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Chromosome (mis)segregation is biased by kinetochore size
Danica Drpic1,2,3, Ana Almeida1,2, Paulo Aguiar2,4, Helder Maiato1,2,5*
1Chromosome Instability & Dynamics Laboratory, Instituto de Biologia Molecular e Celular,
Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal;
2i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen
208, 4200-135 Porto, Portugal
3Graduate Program in Areas of Basic and Applied Biology (GABBA), Instituto de Ciências
Biomédicas Abel Salazar da Universidade do Porto, Rua de Jorge Viterbo Ferreira nº228, 4050-
313 Porto, Portugal
4INEB – Instituto Nacional de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen
208, 4200-135 Porto, Portugal
5Cell Division Group, Experimental Biology Unit, Department of Biomedicine, Faculdade de
Medicina, Universidade do Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal
*Correspondence to: [email protected]
Keywords: kinetochore; mitosis; robertsonian fusion; aneuploidy; meiotic drive; centromere;
CENP-A; congression; merotelic attachments; indian muntjac.
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Summary:
Aneuploidy, the gain or loss of chromosomes, arises through problems in chromosome
segregation during mitosis or meiosis and has been implicated in cancer and developmental
abnormalities in humans [1]. Possible routes to aneuploidy include a compromised spindle
assembly checkpoint (SAC), cohesion defects, centrosome amplification, as well as improper
kinetochore-microtubule attachments [2]. However, none of these established routes takes into
account the intrinsic features of the kinetochore - the critical chromosomal interface with spindle
microtubules. Kinetochore size and respective microtubule binding capacity varies between
different animal and plant species [3-10], among different chromosomes from the same species
(including humans) [3, 11-15], and in response to microtubule attachments throughout mitosis
[16-18]. How kinetochore size impacts chromosome segregation remains unknown. Addressing
this fundamental question in human cells is virtually impossible, because most of the 23 pairs of
chromosomes cannot be morphologically distinguished, while detection of 2-3 fold differences in
kinetochore size is limited by diffraction and cannot be resolved by conventional light
microscopy in living cells. Here we used the unique cytological attributes of female Indian
muntjac, the mammal with the lowest known chromosome number (n=3), to track individual
chromosomes with distinct kinetochore sizes throughout mitosis. We found that chromosomes
with larger kinetochores bi-orient more easily and are biased to congress to the equator in a
motor-independent manner. However, they are also more prone to establish erroneous merotelic
attachments and lag behind during anaphase. Thus, we uncovered an intrinsic kinetochore feature
- size - as an important determinant of chromosome segregation fidelity.
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was notthis version posted March 7, 2018. ; https://doi.org/10.1101/278572doi: bioRxiv preprint
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Results and Discussion:
Kinetochore size is generally determined by the length of α-satellite DNA, the presence of a
CENP-B-box, and the proportional incorporation of CENP-A at centromeres [19-22]. Additional
size changes are due to an expandable module formed by CENP-C and outer kinetochore
proteins involved in SAC signaling, as well as motor proteins, such as CENP-E and cytoplasmic
dynein [12, 17, 23]. In humans, the length of α-satellite DNA arrays at centromeres ranges from
200 kb on the Y chromosome, to >5 Mb on chromosome 18 [24], leading to an 3 fold
variability in the amount of centromeric CENP-A and respective kinetochore size among
different chromosomes [11-14, 19-21]. To investigate the relevance of kinetochore size for
chromosome congression and segregation during mitosis we took advantage from the unique
cytological features of the Indian muntjac (IM), a small deer whose females have the lowest
known chromosome number (n=3) in mammals [25]. As the result of tandem fusions during
evolution, IM chromosomes are large and morphologically distinct (one metacentric and two
acrocentric pairs). Specifically relevant for our purposes, one of the acrocentric chromosomes
resulting from the fusion between chromosomes 3 and X contains an unusually large compound
kinetochore that can bind more than 100 microtubules [5, 25, 26] (Fig. 1A). Like in humans,
kinetochore size among the different IM chromosomes scaled with the amount of CENP-A and
Ndc80/Hec1 and varied 2-3 fold (Fig. 1B,C). To track chromosomes and kinetochores in living
cells we established a culture of hTERT-immortalized primary fibroblasts from a female IM [27]
stably expressing histone H2B-GFP or CENP-A-GFP, respectively. Spindle microtubules were
visualized with 20 nM SiR-tubulin [28], which did not interfere with normal mitotic progression
in this system (Figs. 1D, E and 2A, movies S1 and S2).
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In mammals, the initial contacts between mitotic spindle microtubules and kinetochores
take place during prometaphase of mitosis after nuclear envelope breakdown (NEB). Scattered
chromosomes then align at the spindle equator by a process known as chromosome congression
[29]. When chromosomes are favorably positioned between the spindle poles they establish end-
on kinetochore-microtubule attachments and congress after bi-orientation. In contrast, more
peripheral chromosomes are laterally transported along spindle microtubules by the plus-end-
directed kinetochore motor CENP-E (kinesin-7) [30, 31]. To directly test the implications of
kinetochore size for chromosome congression we treated IM fibroblasts for 1 h with the CENP-E
inhibitor GSK923295 [32] and followed their entry and progression throughout mitosis (Fig. 2B
and movie S3). We found three different scenarios: 1) very few cells whose centrosomes did not
fully separate before NEB showed all chromosomes at the poles (2/28 cells, 6 independent
experiments), in line with our previous observations in human cells [31]; 2) some cells aligned
all their chromosomes at the metaphase plate soon after NEB (6/28 cells, 6 independent
experiments); and 3) most cells showed at least one chromosome, either with a small or large
kinetochore, which remained at the poles (20/28 cells, 6 independent experiments). These data
indicate that any chromosome is competent to use either the CENP-E-dependent or -independent
pathway to congress, regardless of kinetochore size.
Next, we determined the number of chromosomes with small or large kinetochores at the
pole after CENP-E inhibition by immunofluorescence in fixed cells containing only 6
chromosomes (Fig. 3A). Based on these data we constructed a joint probability table involving
two defined random variables representing the number of chromosomes with small and large
kinetochores found at the pole after CENP-E inhibition (table S1). We concluded that: 1) the
number of chromosomes with small and large kinetochores at the pole could be described by a
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binomial distribution (Fig. 3B), meaning that the fate of each individual chromosome was
independent of the other chromosomes of the same class (i.e. with small or large kinetochores);
2) the state of the chromosomes with small kinetochores did not influence the state of the
chromosomes with large kinetochores and vice-versa; and 3) the probability of each individual
chromosome with small kinetochores to stay at the pole was approximately twice the probability
of a chromosome with a large kinetochore (regardless of the number of chromosomes with small
or large kinetochores): 0.19 ± 0.034 vs. 0.11 ± 0.048, respectively (mean ± s.d., n=621 cells, 6
independent experiments, p=0.0067, t test; Fig. 3C).
In human cells, >96% of the chromosomes relying on CENP-E for congression are
normally excluded from the spindle region and locate near one of the spindle poles at NEB [31].
To rule out that the observed bias for IM chromosomes with large kinetochores to align
independently of CENP-E was due to a tendency to localize between the two poles at NEB, we
performed four-dimensional (4D, x,y,z,t) tracking of chromosomes with large kinetochores after
CENP-E inhibition in living cells (n = 23 large kinetochore pairs, 13 cells). We found that 22/23
IM chromosomes with large kinetochores were excluded from the spindle region and were nearly
randomly positioned along the spindle axis at NEB (45% of the kinetochores were closer to the
poles vs. 55% of the kinetochores that were closer to the spindle equator; Fig. 3D and movie S4).
Overall, these data indicate that chromosomes with a larger kinetochore rely less on CENP-E
motor activity and are biased to congress after bi-orientation, despite being randomly located at
the spindle periphery at NEB.
Recent computational modelling predicted that changes in kinetochore size in response to
microtubule attachments affect both error formation and the efficiency of spindle assembly in
human cells [18]. To directly test this prediction and investigate whether chromosomes with
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large kinetochores are more prone to establish erroneous attachments with spindle microtubules
we set up a monastrol treatment/washout assay [33] in IM fibroblasts to inhibit kinesin-5 and
spindle bipolarity, thereby preventing the formation of amphitelic attachments (Fig. 4A, B and
movie S5). After monastrol washout, cells were released into fresh medium with or without the
Aurora B inhibitor ZM447439 (and the proteasome inhibitor MG132 to prevent anaphase onset),
to partially inhibit error correction without interfering with bipolar spindle assembly (fig. S1A-
C), and subsequently fixed. For each cell treated with ZM447439 and MG132, we calculated the
fraction of each chromosome group (with small or large kinetochores) with merotelic
(microtubules from both poles attached to the same kinetochore) or syntelic (microtubules from
the same pole attached to both sister kinetochores) attachments (Fig. 4C). We found an
equivalent low frequency of syntelic attachments for chromosomes with small or large
kinetochores (1.9% vs. 1.4%, respectively; n=207 cells, pool of 3 independent experiments).
However, the frequency of chromosomes with large kinetochores that established erroneous
merotelic attachments was several fold higher when compared with chromosomes with small
kinetochores (7.0% vs. 1.6%, respectively). These results could be explained either by
differences in the formation or in the correction of erroneous merotelic attachments that could
scale with kinetochore size. To distinguish between these possibilities, we measured Aurora B
activity in chromosomes with different kinetochore sizes by quantifying the levels of
phosphorylated Knl1, a bona fide Aurora B substrate at the kinetochores [34]. We were unable to
detect any significant difference in Aurora B activity between chromosomes with small or large
kinetochores (n=76 small KTs, n=63 large KTs, 50 cells, Mann- Whitney test) (Fig. 4D). We
concluded that chromosomes with larger kinetochores are more prone to establish erroneous
merotelic attachments.
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The formation of erroneous merotelic attachments in the early stages of mitosis normally
results in lagging chromosomes during anaphase and are a main cause of aneuploidy in mammals
[35, 36]. Thus, one would predict that chromosomes with large kinetochores tend to lag more in
anaphase when compared with chromosomes with small kinetochores. To test this prediction,
and because IM fibroblasts in anaphase showed a very low natural frequency of lagging
chromosomes (1.5%; n=267 anaphase cells, pool of 3 independent experiments), we promoted
error formation with monastrol as before, and released IM fibroblasts into fresh medium with or
without ZM447439 (Fig. 4A, E, F and movie S5). Immunofluorescence analysis in fixed cells
after monastrol treatment and washout revealed a significant increase in anaphase cells with
lagging chromosomes (5.5%; n=2603 anaphase cells, pool of 9 independent experiments), which
was even more pronounced in the presence of ZM447439 (7.7%, n=1323 anaphase cells, pool of
6 independent experiments) (Fig. 4F). Importantly, we found that 73% of the cells after
monastrol treatment and washout showed at least one lagging chromosome with a large
kinetochore, whereas 30% of the cells showed at least one lagging chromosome with small
kinetochores (n=32 cells) (Fig. 4G). Stimulated Emission Depletion (STED) super-resolution
microscopy confirmed that the large kinetochores on lagging chromosomes were often found
deformed and resulted from merotelic attachments (Fig. 4E). Given that chromosomes with large
kinetochores only represent one third of all chromosomes, our measurements indicate a strong
bias for chromosomes with large kinetochores to lag behind in anaphase. Because chromosome
3+X in female IM is smaller than chromosome 1 (which has a smaller kinetochore), but larger
than chromosome 2 (also with a smaller kinetochore), this works as an internal control to exclude
that the measured bias for chromosome 3+X to lag behind in IM was due to a decrease in
chromosome size [37]. These data directly demonstrate that chromosomes with large
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kinetochores are more prone to establish erroneous merotelic attachments that result in lagging
chromosomes during anaphase.
Taken together, our results show how an intrinsic kinetochore feature - size - impacts
chromosome congression, bi-orientation and error-formation during mitosis. Because
chromosomes with large kinetochores bi-orient and congress more efficiently (fig. S2), our work
explains why certain species with holocentric kinetochores align their chromosomes in the
absence of a CENP-E orthologue [9]. On the other hand, because chromosomes with large
kinetochores also establish more errors (fig. S2), one prediction from our work is that
chromosomes that use the CENP-E pathway for congression should be less prone to
missegregate, explaining why the CENP-E pathway might have emerged during evolution. This
would work as a selective pressure to maintain chromosomes with large kinetochores during
evolution, suggesting that the errors resulting from incorrect merotelic attachments are unlikely
to be propagated. Indeed, our live-cell analysis showed that lagging chromosomes with large
kinetochores are normally resolved during anaphase (Fig. 4A and movie S5), consistent with
previous findings in other systems [36, 38]. Additionally, any potential loss/gain of an autosome
in IM (representing a copy of 1/3 of the genome) might seriously compromise cell viability, in
agreement with previous work with male IM fibroblasts reporting that chromosome
missegregation and aneuploidy was essentially limited to the smallest Y2 chromosome [39]. In
humans, the Y chromosome, which has the smallest centromere with significantly less CENP-A
compared with any other chromosome [19, 21] also missegregates at elevated frequencies [22],
and its loss is the most common somatic alteration in men recently associated with shorter
survival and higher risk of cancer [40]. At the other extreme, CENP-A domain expansion and
overexpression leading to alterations in kinetochore size, structure and function, have been
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linked with chromosome missegregation and genomic instability in human cancer cell models
[20, 41, 42]. Thus, chromosome segregation fidelity might be ensured by a species-specific
optimal kinetochore size.
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Acknowledgments:
We would like to thank António Pereira for assistance with STED microscopy. This work was
supported by CODECHECK grant from the European Research Council, FLAD Life Science
2020, and the Louis-Jeantet Young Investigator Career Award. D.D. established the Indian
muntjac system, performed all the experiments and analyzed the data. A.A. performed the SiR-
tubulin titration in the H2B-GFP line. P.A. constructed the joint probability table, performed
frequency analysis and 4D tracking of kinetochores. H.M. conceived the project, designed
experiments and analyzed the data. D.D. and H.M. wrote the paper. The authors declare no
conflict of interests.
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Figure legends:
Fig. 1. Chromosome and kinetochore size variability in Indian muntjac cells. (A) Normal
karyotype of female Indian muntjac (IM) fibroblasts. Scale bar = 5 μm. (B) Linear centromere
length of IM chromosomes, measured from chromosome spreads (box-whisker plots, left) and
respective quantification of CENP-A (middle) and Ndc80/Hec1 (right) levels at IM kinetochores,
relative to chromosome 3+X. (C) Immunofluorescence of IM chromosome spreads (blue) to
detect CENP-A or Ndc80/Hec1 (green). Scale bars = 5 μm. (D) Mitotic progression from nuclear
envelope breakdown (NEB) until anaphase onset (ANA) in IM fibroblasts is unperturbed by
treatment with 20 nM SiR-tubulin. (E) Live-cell imaging of IM fibroblasts stably expressing
H2B-GFP (green) and treated with 20 nM SiR-tubulin dye (magenta). Scale bar = 5 μm. Time =
h:min.
Fig. 2. Any chromosome can use either the CENP-E-dependent or -independent pathway to
congress, regardless of kinetochore size. (A) Control IM fibroblasts stably expressing CENP-
A-GFP (green) and treated with 20 nM SiR-tubulin (magenta). Scale bar = 5 μm. Time = h:min.
(B) IM fibroblasts stably expressing CENP-A-GFP (green) and treated with 20 nM SiR-tubulin
(magenta) after CENP-E inhibition with 20 nM GSK923295. Green arrowheads indicate
chromosomes with large kinetochores. Scale bars = 5 μm. Time = h:min.
Fig. 3. Chromosomes with a larger kinetochore rely less on CENP-E and are biased to
congress after bi-orientation. (A) Immunofluorescence of an IM fibroblast after CENP-E
inhibition showing chromosomes (DAPI, white in merged image), kinetochores (ACA, white;
green in merged image) and microtubules (α-tubulin, magenta in merged image). Scale bar = 5
μm. (B) Quantification of the number of chromosomes with small or large kinetochores (KTs) at
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14
the pole after CENP-E inhibition by immunofluorescence in fixed cells with 6 chromosomes
only (magenta and green lines) and respective theoretical prediction based on a binomial
distribution (grey bars). (C) Probability of each individual chromosome with small or large
kinetochores (KTs) to stay at the pole upon CENP-E inhibition. (D) Location of chromosomes
with large kinetochores (KTs) after CENP-E inhibition obtained from 4D (x, y, z, t) tracking, to
determine their position relative to the poles at NEB and the forming mitotic spindle (see dashed
box in A for reference). Scale bar = 5 μm.
Fig. 4. Chromosomes with large kinetochores are more prone to establish erroneous
merotelic attachments that result in lagging chromosomes during anaphase. (A) High
spatial and temporal resolution analysis of error correction after monastrol washout in live Indian
muntjac fibroblasts stably expressing CENP-A-GFP (green) and treated with 20 nM SiR-tubulin
(magenta). Dashed box highlights a region with a chromosome with a large kinetochore pair
(indicated by green arrows in the lower panel). Lower panels show corresponding 1.5x zoom
images, plus additional time frames. Note the changes in the conformation of the large
kinetochore pair, which lags behind during anaphase, but eventually segregates to the correct
daughter. Scale bar = 5 μm. Time = min:sec. (B) STED/Confocal image of an IM fibroblast in
prometaphase after monastrol washout and immunofluorescence to reveal microtubules (α-
tubulin, magenta), chromosomes (DAPI, white) and kinetochores (ACA, green). Dashed arrows
indicate one chromosome near each pole with syntelic attachments. (C) Quantification of
erroneous syntelic and merotelic attachments on chromosomes with small or large kinetochores
(KTs) in IM fibroblasts after monastrol washout into Aurora B inhibitor and MG-132. (D)
Quantification of Aurora B activity at kinetochores of IM as inferred by fluorescence intensity
quantification of pKNL1 relative to anti-centromere antibodies (ACA) and normalized for the
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-
15
kinetochore area. There are no statistically significant differences. (E) STED/Confocal image of
an IM fibroblast in anaphase after monastrol washout and immunofluorescence to reveal
microtubules (α-tubulin, magenta), chromosomes (DAPI, white) and kinetochores (ACA, green).
A merotelic attachment on a lagging chromatid containing a large kinetochore is indicated
(dashed box and arrow). Scale bar = 5 μm. The image on the right shows the corresponding 4x
zoom. (F) Frequencies of anaphase cells with lagging chromosomes in control IM fibroblasts,
monastrol washout into MEM medium and monastrol washout into MEM medium with Aurora
B inhibitor. (G) Frequencies of anaphase cells with at least 1 lagging chromosome with small or
large kinetochores (KTs) after monastrol washout, respectively.
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16
Materials and Methods
Cell culture:
Indian muntjac (IM) female fibroblasts were immortalized with human hTERT and were a kind
gift from Jerry W Shay, (University of Texas Southwestern Medical Center, Dallas, Texas) (31).
IM fibroblasts were grown in Minimum Essential Media (MEM) (Gibco, Life Technologies),
supplemented with 20%FBS (Gibco, Life Technologies), 2mM L-Glutamine (Invitrogen) at
37°C in humidified conditions with 5% CO2. To collect IM fibroblasts we used Trypsin (Gibco,
Life Technologies). Because IM fibroblasts tend to become polyploid in culture, for the purpose
of our studies we used only diploid cells, which was controlled by counting the number of
kinetochores (2n=6).
Cell transfection:
IM fibroblasts were transfected either with human H2B-GFP (from Geoff Wahl lab, Addgene
plasmid # 11680) or pSV-IRESneo3-CENP-A-EGFP (kind gift from Patrick Meraldi, University
of Geneva) plasmids using Lipofectamine 2000 (Invitrogen) to generate stable cell lines. For this
purpose, at day 1 cells were seeded in 6-well plates at 60-70% confluence in MEM containing
20% FBS. The day after, cells were washed 3x with PBS and incubated with Optimem medium
(Gibco, Life Technologies) containing Lipofectamine 2000 (Invitrogen) and the respective DNA
for 4h. Optimem with DNA and Lipofectamine 2000 were previously mixed and incubated for
20 min before adding to the cells. After 4 hours Optimem medium was exchanged to MEM
supplemented with 20% FBS and cells were selected with G418 (Merck Millipore) after 48h.
Imunofluorescence:
IM fibroblasts were seeded on poly-L-lysine-coated coverslips 2 days before the experiment.
After fixation with ice-cold methanol (Invitrogen) or 4% paraformaldehyde (Electron
Microscopy Sciences), washed with PBS-0.05%Tween 20 (Sigma-Aldrich) or cytoskeleton
buffer pH 6.1 (274 mM NaCl, 10mM KCl, 2.2 mM Na2HPO4, 0.8mM KH2PO4, 4mM EGTA,
4mM MgCl2, 10mM Pipes, 10mM Glucose). Extraction after paraformaldehyde fixation was
performed using PBS-0.1%Triton (Sigma-Aldrich). Coverslips were incubated with primary
antibodies in blocking solution (10% FBS diluted in PBS-0.05%Tween 20 (Sigma-Aldrich) or in
cytoskeleton buffer pH 6.1) for 1h. The following primary antibodies were used: human anti-
centromere (CREST) 1:2000 (Fitzgerald), mouse anti--tubulin (1:2000; B-512 clone, Sigma-
Aldrich), rabbit anti-pKNL1 (1:1000) (32) (kind gift from Iain Cheeseman, Whitehead Institute,
MIT, Cambridge, MA, USA). Subsequently, cells were washed 3x with PBS-0.05% Tween or
cytoskeleton buffer and incubated 45 min with the corresponding secondary antibodies Alexa-
488, 568 and 647 (Invitrogen) or Abberior STAR 580 and Abberior STAR 635p (Abberior
Instruments) for STED microscopy. For STED microscopy, both primary and secondary
antibodies were used at 1:100 concentrations. After adding 1 μg/ml 4´6´-Diamidino-2-
phenylindole (DAPI) (Sigma Aldrich) for 5 min, coverslips were washed in PBS and sealed on
glass slides using mounting medium (20 mM Tris pH8, 0.5 N-propyl gallate, 90% glycerol).
Fixed image analysis and acquisition:
Image acquisition (0.22 m thin Z stacks) was performed on a Zeiss AxioObserver Z1 wide-field
microscope equipped with a plan-apochromat (1.46 NA 60x) DIC objective and a cooled CCD
(Hamamatsu Orca R2). We used Autoquant X (Media Cybernetics) for image blind
deconvolution and Fiji (ImageJ) for attachment quantifications and KT tracking. Adobe
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Photoshop CS4 and Adobe Illustrator (Adobe Systems) were used for histogram adjustments and
image preparation for publication. Merotelic attachments were defined by the position of the
kinetochores (bent or parallel to the spindle axis) and the respective microtubules (defined
through Z stacks). pKNL1 levels were analyzed using ROI manager in Fiji (ImageJ), normalized
for the area of each kinetochore. The pKNL1 levels were corrected for the background and
normalized to CREST levels (also corrected for the background).
STED super-resolution microscopy:
For STED imaging we used a pulsed gated-STED microscope (Abberior Instruments) with
excitation wavelengths at 561 nm and 640 nm doughnut-depleted with a single laser at 775 nm.
All acquisitions were performed using a 1.4 NA oil-immersion and a pixel size set to 35 nm.
Chromosome spreads:
IM fibroblasts were incubated with 3.3 μM of nocodazole (Sigma-Aldrich) for 6-7 hours, then
trypsinized and centrifuged for 5 min at 1200 rpm. The pellet was resuspended in 500 μL of the
supernatant, and a hypotonic solution (medium:water 1:1 and 3.3 μM nocodazole) was added
drop by drop until the final volume of 5 mL. The mixture was incubated at 37ºC for 20 min.
After centrifugation the supernatant was discarded and the cells were fixed with Carnoy
(methanol (AppliChem Panreac): acetic acid (Milipore Corporation) - 3:1) solution and kept
overnight at -20ºC. The following day, the Carnoy fixation was repeated and then the cells were
spread drop-by-drop onto the slide. DNA was counterstained with 1 μg/ml DAPI (Sigma-
Aldrich) for 10 min and the preparations were mounted on slides using Mounting Media (20 mM
Tris pH8, 0.5 N-propyl gallate, 90% glycerol). For chromosome spreads with antibody staining
IM fibroblasts were incubated with 3.3 μM nocodazole for 6-7 hours, then trypsinized and
centrifuged for 5 min at 1200 rpm. The pellet was resuspended in 500 μL of the hypotonic
solution containing sodium citrate (Sigma-Aldrich) and FBS (Sigma-Aldrich) and incubated for
30 min at 37ºC. Cells were then placed on glass slides using a cytospin 4 centrifuge (Thermo
Scientific). Glass slides containing chromosome spreads were fixed with 4% paraformaldehyde
and immunofluorescence was performed as indicated.
Live-cell imaging:
IM fibroblasts stably expressing human CENP-A-GFP or H2B-GFP were plated on fibronectin
(Sigma-Aldrich) coated 35 mm glass-bottom dishes (14 mm, No 1.5, MatTek Corporation) 2
days before imaging. Before live-cell imaging, cells were cultured in Leibovitz´s-L15 medium
(Gibco, Life Technologies). For tubulin staining, we used 20 nM SiR-tubulin cell-permeable dye
(33) (Spirochrome) and incubated cells for 6-12 h. Live-cell imaging was performed on a
temperature-controlled Nikon TE2000 microscope equipped at the camera port with a modified
Yokogawa CSU-X1 spinning-disc head (Solamere Technology), an FW-1000 filter-wheel (ASI)
and an iXon+ DU-897 EM-CCD (Andor). The excitation optics is composed of two sapphire
lasers at 488 nm and 647 nm (Coherent), which are shuttered by an acousto-optic tunable filter
(Gooche&Housego, model R64040-150) and injected into the Yokogawa head via a polarization-
maintaining single-mode optical fiber (OZ optics). Sample position is controlled by a motorized
SCAN-IM stage (Marzhauser) and a 541.ZSL piezo (Physik Instrumente). The objective was an
oil-immersion 60x 1.4 NA Plan-Apo DIC CFI (Nikon, VC series), yielding an overall (including
the pinhole-imaging lens) 190 nm/pixel sampling. A 1.5x tube lens (optivar) was also used (126
nm/pixel sampling). Eleven 1 μm separated z-stacks were acquired every 2 min while recording
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IM fibroblasts stably expressing H2B-GFP. For kinetochore tracking we used IM fibroblasts
stably expressing CENP-A-GFP, recorded at 30 sec or 60 sec interval and 0.75 μm separated z-
stack. The system was controlled by NIS-Elements via a DAC board (National Instruments, PCI-
6733).
Error formation assay:
IM fibroblasts were seeded on poly-L-lysine-coated (Sigma-Aldrich) coverslips 2 days before
the experiment. Initially, cells were incubated for 12h with 100 μM monastrol (Tocris
bioscience) and after that washed out into MEM medium or MEM containing 1μM Aurora B
inhibitor (ZM447439, Selleckchem.com) or MEM containing 1μM Aurora B inhibitor and 20
μM MG-132, a proteasome inhibitor (Calbiochem), based on previous reports (34). When
indicated, cells were fixed with ice-cold methanol (Invitrogen) for 4 min at -20 °C or 4%
paraformaldehyde (Electron Microscopy Sciences) for immunofluorescence analysis.
CENP-E inhibition:
To inhibit CENP-E, IM fibroblasts were treated with 20 nM GSK923295 (35)
(Selleckchem.com) 1h before fixation or live-cell imaging.
Frequency analysis and joint probability tables:
Custom-made scripts were developed in MATLAB 8.1 (The MathWorks Inc.) to perform the
frequency analysis for the number of chromosomes with small and large kinetochores staying at
the pole upon CENP-E inhibition. Joint probability tables were calculated for six independent
sets of mitotic cells, each with at least 100 cells. The tables were used to calculate the marginal
and the conditional probabilities of the number of chromosomes with small/large kinetochores
found at the pole. The random variables considered for the joint probability table were ‘S’, for
the number of chromosomes with small kinetochores that stay at the pole, and ‘L’, for the
number of chromosomes with large kinetochores that stay at the pole. Binomial distributions
were fitted to both random variables, using information about the number of independent
components (2 for large kinetochores, and 4 for small kinetochores) and the respective
experimental values. All data are represented as the mean ± s.d.
Kinetochore tracking:
Live-cell imaging of IM fibroblasts stably expressing CENP-A-GFP was performed as indicated,
every 60 sec, and analyzed after CENP-E inhibition using TrackMate Tool in Fiji (Image J).
Initial kinetochore and pole positions at nuclear envelope breakdown were manually tracked in
four dimensions (x,y,z,t) using Manual Tracking Tool. Further analyses and plotting were
performed using MATLAB to assess the initial position of the chromosomes with large
kinetochores relative to the spindle and spindle poles/equator. Data from different cells was
pooled together by applying geometric affine transformations (without shear) to generate overlap
for the poles location. Initial positions of the chromosomes with large kinetochores were plotted
on a standardized geometrical representation of the mitotic spindle.
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Fig. S1. Titration of the Aurora B inhibitor in IM fibroblasts. Immunofluorescence images of
IM fibroblasts with or without the Aurora B inhibitor ZM447439 at different concentrations to
evaluate the impact on spindle formation and Aurora B activity. In the merged images,
kinetochores are shown with ACA (green), Aurora B activity is revealed by pH3(S10) (white),
microtubules by α-tubulin (red), and chromosomes with DAPI (blue). Scale bars = 5 μm.
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20
Fig. S2. Proposed model for how kinetochore size impacts chromosome congression, bi-
orientation and error-formation based on the findings in Indian muntjac. Chromosomes
with larger kinetochores would tend to congress and bi-orient independently of CENP-E motor
activity. However, they are also more prone to establish erroneous merotelic attachments that
would lead to the formation of lagging chromosomes during anaphase and potential
missegregation.
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Table S1. Conditional probability table for the random variables representing the number
of chromosomes with small and large kinetochores found at the pole. This table summarizes
the experimental data obtained in the CENP-E inhibition experiments. Shaded values correspond
to the marginal distributions.
# small KTs
0 1 2 3 4
# la
rge
KT
s
0 0.18 0.44 0.11 0.02 0.00 0.76
1 0.10 0.08 0.04 0.00 0.00 0.22
2 0.00 0.01 0.00 0.00 0.00 0.02
0.29 0.53 0.15 0.02 0.00 1.00
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Movie legends:
Movie S1. Mitosis in Indian muntjac fibroblasts. Live-cell imaging of an hTERT-
immortalized IM fibroblast stably expressing H2B-GFP to visualize the chromosomes (green)
and treated with 20 nM SiR-tubulin dye to label spindle microtubules (magenta). Time = h:min.
Movie S2. Tracking individual chromosomes with distinct kinetochore size in Indian
muntjac fibroblasts. Live-cell imaging of a control hTERT-immortalized IM fibroblast stably
expressing CENP-A-GFP to visualize the kinetochores (green) and treated with 20 nM SiR-
tubulin dye to label spindle microtubules (magenta). Time = h:min.
Movie S3. Any chromosome can use either the CENP-E-dependent or -independent
pathway to congress, regardless of kinetochore size. Live-cell imaging of hTERT-
immortalized IM fibroblasts stably expressing CENP-A-GFP to visualize the kinetochores
(green) and treated with 20 nM SiR-tubulin dye to label spindle microtubules (magenta), after
CENP-E inhibition with 20 nM GSK923295. Time = h:min.
Movie S4. Quantitative mapping of the positions of chromosomes with large kinetochores
relative to the spindle pole, the metaphase plate and the forming spindle (normalized as an
‘ellipsoid’) obtained by four-dimensional (x, y, z, t) tracking of CENP-E-inhibited cells. Note that most kinetochore pairs (all green dots) occupy a position at the spindle periphery, but
their distribution relative to the pole/equator (red dot) is random. One exceptional kinetochore
pair that fell inside the spindle region is also indicated (black dot).
Movie S5. High spatial and temporal resolution analysis of error correction in Indian
muntjac fibroblasts. Monastrol treatment and washout in a live hTERT-immortalized Indian
muntjac fibroblast stably expressing CENP-A-GFP to visualize the kinetochores (green) and
treated with 20 nM SiR-tubulin to label spindle microtubules (magenta). Note the difference
between chromosomes with a small or large kinetochore pair. One chromosome with a large
kinetochore pair can be seen to change conformation and lag behind during anaphase for a short
time, but eventually resolves and segregates to the correct daughter. Time = min:sec.
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H2B-GFPSiR-tubulin
00:00 00:18 00:31 00:55 01:18
Figure 1 - Drpic et al.,
Chr1 Chr2 Chr3+X0
1
2
3
4
cent
rom
ere
leng
th (µ
m)
0.0
0.5
1.0
Nor
mal
ized
Ndc
80/H
ec1
leve
ls a
t kin
etoc
hore
s
Nor
mal
ized
CEN
P-A
leve
ls a
t kin
etoc
hore
s
0.0
0.5
1.0
Chr1 Chr2 Chr3+X Chr1 Chr2 Chr3+X
H2B-GFP+ SiR-tub
H2B-GFP20
40
60
80
NEB
-AN
A (m
in)
A Chr3+X
Chr2Chr1
Chr1
Chr3+X
Chr2
B
D
E
CCENP-ADAPI
Ndc80/Hec1DAPI n.s.
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-
00:00 00:06 00:31 00:45 00:49
SiR-tubulinCENP-A-GFP
CEN
P-E
inhi
bitio
n (2
0 nM
GSK
9232
95)
00:00 00:05
00:00
00:00 00:04
00:05
00:08 00:17 00:25
00:50 01:14 01:18
00:25 00:40 00:50
A
B
Con
trol
Figure 2 - Drpic et al.,
All at the poles(2/28)
None at the poles(6/28)
Few at the poles(20/28)
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-
ACA α-tubulinDAPI
A
Prob
abilit
y of
eac
h in
divi
dual
chro
mos
ome
to s
tay
at th
e po
le
Small KTs Large KTs0.00
0.05
0.10
0.15
0.20
0.25 p=0.0067
Binomial (bars) vs. Data (lines)
CEN
P-E
inhi
bitio
n
20 nM GSK923295
ACA
B
C
Prob
abilit
y di
strib
utio
n
0 1 2 3 4 0 1 2
0.80.6
0.0 0.0#Small KTs at poles #Large KTs at poles
0.3 0.4
D
Figure 3 - Drpic et al.,
Normalized half-spindle length
Half-
spind
le wi
dth
Hal
f-spi
ndle
wid
th
PoleEqua
tor
Large KT in
Large KT out(closer to pole)
Large KT out(closer to equator)
Spindle ellipsoid
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-
CENP-A-GFPSiR-tubulin
00:00 03:00 08:30 15:00 17:0000:00 03:00 8:30 15:00 17:0002:00 04:30 13:30 15:30 16:30
AM
onas
trol w
asho
ut
0
2
4
6
8
10
ControlMonastrol washout
ZM447439%
of a
naph
ase
cells
with
lagg
ing
chro
mos
omes
Syntelic Merotelic Syntelic Merotelic% o
f chr
omos
omes
with
erro
neou
s at
tach
men
ts
0
2
4
6
8
0.0
0.5
1.0
Fluo
resc
ence
pKN
L1/ A
CA
(A.U
.)
Small KTs Large KTs
B Cα-tubulin (STED)DAPIACA
α-tubulin (STED)DAPIACA
D
E
G
Small KTs Large KTs
n.s.
F
MEM
0
20
40
60
80
Small KTs Large KTs
% o
f ana
phas
e ce
llsw
ith >
=1 la
ggin
g ch
rom
osom
e(M
onas
trol w
asho
ut)
Figure 4 - Drpic et al.,
ZOOM 4X
Mon
astro
l was
hout
Mon
astro
l was
hout
DAPI
Syntelic
Syntelic
Merotelic
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