Dynein counteracts PEFs to bring peripheral chromosomes to
the poles and prevent stabilization of end-on kinetochore–microtubule
attachments. (a) Representative examples of CENP-E-inhibited cells depleted
either of DHC alone (upper panel; n = 12 cells from 3 independent
experiments), or of DHC and chromokinesins (KK; lower panel; n=22 cells
from 2 independent experiments). Arrows highlight robust k-fibres on polar
chromosomes. Arrowheads indicate positions of chromosome arms ejected
from the poles. Scale bars, 5 µm. Time, h:min:s. (b) U2OS cells treated
with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC) and immunostained
for DNA (DAPI), kinetochores (ACA) and centrioles (centrin). Maximum￾intensity projection images of representative examples for each condition
are shown. Numbers indicate mean kinetochore-to-pole distances ± s.d.
of the pooled data from 3 independent experiments. All experimental
conditions were reproduced by 3 independent experiments. Scale bars, 5 µm.
(c) Quantification of kinetochore (KT)-to-pole distances in STLC-treated cells
under different conditions; n(Control) = 5,860 kinetochores from 47 cells;
n(CENP-E inh.) = 7,858 kinetochores from 70 cells; n(Kid RNAi) = 6,655
kinetochores from 48 cells; n(Kif4A RNAi) = 5,784 kinetochores from 45
cells; n(KK RNAi) = 5,063 kinetochores from 41 cells; n(DHC RNAi) = 5,087
kinetochores from 40 cells. Error bars represent s.d. Asterisks indicate
Mann–Whitney U-test significance values: P < 0.001 relative to control.
(d) Model illustrating the relationship between polar ejection forces (PEF)
and poleward forces (PF) and their individual contributions to chromosome
positioning in each experimental scenario.
Overexpression of the Kid chromokinesin orthologue Nod in
Drosophila S2 cells stabilizes end-on kinetochore–microtubule
attachments, suggesting that PEFs on chromosome arms normally
contribute to k-fibre maturation22. Moreover, the dynein co￾factor at kinetochores Spindly has been shown to oppose PEFs
by counteracting Kid in human cells23. To investigate whether
PEFs on chromosome arms accounted for the stabilization of
end-on kinetochore–microtubule attachments observed on polar
chromosomes in the absence of DHC, we co-depleted both human
chromokinesins (Kid and Kif4A; hereafter referred as KK) and DHC
by RNAi, while simultaneously inhibiting CENP-E motor activity.
We were unable to detect robust k-fibres associated with polar
chromosomes, which remained in close proximity to centrosomes
(Fig. 3a and Supplementary Fig. 1a and Video 3). Notably, several
chromosomes were able to align at the cell equator after functional
perturbation of all three classes of motors, further supporting the
© 2014 Macmillan Publishers Limited. All rights reserved.
Figure 4 Stabilization of kinetochore–microtubule attachments on polar
chromosomes leads to random ejection. (a) Immunofluorescence images
of U2OS cells stained for DNA (DAPI), kinetochores (ACA), α-tubulin and
MAD1. Maximum-intensity projection images of representative examples are
shown for each condition. Insets indicate representative kinetochore (KT)
pairs and the respective fluorescence intensity of ACA (red) and MAD1
(green) are shown as line-scan plots. At least 2 independent experiments
were performed per condition. Scale bar, 5 µm (main panels) and 1 µm
(insets). (b) Quantification of MAD1 detection on kinetochore pairs from polar
chromosomes under different conditions. n(CENP-E inh.) = 191 kinetochore
pairs from 26 cells; n(DHC RNAi + CENP-E inh.) = 312 kinetochore pairs
from 29 cells; n(10 nM taxol + CENP-E inh.) = 171 kinetochore pairs from
13 cells; n(100 nM nocodazole + CENP-E inh.) = 222 kinetochore pairs from
12 cells; n(Aurora A inh. + CENP-E inh.) = 304 kinetochore pairs from
42 cells; n(Aurora B inh. + CENP-E inh.) = 556 kinetochore pairs from
24 cells. Asterisks indicate z-test significance values: P < 0.001 relative
to CENP-E inhibition alone. All conditions were reproduced by at least 2
independent experiments.
© 2014 Macmillan Publishers Limited. All rights reserved.
00:00 00:01 00:09 00:20 00:24
00:00 00:04 00:10 00:16 00:22
CENP-E inh.
00:00 00:01 00:02 00:04 00:05
CENP-E inh.
CENP-E inh. CENP-E inh.
No movement
Towards cortex
d Control CENP-E inh.
CENP-E inh. + Chromokinesins RNAi
Figure 5 CENP-E is critical to bias polar chromosome movement exclusively
towards the cell equator. (a) Illustration of laser microsurgery experimental
setting showing the typical position of the cut on the chromosome
arm. (b) Chromosome arms of CENP-E-inhibited U2OS-H2B–GFP/mCherry–
α-tubulin cells were severed by laser microsurgery and recorded by
spinning-disc confocal microscopy (upper 2 panels; n = 33 cells from
16 independent experiments). In the lowest panel chromokinesins were
depleted simultaneously with CENP-E inhibition (n = 16 cells from 6
independent experiments). Arrowheads indicate the site of laser microsurgery;
white arrows follow the acentric chromosome fragment. Kymographs
represent 1 min time frames containing the acentric chromosome fragments
corresponding to the regions outlined by the yellow dotted lines marked
on the time-lapse stills on the left. Red dotted lines and arrows
highlight the direction of movement by acentric fragments. Scale bars,
5 µm. Time = h:min. (c) Quantification of the movements by acentric
chromosome fragments after laser microsurgery. (d) Model illustrating
peripheral chromosome congression in human cells based on the functional
coordination of three distinct motor protein activities. In upper left panel,
arrows indicate the direction of motion by dynein (orange), CENP-E (grey)
and chromokinesins (purple) during peripheral chromosome congression.
Peripheral chromosomes are first brought to the spindle pole by dynein.
At the pole, CENP-E becomes dominant over dynein at kinetochores
and directs chromosomes towards the cell equator, in coordination
with chromokinesins at chromosome arms. Grey dashed line depicts an
imaginary border line between peripheral and central chromosomes. Acentric
chromosome fragments generated by laser microsurgery on chromosome
arms are able to move either towards the cell equator or towards the
cell cortex (upper right panel). The dominant activity of CENP-E prevents
chromokinesins from working in the wrong direction and directs the
chromosomes exclusively towards the metaphase plate (lower left panel).
Dynein prevents chromokinesin-mediated PEFs from stabilizing end-on
kinetochore–microtubule attachments and from random ejection of polar
chromosomes, including away from the metaphase plate towards the cell
cortex (lower right panel).
© 2014 Macmillan Publishers Limited. All rights reserved.
existence of motor-independent mechanisms, probably relying on
favourable chromosome positioning after NEB and immediate
bi-orientation. To further test whether dynein counteracts ejection
forces on polar chromosomes, we first confirmed the presence
of dynein (and CENP-E) on polar chromosomes after CENP-E
inhibition (Supplementary Fig. 3a,b). Second, we perturbed the
function of each motor protein in a monopolar spindle configuration
generated after inhibition of kinesin-5, which is necessary for
prophase centrosome separation24. We found that both CENP-E and
each chromokinesin were individually required for chromosome
ejection from the monopole (Fig. 3b,c), in agreement with recent
findings11, but contrary to another report suggesting that Kif4A
counteracts Kid-mediated ejection forces25. Moreover, DHC was
required for the poleward force that brings chromosomes close
to the monopole (Fig. 3b,c). Thus, dynein-mediated poleward
force normally counteracts CENP-E and chromokinesin-mediated
PEFs (Fig. 3d).
To confirm that dynein normally prevents the formation of stable
end-on kinetochore–microtubule attachments on polar chromosomes
we performed immunofluorescence in fixed U2OS cells using specific
antibodies against the spindle-assembly checkpoint protein MAD1,
which marks unattached kinetochores. MAD1 was enriched on both
kinetochores from dispersed chromosomes at NEB and undetectable
on bi-oriented chromosomes in control metaphase cells (Fig. 4a). After
inhibition of CENP-E, most polar chromosomes accumulated MAD1
on both kinetochores, as opposed to bi-oriented chromosomes at the
metaphase plate (Fig. 4a,b). This is fully consistent with previous
electron microscopy reconstructions of polar chromosomes, whose
kinetochores showed no or reduced end-on binding to microtubules
after functional perturbation of CENP-E (refs 26,27). Importantly,
CENP-E inhibition in DHC-depleted cells decreased the number
of polar chromosomes with MAD1 on both kinetochores from the
pair and increased the number of polar chromosomes with the
distal kinetochore positive for MAD1 or with both kinetochores
from the pair negative for MAD1, indicating that dynein-mediated
poleward motion prevents the establishment of stable monotelic and
syntelic attachments on polar chromosomes (Fig. 4a,b). To further
investigate the effect of end-on kinetochore–microtubule attachments
on the ejection of polar chromosomes, we combined the inhibition of
CENP-E with inhibition of Aurora kinases (A or B), or with treatment
with nanomolar doses of nocodazole or taxol, all of which have
been shown to stabilize kinetochore–microtubule attachments28–32
All of these conditions consistently increased the number of polar
chromosomes with no detectable MAD1 on both kinetochores from
the pair or with MAD1 accumulation at the distal kinetochore alone
(Fig. 4a,b). Curiously, in all cases, except after Aurora A inhibition,
chromosomes were ejected from the spindle pole, as observed after
DHC depletion. This might reflect an additional role for Aurora A in
the activation of CENP-E on polar chromosomes33. Taken together,
these experiments demonstrate that dynein prevents stabilization
of immature/incorrect end-on kinetochore microtubule attachments
that otherwise would lead to the ejection of polar chromosomes.
The previous experiments strongly suggest that CENP-E, not
arm-ejection forces mediated by chromokinesins, is critical to bias
polar chromosome movement towards the cell equator, implying
that CENP-E must be dominant over chromokinesins. To investigate
the functional relationship between CENP-E and chromokinesins
during congression of polar chromosomes we implemented a laser
microsurgery-based live-cell assay to physically separate chromosome
arms from their respective kinetochores, and combined this with
CENP-E inhibition, with or without chromokinesin depletion
(Fig. 5a,b). Accordingly, we first inhibited CENP-E motor activity
to generate polar chromosomes and then used laser microsurgery
to sever chromosome arms away from the kinetochore region and
then tracked the movement of the resulting chromosome fragments
(with and without kinetochore). We found that, contrary to the
kinetochore-containing fragments, which remained ‘locked’ at the
poles by dynein-mediated pulling forces, acentric (that is, without
kinetochore) chromosome arms were able to be ejected from the
pole with a measured peak velocity of 0.86±0.27 µm min−1
(mean ±
s.d.; n=8 chromosomes from 8 cells; ∼30% slower than congressing
chromosomes in unperturbed mitosis) and congressed to the cell
equator in 64% of the cases (Fig. 5b,c and Supplementary Video 5).
In the other 36% of the cells acentric chromosome fragments
failed to congress towards the equator and either moved towards
the cell cortex or remained stalled with no discernible directed
motion (Fig. 5b,c and Supplementary Video 5). Importantly, only
19% of acentric chromosome fragments managed to align at the
cell equator after co-depletion of chromokinesins (probably by
the action of microtubule polymerization15) and all of the others
(81%) failed to move (Fig. 5b,c and Supplementary Video 5). These
experiments demonstrate that chromokinesin-mediated PEFs are
active on polar chromosomes, but kinetochore forces are dominant
to move peripheral chromosomes poleward (by dynein) and,
subsequently, to eject polar chromosomes exclusively towards the
equator (by CENP-E).
Overall, this study establishes the precise functional hierarchy
between three distinct motor protein families, explaining how
peripheral polar chromosomes that cannot bi-orient immediately after
NEB align at the equator in human cells (Fig. 5d). Chromosome
bi-orientation is limited by topological constraints associated with
incomplete centrosome separation at NEB (refs 34,35), chromosome
shielding14 and/or the degree of cell flattening during mitosis36. Here
we highlight the role of dynein in preventing the stabilization of end￾on kinetochore–microtubule attachments (and consequent reduction
of CENP-E from kinetochores37) on initial lateral interaction of
peripheral chromosomes with astral microtubules. We propose that
this role serves at least three key purposes: to prevent random
ejection of polar chromosomes due to the action of chromokinesins on
chromosome arms; to allow CENP-E to be processive and slide polar
chromosomes along pre-existing spindle microtubules; and to prevent
the formation of erroneous kinetochore–microtubule attachments
and/or facilitate their correction. This role of dynein might be
associated with the inhibition of the Ndc80 complex required for
end-on kinetochore–microtubule attachments, as shown recently in
Caenorhabditis elegans38, and/or due to local Aurora (A and B) kinase
activities on polar chromosomes, as shown in the present study.
In agreement, it has been previously shown that Aurora A and B
phosphorylate a conserved residue on CENP-E motor domain that
is required for the functional switch responsible for the alignment of
polar chromosomes33, with possible implications for the conversion
of lateral to end-on kinetochore–microtubule attachments39. Our
© 2014 Macmillan Publishers Limited. All rights reserved.
findings further demonstrate that CENP-E is the critical motor that
ensures the correct direction of motion required for congression
of polar chromosomes towards the equator. This might involve
the recognition of specific features associated with the intrinsic
diversity of microtubule subpopulations in the spindle (for example,
individual versus bundles or dynamic versus stable microtubules).
Arm-ejection forces mediated by chromokinesins may instead be
important for arm orientation and to work in coordination with
microtubule-depolymerizing kinesins, such as kinesin-8 (ref. 25),
to maintain aligned chromosomes at the equator (Supplementary
Fig. 1; see also refs 11,16,40). This would be consistent with the
concomitant reduction of dynein and CENP-E from kinetochores
on microtubule binding37, allowing chromokinesins to overcome
kinetochore motors and stabilize end-on microtubule attachments on
bi-oriented chromosomes22,33
Methods and any associated references are available in the online
version of the paper.
Note: Supplementary Information is available in the online version of the paper
We thank A. Pereira for the development of image analysis tools, M. Barisic
for exceptional technical help and R. Gassmann for the critical reading of this
manuscript S.G. is supported by FWF DK W 1101 ‘MCBO’. H.M. is financially
supported by FEDER through the Operational Competitiveness Programme—
COMPETE and by National Funds through FCT—Fundação para a Ciência e
a Tecnologia under the project FCOMP-01-0124-FEDER-015941 (PTDC/SAU￾ONC/112917/2009), the Human Frontier Science Program and the 7th framework
program grant PRECISE from the European Research Council.
M.B. designed, performed and analysed experiments. P.A. performed image analysis.
S.G. provided reagents. H.M. designed and analysed experiments, and coordinated
the work. H.M. and M.B. wrote the manuscript.
The authors declare no competing financial interests.
Published online at
Reprints and permissions information is available online at
1. Walczak, C. E., Cai, S. & Khodjakov, A. Mechanisms of chromosome behaviour during
mitosis. Nat. Rev. 11, 91–102 (2010).
2. Rieder, C. L. & Alexander, S. P. 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 (1990).
3. Li, Y., Yu, W., Liang, Y. & Zhu, X. Kinetochore dynein generates a poleward
pulling force to facilitate congression and full chromosome alignment. Cell Res. 17,
701–712 (2007).
4. Yang, Z., Tulu, U. S., Wadsworth, P. & Rieder, C. L. Kinetochore dynein is required
for chromosome motion and congression independent of the spindle checkpoint.
Curr. Biol. 17, 973–980 (2007).
5. Vorozhko, V. V., Emanuele, M. J., Kallio, M. J., Stukenberg, P. T. & Gorbsky, G. J.
Multiple mechanisms of chromosome movement in vertebrate cells mediated through
the Ndc80 complex and dynein/dynactin. Chromosoma 117, 169–179 (2008).
6. Kapoor, T. M. et al. Chromosomes can congress to the metaphase plate before
biorientation. Science 311, 388–391 (2006).
7. Cai, S., O’Connell, C. B., Khodjakov, A. & Walczak, C. E. Chromosome congression
in the absence of kinetochore fibres. Nat. Cell Biol. 11, 832–838 (2009).
8. Antonio, C. et al. Xkid, a chromokinesin required for chromosome alignment on the
metaphase plate. Cell 102, 425–435 (2000).
9. Funabiki, H. & Murray, A. W. The Xenopus chromokinesin Xkid is essential for
metaphase chromosome alignment and must be degraded to allow anaphase
chromosome movement. Cell 102, 411–424 (2000).
10. Rieder, C. L., Davison, E. A., Jensen, L. C., Cassimeris, L. & Salmon, E. D. 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 (1986).
11. Wandke, C. et al. Human chromokinesins promote chromosome congression and
spindle microtubule dynamics during mitosis. J. Cell Biol. 198, 847–863 (2012).
12. Poirier, C. C., Zheng, Y. & Iglesias, P. A. Mitotic membrane helps to focus and stabilize
the mitotic spindle. Biophys. J. 99, 3182–3190 (2010).
13. Civelekoglu-Scholey, G., Tao, L., Brust-Mascher, I., Wollman, R. & Scholey, J. M.
Prometaphase spindle maintenance by an antagonistic motor-dependent force
balance made robust by a disassembling lamin-B envelope. J. Cell Biol. 188,
49–68 (2010).
14. Magidson, V. et al. The spatial arrangement of chromosomes during prometaphase
facilitates spindle assembly. Cell 146, 555–567 (2011).
15. Brouhard, G. J. & Hunt, A. J. Microtubule movements on the arms of mitotic
chromosomes: polar ejection forces quantified in vitro. Proc. Natl Acad. Sci. USA
102, 13903–13908 (2005).
16. Levesque, A. A. & Compton, D. A. The chromokinesin Kid is necessary for
chromosome arm orientation and oscillation, but not congression, on mitotic spindles.
J. Cell Biol. 154, 1135–1146 (2001).
17. Rieder, C. L. & Salmon, E. D. Motile kinetochores and polar ejection forces dictate
chromosome position on the vertebrate mitotic spindle. J. Cell Biol. 124, 223–233
18. Wood, K. W. et al. Antitumor activity of an allosteric inhibitor of centromere￾associated protein-E. Proc. Natl Acad. Sci. USA 107, 5839–5844 (2010).
19. Gassmann, R. et al. Removal of Spindly from microtubule-attached kinetochores
controls spindle checkpoint silencing in human cells. Genes Dev. 24,
957–971 (2010).
20. Lombillo, V. A., Nislow, C., Yen, T. J., Gelfand, V. I. & McIntosh, J. R. Antibodies to the
kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent
motion of chromosomes in vitro. J. Cell Biol. 128, 107–115 (1995).
21. Gudimchuk, N. et al. Kinetochore kinesin CENP-E is a processive bi-directional
tracker of dynamic microtubule tips. Nat. Cell Biol. 15, 1079–1088 (2013).
22. Cane, S., Ye, A. A., Luks-Morgan, S. J. & Maresca, T. J. Elevated polar ejection forces
stabilize kinetochore-microtubule attachments. J. Cell Biol. 200, 203–218 (2013).
23. Barisic, M. et al. Spindly/CCDC99 is required for efficient chromosome congression
and mitotic checkpoint regulation. Mol. Biol. Cell 21, 1968–1981 (2010).
24. Tanenbaum, M. E., Macurek, L., Galjart, N. & Medema, R. H. Dynein, Lis1 and
CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle
assembly. EMBO J. 27, 3235–3245 (2008).
25. Stumpff, J., Wagenbach, M., Franck, A., Asbury, C. L. & Wordeman, L. Kif18A and
chromokinesins confine centromere movements via microtubule growth suppression
and spatial control of kinetochore tension. Dev. Cell 22, 1017–1029 (2012).
26. Putkey, F. R. et al. Unstable kinetochore-microtubule capture and chromosomal
instability following deletion of CENP-E. Dev. Cell 3, 351–365 (2002).
27. McEwen, B. F. et al. CENP-E is essential for reliable bioriented spindle attachment,
but chromosome alignment can be achieved via redundant mechanisms in
mammalian cells. Mol. Biol. Cell 12, 2776–2789 (2001).
28. Yang, Z., Kenny, A. E., Brito, D. A. & Rieder, C. L. Cells satisfy the mitotic checkpoint
in Taxol, and do so faster in concentrations that stabilize syntelic attachments. J. Cell
Biol. 186, 675–684 (2009).
29. Vasquez, R. J., Howell, B., Yvon, A. M., Wadsworth, P. & Cassimeris, L. Nanomolar
concentrations of nocodazole alter microtubule dynamic instability in vivo and
in vitro. Mol. Biol. Cell 8, 973–985 (1997).
30. Cimini, D., Wan, X., Hirel, C. B. & Salmon, E. D. Aurora kinase promotes turnover of
kinetochore microtubules to reduce chromosome segregation errors. Curr. Biol. 16,
1711–1718 (2006).
31. Hauf, S. et al. The small molecule Hesperadin reveals a role for Aurora B in
correcting kinetochore-microtubule attachment and in maintaining the spindle
assembly checkpoint. J. Cell Biol. 161, 281–294 (2003).
32. Bakhoum, S. F., Kabeche, L., Murnane, J. P., Zaki, B. I. & Compton, D. A. DNA
damage response during mitosis induces whole chromosome mis-segregation. Cancer
Discov. (2014).
33. Kim, Y., Holland, A. J., Lan, W. & Cleveland, D. W. Aurora kinases and protein
phosphatase 1 mediate chromosome congression through regulation of CENP-E. Cell
142, 444–455 (2010).
34. Silkworth, W. T., Nardi, I. K., Paul, R., Mogilner, A. & Cimini, D. Timing of centrosome
separation is important for accurate chromosome segregation. Mol. Biol. Cell 23,
401–411 (2012).
35. Kaseda, K., McAinsh, A. D. & Cross, R. A. Dual pathway spindle assembly increases
both the speed and the fidelity of mitosis. Biol. Open 1, 12–18 (2012).
36. Lancaster, O. M. et al. Mitotic rounding alters cell geometry to ensure efficient bipolar
spindle formation. Dev. Cell 25, 270–283 (2013).
37. Hoffman, D. B., Pearson, C. G., Yen, T. J., Howell, B. J. & Salmon, E. D. Microtubule￾dependent changes in assembly of microtubule motor proteins and mitotic spindle
checkpoint proteins at PtK1 kinetochores. Mol. Biol. Cell 12, 1995–2009 (2001).
38. Cheerambathur, D. K., Gassmann, R., Cook, B., Oegema, K. & Desai, A.
Crosstalk between microtubule attachment complexes ensures accurate chromosome
segregation. Science 342, 1239–1242 (2013).
39. Shrestha, R. L. & Draviam, V. M. Lateral to end-on conversion of chromosome￾microtubule attachment requires kinesins CENP-E and MCAK. Curr. Biol. 23,
1514–1526 (2013).
40. Takagi, J., Itabashi, T., Suzuki, K. & Ishiwata, S. Chromosome position at the spindle
equator is regulated by chromokinesin and a bipolar microtubule array. Sci. Rep. 3,
2808 (2013).
© 2014 Macmillan Publishers Limited. All rights reserved.
DOI: 10.1038/ncb3060 M E T H O D S
Cell culture, RNAi and western blot analysis. A U2OS cell line stably
expressing mCherry–α-tubulin (gift from R. Medema, NKI, Amsterdam, The
Netherlands) was transfected with GFP–CENP-A plasmid (gift from P. Meraldi,
University of Geneva, Switzerland) using Lipofectamine 2000 to generate a
new stable cell line. Stable integrants were selected using puromycin. A U2OS
cell line stably expressing H2B–GFP and mCherry–α-tubulin was generated
as previously described23. Both stable and parental U2OS cell lines were
grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with
10% fetal bovine serum (FBS; Invitrogen) at 37 ◦C in humidified conditions
with 5% CO2
. The RNAi-resistant Spindly mutant was generated by site￾directed mutagenesis (Quick-Change, Stratagene) using the following primers:
TGCCTTTACTATT (changed nucleotides are underlined).
For RNA interference (RNAi) experiments, cells were transfected at 30–50%
confluency using Lipofectamine 2000 with 50 nM siRNAs for 48 h: hKID 50
(refs 11,41), KIF4A 50
(ref. 11), DHC1 50
(refs 23,42,43), Spindly: 50
(ref. 23) and
control (Luciferase): 50
. Depletion efficiency
was monitored by western blotting using the following antibodies: rabbit anti￾KIF4A, diluted 1:1,000 (ref. 11); mouse anti-hKID (clone 8C12), diluted 1:1,000
(ref. 44); rabbit anti-dynein heavy chain 1, diluted 1:300 (HPA003742, Sigma);
mouse anti-α-tubulin, diluted 1:10,000 (clone B512, Sigma) and rat anti-CLASP1,
diluted 1:100 (ref. 45). Spindly depletion by RNAi was performed as previously
described23. HRP-conjugated secondary antibodies, diluted 1:10,000 (Amersham),
were visualized using the ECL system (Pierce).
Drug treatments. To inhibit CENP-E, 20 nM GSK-923295 (MedChemexpress) was
added to the media and cells were either immediately filmed or fixed after 2 h. For
Aurora A and B kinase inhibition 250 nM MLN-8054 (Selleck Chemicals) and 4 µM
ZM447439 (AstraZeneca) were added respectively to the cells 2 h before fixation. To
stabilize microtubules 10 nM taxol (Cytoskeleton) and 100 nM nocodazole (Sigma)
were added respectively to the cell culture media 2 h before fixation. 5 µM S-trityl￾L-cysteine (STLC; Sigma) was added to the cell culture media 2 h before fixation to
induce monopolar spindles by inhibiting Eg5.
Immunofluorescence microscopy. U2OS cells were grown on glass coverslips
and fixed by ice-cold methanol for 4 min at −20 ◦C. Primary antibodies used were:
mouse anti-α-tubulin 1:2,000 (clone B512, Sigma), rabbit anti-MAD1 (generated
against full-length MAD1, 1:1,000; provided by P. Meraldi, University of Geneva,
Switzerland), rabbit anti-centrin (provided by I. Cheeseman, Whitehead Institute
for Biomedical Research, Cambridge, Massachusetts, USA), rabbit anti-dynein heavy
chain 1, diluted 1:100 (HPA003742, Sigma), rabbit anti-CENP-E 1:400 (Santa Cruz)
and human anticentromere antibodies (ACA) 1:5,000 (provided by B. Earnshaw,
Wellcome Trust Centre for Cell Biology, University of Edinburgh, UK). Alexa Fluor
488, 568 and 647 (Invitrogen) were used as secondary antibodies (1:1,000). DNA
was counterstained with DAPI (1 µg ml−1
; Sigma-Aldrich). Images were acquired on
an AxioImager Z1 (×100 Plan-Apochromatic oil differential interference contrast
objective lens, 1.46 NA) equipped with a CCD (charge-coupled device; ORCA￾R2, Hamamatsu) camera operated by Zen software (Zeiss). Blind deconvolution of
images was performed using Autoquant X software (Media Cybernetics). Images
were processed in Photoshop CS4 (Adobe) and represent maximum-intensity
projections of a deconvolved stack. Kinetochore-to-pole distances were measured
using a custom routine written in MATLAB 8.1 (The Mathworks) to determine the
three-dimensional distance between the centroids of a kinetochore and the midpoint
between the centrioles.
Live-cell imaging. U20S-H2B–GFP/mCherry–α-tubulin cells and U20S-GFP–
CENP-A/mCherry–α-tubulin cells were cultured in 35 mm glass-bottomed dishes
(14 mm, No. 1.5, MatTek Corporation). Before imaging, cell culture medium was
changed to L15 (Invitrogen). Imaging was performed in a heated chamber (37 ◦C)
using a ×100 1.4 NA Plan-Apochromatic DIC objective mounted on an inverted
microscope (TE2000U; Nikon) equipped with a CSU-X1 spinning-disc confocal
head (Yokogawa Corporation of America) provided with two laser lines (488 nm
and 561 nm). Images were taken with an iXonEM+ electron-multiplying CCD
camera (Andor Technology). Eleven 1 µm-separated z-planes covering the entire
volume of the mitotic spindle were collected every 10 s, up to 2 min, as indicated.
All images represent maximum-intensity projections of z-stacks. Image processing
was performed in ImageJ (ref. 46).
Kinetochore tracking. Kinetochore and pole positions were tracked in three￾dimensional space using the TrackMate tool in ImageJ46. A total of 54 kinetochore
pairs from 5 cells were used to assess the original positions of the chromosomes
that failed to congress on CENP-E inhibition. To combine the data from different
cells, the kinetochore positions were corrected for the spindle movement and placed
in the polar coordinate system (applying the appropriate transformation to match
the axes of the poles to the Ox axis and placing the midpoints of the poles at the
origin). In addition to rotation and translation, a scaling factor was also applied,
normalizing for the pole-to-midpoint distance. Given the symmetry of the spindle,
all data points were represented in the first octant. The surface of an ellipsoid
of rotation, approximating the spindle region, was combined with the data point
representation. The ratio between the major axis and the (equal) minor axis of the
ellipsoid was estimated from CENP-E-inhibited cells in the initial stage of bipolar
spindle formation. Analysis and plotting were performed using custom-made scripts
developed in MATLAB 8.1 (The MathWorks).
Laser microsurgery. Severing of chromosome arms was carried out by 3–9 series
(10 Hz repetition rate) of second-harmonic single spatial mode, 532 nm pulses of
a Nd:YAG laser (either an ULTRA-CFR TEM00 from Quantel or an FQ-500-532
from L4light). The pulse width was 8–10 ns and the pulse energy used was 1.5–2 µJ.
A detailed description of the laser microsurgery has been published47. Imaging
and laser focusing were performed using a ×100 1.4 NA plan-Apochromatic DIC
objective on a Nikon TE2000U inverted microscope equipped with a Yokogawa
CSU-X1 spinning-disc confocal head and an iXonEM+ electron-multiplying CCD
camera. Images were acquired every 1 min. Spindle rotation was compensated using
a custom routine written in MATLAB 8.1 (The Mathworks) to allow the tracking of
severed acentric chromosomal fragments.
Velocity measurements. The velocity of chromosomal fragments after laser
microsurgery, and the velocity of chromosome congression in control cells, were
estimated using conventional kymograph analysis48. As velocities vary during
congression, the presented values correspond to the peak velocities.
Statistical analysis. Statistical analysis was performed using SigmaStat 3.5 software.
All data represent the mean ± s.d. Statistical significance of differences between
the population distributions was determined by the Mann–Whitney two-tailed test,
except for Fig. 4b for which a z-test was used to statistically compare the difference
between population proportions. A statistical difference with a P value < 0.001 is
presented with a single asterisk (∗).
41. Wolf, F., Wandke, C., Isenberg, N. & Geley, S. Dose-dependent effects of stable cyclin
B1 on progression through mitosis in human cells. EMBO J. 25, 2802–2813 (2006).
42. Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T. & Weber, K. Identification of
essential genes in cultured mammalian cells using small interfering RNAs. J. Cell
Sci. 114, 4557–4565 (2001).
43. Draviam, V. M., Shapiro, I., Aldridge, B. & Sorger, P. K. Misorientation and reduced
stretching of aligned sister kinetochores promote chromosome missegregation in
EB1- or APC-depleted cells. EMBO J. 25, 2814–2827 (2006).
44. Wandke, C. & Geley, S. Generation and characterization of an hKid-specific
monoclonal antibody. Hybridoma (Larchmt) 25, 41–43 (2006).
45. Maffini, S. et al. Motor-independent targeting of CLASPs to kinetochores by
CENP-E promotes microtubule turnover and poleward flux. Curr. Biol. 19,
1566–1572 (2009).
46. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of
image analysis. Nat. Methods 9, 671–675 (2012).
47. Pereira, A. J., Matos, I., Lince-Faria, M. & Maiato, H. Dissecting mitosis with
laser microsurgery and RNAi in Drosophila cells. Methods Mol. Biol. 545,
145–164 (2009).
48. Pereira, A. J. & Maiato, H. Improved kymography tools and its applications to mitosis.
Methods 51, 214–219 (2010).
© 2014 Macmillan Publishers Limited. All rights reserved.
DOI: 10.1038/ncb3060
Control KID RNAi
Control DHC RNAi
Control KK RNAi
CENPE inh.+
Control KK RNAi
Control KK RNAi
Supplementary Figure 1 RNAi and live cell imaging phenotypes. a) Protein
lysates obtained 48 h after siRNA transfection (50 nM) were immunoblotted
for the target proteins and α-tubulin was used as loading control. Due to the
large size of DHC (~500 kDa), CLASP1 (~160 kDa) was used as a loading
control in the lowest panel. n(Kid RNAi)=1 experiment; n(Kif4A RNAi)=1
experiment; n(DHC RNAi)=1 experiment (due to limited antibody supply); n(KK
RNAi)=2 experiments; b) Chromokinesins Kid and Kif4A were depleted by
RNAi in U2OS-H2B-GFP/mCherry-α-tubulin cells with or without the presence
of CENP-E inhibitor. Data was then acquired by live-cell imaging. n(control)=4
cells imaged with 10 sec interval (2 experiments) plus 9 cells imaged with 2
min interval (3 experiments); n(KK RNAi)=16 cells (3 experiments); n(KK RNAi
+ CENP-E inh)=5 cells (1 experiment). Scale bar = 5 mm. Time = h:min.
© 2014 Macmillan Publishers Limited. All rights reserved.
00:00 00:10 00:30 00:50 01:10 01:30
00:00 00:10 00:30 00:50 01:10 01:30
Sup. Figure 2 - Barisic et al., 2014
Supplementary Figure 2 Preventing loading of Dynein to kinetochores
stabilizes microtubule attachments and leads to ejection of polar
chromosomes. RNAi resistant GFP-Spindly wild-type (WT) or F258A
mutant were expressed in Spindly depleted U2OS cells stably expressing
mCherry-α-tubulin. Data was then acquired by live-cell imaging. There
were 5.1±2.5 (mean±SD) chromosomes ejected from the spindle in cells
expressing GFP-Spindly (F258A) (n=16 cells), as oposed to none in cells
expressing GFP-Spindly (WT) (n=5 cells). Both conditions were reproduced
in 2 independent experiments. Scale bar = 5 mm, except in inset = 1 mm.
Time = h:min.
© 2014 Macmillan Publishers Limited. All rights reserved.
CENP-E inh. CENP-E inh.
Sup. Figure 3 - Barisic et al., 2014
Supplementary Figure 3 Both CENP-E and Dynein remain on kinetochores
from polar chromosomes after CENP-E inhibition. a) U2OS cells immuno￾stained for DNA (DAPI), kinetochores (ACA), α-tubulin and CENP-E.
Maximum intensity projection images of representative examples are shown.
n(CENP-E inh)= 5 cells (1 experiment). Scale bar = 5 mm. b) U2OS cells
immuno-stained for DNA (DAPI), kinetochores (ACA), α-tubulin and DHC.
Maximum intensity projection images of representative examples in each
indicated condition are shown. n(CENP-E inh)= 5 cells (1 experiment);
n(CENP-E inh + DHC RNAi)= 5 cells (1 experiment; due to limited antibody
supply). Scale bar = 5 mm.
© 2014 Macmillan Publishers Limited. All rights reserved.
Sup. figure 4 - Barisic et al., 2014
Supplementary Figure 4 Original Western blot scans. Full scans of Western blots corresponding to Supplementary Figure 1. White lines represents positions
where the original membrane was cut. Dashed white lines represent the areas used in Supplementary Figure 1.
© 2014 Macmillan Publishers Limited. All rights reserved.
Supplementary Video Legends
Supplementary Video 1 – Congression of only a subgroup of chromosomes depends on Dynein and CENP-E.
Spinning disk confocal time-lapse imaging of chromosome congression in a control U2OS H2B-GFP/mCherry-α-tubulin cell recorded every 10 sec (video on
the left) and compared to a cell treated with 20 nM of CENP-E inhibitor GSK923295 (added prior to imaging and before mitotic entry; video in the middle)
and to a cell transfected with DHC specific siRNA for 48h (video on the right), both recorded every 2 min. Spinning-disk confocal time-lapse imaging was
performed every 10 sec. Time=min:sec.
Supplementary Video 2 – Kinetochore tracking of peripheral chromosomes relying on CENP-E motor activity for congression.
Representative videos of chromosome congression and respective kinetochore tracking in a control U2OS GFP-CENP-A/mCherry-α-tubulin cell (on the left),
compared to a cell treated with 20 nM of CENP-E inhibitor GSK923295 prior to imaging and before mitotic entry (on the right). Spinning-disk confocal time￾lapse imaging was performed every 10 sec. Each tracked kinetochore pair is indicated with a different color. Time=min:sec.
Supplementary Video 3 – Dynein counteracts PEFs to prevent stabilization of end-on kinetochore-microtubule attachments and random ejection of polar
chromosomes ZM 447439.
Representative videos of a U2OS H2B-GFP/mCherry-α-tubulin cell transfected with DHC specific siRNA for 48h and treated with 20 nM of CENP-E
inhibitor GSK923295 prior to imaging and before mitotic entry (on the left), compared to a similarly treated cell, which was additionally transfected with
chromokinesins (Kid+Kif4a) specific siRNAs for 48h (on the right). Spinning-disk confocal time-lapse imaging was performed every 2 min. Time=h:min.
Supplementary Video 4 – Preventing Dynein localization at kinetochores leads to random ejection of polar chromosomes.
Representative videos of a U2OS cell expressing mCherry-α-tubulin and either RNAi-resistant GFP-Spindly wild-type (on the left) or GFP-Spindly F258A
mutant (on the right), transfected with Spindly specific siRNA for 48h and treated with 20 nM of CENP-E inhibitor GSK923295 prior to imaging and before
mitotic entry. Spinning-disk confocal time-lapse imaging was performed every 2 min. Time=h:min.
Supplementary Video 5 – The motion of acentric fragments from polar chromosomes depends on chromokinesins-mediated PEFs and can be directed both
towards the cell equator and the cortex.
Laser microsurgery on chromosome arms of CENP-E inhibited (20 nM GSK923295) U2OS-H2B-GFP/mCherry-α-tubulin cells (on the left and in the middle)
compared to similarly treated cells that were additionally depleted of chromokinesins Kid and Kif4A by 48h RNAi (on the right). Spinning-disk confocal time￾lapse imaging was performed every 1 min. Time=h:min.
© 2014 Macmillan Publishers Limited. All rights reserved.