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Received 5 Dec 2014 | Accepted 10 Jun 2015 | Published 15 Jul 2015 DOI: 10.1038/ncomms8784 OPEN An actin-dependent spindle position checkpoint ensures the asymmetric division in mouse oocytes

Aı¨cha Metchat1, Manuel Eguren1, Julius M. Hossain1, Antonio Z. Politi1,Se´bastien Huet1 & Jan Ellenberg1

Faithful chromosome segregation, during meiosis, is of critical importance to prevent aneuploidy in the resulting embryo. In mammalian oocytes, the segregation of homologous chromosomes takes place with the spindle located at the cell’s periphery. The spindle is often assembled close to the centre of the cell, which necessitates the actin network for spindle transport to the cell cortex. In this study, we investigate how the segregation of chromosomes is coordinated with the positioning of the metaphase I spindle. We develop different assays to perturb the spindle’s position and to delay its relocation to the cell periphery. We find that anaphase is delayed until the spindle is positioned in close proximity with the oocyte cortex. We further show that the metaphase arrest is dependent on a functional actin network, in addition to the spindle assembly checkpoint. Our work provides the first evidence for the existence of a functional spindle position checkpoint.

1 European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany. Correspondence and requests for materials should be addressed to A.M. ([email protected]).

NATURE COMMUNICATIONS | 6:7784 | DOI: 10.1038/ncomms8784 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE RETRACTED NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8784

ccurate chromosome segregation in mammalian oocytes determine whether the spindle kept the same orientation during requires chromosomes to achieve biorientation through its relocation, we defined the spindle axis using the principal Athe attachment of homologous kinetochores to the component of segmented spindle voxels and measured the opposite poles of the meiotic microtubule spindle1–4. Errors in rotational movement of the spindle axis from the onset of segregation of chromosomes during the first meiotic division, migration to its arrival at the cortex (o5 mm; Fig. 1h,i and result in the generation of aneuploid embryos and cause Supplementary Fig. 1). The average rotational movement of pregnancy loss or severe birth defects such as Down’s spindle orientation was 16.44°, indicating that spindle orientation syndrome5–7. Similar to mitosis, the fidelity of chromosome does not change considerably during its relocation to the cortex segregation during oocyte meiosis is ensured by the spindle (Fig. 1i). This finding is supported by previous observations that assembly checkpoint (SAC) which monitors kinetochore– the spindle is pulled to the cortex through an interaction between microtubule attachment2,3,8–17. During meiosis I, all the actin network and the closest spindle pole18–20,28.To chromosomes usually achieve a stable biorientation 2–3 h before determine whether the time of anaphase onset depends on the the onset of anaphase1,4. During this delay, a dynamic actin initial position of the spindle, we analysed the correlation between network spanning throughout the large cytoplasm moves the the initial distance between the spindle surface and the cortex and spindle from the centre toward the cortex of the oocyte18–23. The the time of chromosome segregation. We found that the initial onset of anaphase coincides with the stable anchoring of spindle distance from the cortex correlates with the time delay of the spindle, which allows the oocyte to divide asymmetrically anaphase (r ¼ 0.78; Fig. 1j). (Fig. 1a). The spindle checkpoint protein Mad2 remains evident During meiosis I, anaphase entry is activated by anaphase- at kinetochores in late metaphase I, despite the fact that promoting complex (APC/C) that targets the key meiotic correct attachment of kinetochore fibres to the chromosome is substrates for degradation, including Securin11,12,29–32.To achieved before spindle relocation1,4. These events suggest that understand the link between APC/C-mediated Securin chromosome biorientation may not be the sole mechanism that is degradation and spindle position, we quantitatively measured monitored to prevent precocious chromosome segregation in the Securin degradation during spindle relocation by live-cell oocytes. We hypothesized that SAC senses the spindle position in imaging of oocytes expressing Securin-EGFP and H2B-mCherry. an actin-dependent manner to ensure the asymmetric division Interestingly, Securin degradation started only when the during meiosis I. chromosome was located close to the cortex (distance between Here, we set out different assays to analyse whether the onset of the centre of mass of chromosome and the cortex inferior to chromosome segregation is linked to spindle position in mouse 20 mm, Fig. 1k,l). These data support the hypothesis that oocytes. Using these assays, we demonstrate the existence of a homologous chromosome segregation is coordinated with the mechanism similar to the so-called ‘spindle position checkpoint’ asymmetric spindle position. (SPC) in yeast24, which prevents chromosome segregation until the spindle is positioned in the cortical position. Functional SPC during meiosis I. It has long been proposed that cortical positioning is not required for chromosome segregation Results during meiosis I. Depolymerization of the actin network by Quantitative analysis of spindle relocation. To quantitatively cytochalasin D (CD), which inhibits spindle relocation, does not determine how the spindle is relocated from the centre to the affect the timing of chromosome segregation21,33. We obtained cortex for chromosome segregation, we performed four- similar results using low-dose CD or latrunculin from metaphase dimensional live-cell imaging of oocytes, in which microtubules I onward (Supplementary Figs 2a,b and 3a). Interestingly, were labelled with MAP4-GFP1,25, from the metaphase to actin-filament stabilization by jasplakinolide also had no effect anaphase stages using confocal microscope imaging (at 7-min on the timing of chromosome segregation compared with intervals; Fig. 1b). To analyse our data, we developed an CD/latrunculin treatment, consistent with the findings of other automated three-dimensional (3D) computational pipeline that reports on actin network perturbations21,33–35 (Supplementary detects the spindle on the basis of MAP4-green fluorescent Fig. 3b). To examine how APC/C is activated in CD-treated protein (GFP) signal and uses the cytoplasmic background to oocytes; we recorded the destruction of Securin at late metaphase precisely define the oocyte surface. This allowed us to measure the stage. We found that the dynamics of Securin degradation in absolute distance between the spindle surface and the closest CD-treated oocytes was comparable with the control oocytes cortex, as well as the speed of relocation (Fig. 1c,d; Supplementary (Supplementary Fig. 2c). Fig. 1 and Supplementary Video 1). In addition, we computed the We hypothesized that the dynamic actin network is the key orientation of the spindle in 3D with respect to its initial sensor of spindle position. To test this hypothesis, we first orientation and the oocyte surface. Consistent with previous depolymerized the actin filaments in metaphase I using low-dose studies, the spindle formed in the centre of the oocyte (Fig. 1e). CD, then removed the drug to nucleate the actin network de novo The metaphase I spindle is occasionally formed closer to the immediately before the expected time of chromosome segrega- cortex because of the slight asymmetric position of the germinal tion, that is, B7 h after germinal vesicle breaks down (GVBD; vesicle26. At the onset of chromosome segregation, the shortest termed ‘SPC assay I’; Supplementary Fig. 4a). We observed that in distance between the spindle surface and the cortex was CD-washout conditions, chromosome segregation was always 3.06±1.27 mm (mean value±s.d.). This distance corresponded preceded by the relocation of the spindle to the cortex (20/20 to the enriched actin network between the cortex and the spindle oocytes), with a significant delay in anaphase onset (1 h 34 min) surface27 (Fig. 1e). The meiotic spindle relocated to the cortex compared with control oocytes (Fig. 2a–c; Supplementary through three different phases (phases I, II and III). During phase Fig. 4b). In oocytes treated with CD long term, that is, in the I, the spindle started migrating at a low but progressively complete absence of the actin network, chromosome segregation increasing speed from the centre to a location 9 mm away from its occurred at the same time as in control oocytes. final position. During phase II, at 9 and 5 mm from its final It has been shown in zebrafish oocytes that Cyclin B level is position, the spindle migrated at its maximum speed. Upon dependent on the actin network36. To investigate the actin- arrival at the cortex in phase III, the displacement speed of the depolymerization effect on the endogenous level of Cyclin B, we spindle decreased significantly (Fig. 1f,g). Next, to quantitatively immunostained Cyclin B in CD-treated oocytes immediately after

2 NATURE COMMUNICATIONS | 6:7784 | DOI: 10.1038/ncomms8784 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8784 RETRACTED ARTICLE

a Germinal vesicle Metaphase-I Anaphase-I Metaphase-II stage stage stage stage

~1 h

Spindle formation Spindle migration Chromosome Polar body and segregation extrusion chromosome biorientation b cd MAP4-EGFP 0:00 1:38 2:20 3:51 Distance from Trajectory the cortex e f Phase g I II III Phase I II III 20 0.12 0.12

Initial distance )

12 –1 Final distance 15 –1 m) µ m s µ 8 10 Chromosome 0.06 m min 0.06 segregation µ

Frequency 4 5 Speed in Speed position ( 0 Speed ( 0 Distance from the final 0 0 0 5 10 15 20 25 30 35 0 100 200 300 400 1412 108 6 4 2 0 Cortex Center Distance in µm Time in min Distance from the final position (µm) hijPhase I and II during the relocation 12 6 Pearson r = 0.78 11

4 10 9 2 8 Frequency Time in hours 7 0 6 Orientation 02040 60 80 4 9 14 19 Angle in degree Distance in µm

k Metaphase-I PBE l 1.2

0.8

0.4

Relative level Spindle

of Securin-EGFP migration Securin-GFP H2B-mcherry 0 0 120 240 360 480 Time in min

Cortical position

Figure 1 | Complete 3D spindle movement tracking during meiosis I. (a) Schematic representation of meiosis I in mouse oocytes. (b) Time-lapse imaging of the spindle migration in live oocyte expressing MAP4-GFP (spindle in red). Scale bar, 10 mm. (c,d) Image reconstructed in 3D with Amira software representing segmentation of the spindle (in green) and the cortex (in white). (e) Histogram representing initial and final distance of spindle from the cortex in oocytes. (f) Average of spindle displacements (time alignment by cross correlation). The distance of the spindle surface from the closest cortex is plotted over time. The colour code showed the speed of displacement (in grey, the s.d.). The speed scale of spindle displacement is indicated by the colour bar. (g) The mean of the spindle speed was plotted over distance from the cortex (43 independent experiments, n ¼ 21). (h) Three-dimensional reconstruction with Amira image of oocyte showing the orientation-deviation measurement during spindle relocation. (i) Histogram of angle deviations of spindle orientation during relocation (n ¼ 14). (j) The time of chromosome segregation is plotted over the initial distance of the spindle from the cortex (Pearson coefficient of correlation (r) ¼ 0.78 and Po0.001). (k) Time-lapse imaging of live oocyte expressing Securin-GFP and H2B-mCherry during spindle migration (securin in green, chromosomes in red), scale bar, 10 mm. (l) Average of normalized Securin-GFP fluorescence from metaphase stage to anaphase. The line corresponds to the time of the cortical positioning (in grey, the s.d., two independent experiments, n ¼ 6). Data are mean, with error bars displaying s.d. P-values were calculated with Student’s test (nonparametric test, two tailed).

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a Germinal vesicle Metaphase-I stage stage

Chromosome segregation

CD

CD ? Washout b c

P<0.001

CD 13

0:00:00 0:24:04 0:31:04 1:55:39 2:02:40 12 P=0.8 11

(hours) 10 Control 0:00:00 0:24:04 0:31:04 1:55:39 2:02:40 9 Time of anaphase 8 CD CD Control CD washout washout 0:00:00 0:24:04 0:31:04 1:55:39 2:02:40 h:m:s

Figure 2 | SPC assay I. (a) Two hours after GVBD, maturing oocytes were transferred into a medium containing 0.5 mgmlÀ 1 of CD. At the expected time of chromosome segregation (7 h after GVBD), oocytes expressing H2B-EGFP were washed out from CD and transferred to the microscope for 4D live-cell imaging to record the time of anaphase. (b) Time-lapse imaging of chromosome segregation in control, CD-treated and CD-treated washout oocytes. Scale bar, 10 mm. (c) Time of chromosome segregation (GVBD-anaphase) in CD-treated (three independent experiments, n ¼ 24, Po0.001), CD-treated washout (three independent experiments, n ¼ 20, P ¼ 0.8) and control oocytes (three independent experiments, n ¼ 20). Data are mean, with error bars displaying s.d. P-values were calculated with Student’s test (nonparametric test, two tailed). the drug washout in SPC assay I. Cytoplasmic Cyclin B level in the spindle (termed ‘SPC assay III’; Supplementary Fig. 6a; CD-treated oocytes was similar to control oocytes, showing that Fig. 3a,b and Supplementary Video 2). The computational the delay of anaphase onset was not a consequence of Cyclin B pipeline, described in Supplementary Fig. 1, was used to analyse level perturbation at late metaphase stage (Supplementary spindle displacement. After centrifugation, the spindle was Fig. 4c,d). This result shows that repolymerization of the actin displaced from the centre to a random position in the cytoplasm. network delays chromosome segregation until the spindle reaches During SPC assay III, the spindle first moved to the cortex, an the cortex (Fig. 2b and Supplementary Fig. 4b). event followed by chromosome segregation (42/43 oocytes, To examine whether reassembly of the actin network inhibits Fig. 3c). Movement of the spindle toward the cortex occurred in chromosome segregation, we developed an assay for F-actin three phases (phases I, II and/or III) depending on their initial regrowth before and after spindle migration in synchronized distance from the cortex. When the distance from the cortex was oocytes (designed ‘SPC assay II’). Oocytes were transferred before large (11.05±1.17 mm), movement occurred successively through or after spindle relocation to medium enriched with CD phases I, II and III, or I and II. When the distance between the (Supplementary Figs 2a–5a). In addition, we used the proteasome spindle and its final position was 6.54±2.05 mm, the movement inhibitor MG132 to prevent chromosome segregation during CD of the spindle started directly from phase II followed by phase III. treatment. The timing and location of chromosome segregation In oocytes in which the spindle was located close to the cortex was monitored after drug washout using live-cell imaging. (3.85±1.02 mm), relocation only occurred via phase III (Fig. 3d). Chromosome segregation occurred earlier when the spindle was The maximum speed of migration was higher after MG132/CD already positioned at the cell periphery. For oocytes treated with washout, which explained the shorter duration of spindle CD before spindle relocation, the onset of anaphase only occurred relocation (Figs 1f and 3d). Analysis of spindle orientation when the spindle reached the cortex, causing a significant showed that the spindle maintained its initial orientation during time delay (Supplementary Fig. 5b,c). These results indicate relocation (Fig. 3e). As before, we verified whether the endo- that the repolymerization of actin filaments does not directly genous level of Cyclin B was affected by the centrifugation. affect chromosome segregation. It should be noted that these The level of Cyclin B in centrifuged oocytes was similar to that in assays (SPC assays I and II) were conducted without micro- non-centrifuged oocytes (Supplementary Fig. 6b). These data manipulating the oocytes. Therefore, the time delay observed was collectively show that the initial position and orientation of the not due to artifactual spindle disruption. Taken together, these spindle defines its subsequent speed and direction of motion. data showed that an F-actin-dependent mechanism delays the onset of anaphase until the spindle is correctly positioned near Actin network in SPC assay. To better understand the effect of the cortex. actin filaments on the timing of chromosome segregation and the correct reconstruction of the meshwork in CD-washout oocytes, Spindle motion and orientation in SPC assay. To further study we imaged F-actin in live oocytes using the actin-filament the relationship between spindle displacement and chromosome marker EGFP-Utrophin after CD-washout (Supplementary segregation, we developed an assay on the basis of oocyte cen- Video 3)18–20,37. Reassembly of actin filaments was first trifugation using Maro chamber in order to shift the position of detected on the spindle surface 10 min after drug washout and

4 NATURE COMMUNICATIONS | 6:7784 | DOI: 10.1038/ncomms8784 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8784 RETRACTED ARTICLE

a Germinal vesicle Metaphase-I stage stage Centrifugation

CD/MG132 CD Chromosome segregation MG132 Washout

Spindle shift

b Chromosome Polar body c Metaphase I segregation extrusion

25 Initial distance 20 Final distance 15

0:00 2:08 2:15 3:08 10 EB3mcherry Frequency - 5 0 0 5 10 15 20 25 30 35

H2B GFP Cortex Center Distance from the cortex

Cortical position Central position 0:00 0:48 0:53 1:18 (µm) h:m d Phase I, II, III Phase II and III e or I, II or only II Phase III

20 0.7 Phase I and II 20 15 –1 15 m s µ

m) 0.35 µ 10 10 ( Distance 5 5 Frequency Speed in

from the final position 0 0.0 0 0 0 0 0 10 20 30 40 50 60 70 80 90 100 200 300 400 100 200 300 400 100 200 300 400 Angle in degrees Time in min

Figure 3 | SPC assay III. (a) Schematic representation of the SPC assay III. Two hours after GVBD, the oocytes were transferred into a medium containing 0.5 mgmlÀ 1 of CD. At the expected time of chromosome segregation (7 h after GVBD), oocytes were transferred into medium containing 4 mM MG132 and 0.5 mgmlÀ 1 of CD, centrifuged for 1 h at 1,000 r.p.m. at 37 °C. After the spindle shift, oocytes were immediately removed from CD and MG132 and transferred to the microscope for 4D live-cell imaging (details are shown in Supplementary Fig. 6a). (b) Time-lapse imaging of oocytes expressing EB3-mCherry and H2B-EGFP in the SPC assay III. On the top panel, the spindle was initially localized in the centre and on the bottom panel the spindle was localized close to the cortex. Scale bar, 10 mm. (c) Histogram representing initial (at the drugs washout) and final distance (at anaphase onset) of spindles from the cortex in oocytes after CD/MG132 washout in SPC Assay III (43 independent experiments, n ¼ 43). (d) Quantitative analysis of the spindle displacement in the SPC assay III. (e) Histogram representing the angle deviation of the spindle orientation during the relocation.

proceeded to the spindle’s interior after a further 10 min from the centromeric regions to the chromosome arms (Supplementary Fig. 7a,b). Maximal length of actin filaments (Supplementary Fig. 9). was achieved 20 min after drug washout (Supplementary Fig. 7c). The density of F-actin was higher in CD-washout oocytes than in control oocytes. Whether actin was functional after drug washout SPC requires the SAC activity. The SAC regulates chromosome was investigated by tracking vesicles labelled with Rab11a- segregation by monitoring the kinetics of kinetochore biorienta- mCherry34,38. The maximum speed of vesicle displacement was tion and the correct attachment of kinetochore fibres3,8,39,40. achieved 20 min after CD-washout, indicating that the actin Our data showed that the binding of SAC proteins persists even network is almost instantly reconstituted and functional during after chromosome biorientation during spindle relocation, SPC assay (Supplementary Video 4; Supplementary Fig. 8a,b). consistent with the findings of previous studies1,2,4,40.To Our group and others have shown that the spindle motion investigate whether metaphase arrest in the SPC assay was requires direct interaction between F-actin filaments and the owing to SAC activity, we used Cdc20 with a point mutation, spindle poles. Using high-resolution imaging, we could observe Cdc20R132A, which cannot bind to Mad2 (refs 31,41,42) (Fig. 4a that long actin filaments were present within the spindle. On the and Supplementary Fig. 10). In Cdc20R132A-injected oocytes, basis of these observations, we addressed the question whether chromosome segregation occurred before or during spindle these actin filaments, named spindle actin filaments, connect relocation, resulting in the formation of a large polar-body or directly with chromosomes during the spindle motion. To this symmetric division (Fig. 4b–e). Next, to inhibit the SAC activity end, we imaged F-actin and chromosomes during the spindle from metaphase I onward, we used reversine, a small molecule migration in fixed oocytes using very high-resolution imaging. inhibitor of SAC, during centrifugation in the last step of the SPC We observed several actin filaments surrounding chromosomes assay. Reversine inhibits the kinase Mps1, a known positive

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a Germinal vesicle Metaphase-I stage stage Centrifugation Cdc20R132A

CD CD/MG132 Chromosome segregation MG132 Washout

Spindle shift b Chromosome Polar body Spindle position Metaphase segregation extrusion at anaphase Control

0:10 5:20 5:26 5:47 5:20 EB3mcherry - 3D proejction R132A H2B GFP Cdc20

0:10 1:36 1:42 3:01 1:36 h:m (time after druges washout) c d e

20 Cdc20R132A 10 25 P<0.0001 P=0.0462 P<0.0001 Control 15 20 8 15 6 m) m)

µ 10 µ ( ( 10 4 (hours) 5 5 2 Time of anaphase 0 0 Distance from the cortex

0 Distance from the cortex 0 200 400 600 R132A R132A R132A Time in min Control

Cdc20

Initial distanceFinal control distance control

Initial distanceFinal Cdc20 distance Cdc20 fgh20 Reversine 25 P<0.0001 P<0.0001 P=0.31 6 15 Control 20 m) µ m) 10 µ 15 4 10 cortex ( (hours)

cortex ( 2 5 5 Distance from the Distance from the

0 Time of anaphase 0 0 0 100 200 300 Time in min Control Reversine

Initial distanceFinal control distance control Initial distanceFinal reversine distance reversine Figure 4 | SAC depletion inhibits SPC. (a) Protocol of SAC inhibition by Cdc20R132A microinjection combined with the SPC assay. (b) Time-lapse imaging of Cdc20R132A-injected and control oocytes expressing EB3-mCherry (microtubule) and H2B-EGFP (chromosome) after CD/MG132 washout. Three- dimensional projection of the oocyte (white) and spindle (green) with Amira software from oocytes at the onset of anaphase. (c) Quantitative analysis of the spindle displacement, (d) initial (at the drug washout) and final distances (at anaphase onset) from the cortex (Po0.0001 in control oocytes, P ¼ 0.0462 in Cdc20R132A-injected oocytes) and (e) time of anaphase after the drugs washout in control (n ¼ 15, in black) and Cdc20R132A-injected oocytes (three independent experiments, n ¼ 18, in red, Po0.0001). (f) Dynamic of spindle relocation, (g) initial and final distance from the cortex (Po0.0001 in control oocytes and P ¼ 0.31 in Reversine-treated oocytes) and (h) time of anaphase after the drugs washout (two independent experiments, n ¼ 8, in black) in control and in reversine-treated oocytes (two independent experiments, n ¼ 8, in red; Po0.0001). Scale bar, 10 mm. Data are mean, with error bars displaying s.d. P-values were calculated with Student’s test (nonparametric test, two tailed).

6 NATURE COMMUNICATIONS | 6:7784 | DOI: 10.1038/ncomms8784 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8784 RETRACTED ARTICLE regulator of the SAC upstream of Mad2 during meiosis and In phase IV, chromosomes are stably bioriented and positioned mitosis (Supplementary Fig. 10)2,14,15,43–45. In reversine-treated on the metaphase plate but continue to show low-amplitude oocytes, chromosomes segregated before or at the onset of spindle oscillations. At this stage, Mad2 binds the kinetochore and the relocation, resulting in the formation of a large polar-body or spindle starts migration toward the cortex. We speculate that symmetric division, similar to Cdc20R132A-injected oocytes force-driven spindle movement maintains SAC activity on (Fig. 4f–h; Supplementary Video 5). kinetochores, creating a ‘wait-anaphase’ signal. At the cortical To confirm these results, we used dominant negative Mad2DC position, the spindle stops moving; subsequently, all forces to directly prevent Mad2 activity on kinetochores (Supplementary applied to the spindle pole are equal, and chromosome oscillation Fig. 11). After its recruitment to kinetochores, Mad2DC binds to is interrupted. Next, Mad2 completely dissociates from the the endogenous Mad1–Mad2 core complex, inhibiting Mad2- kinetochores, and APC/C mediates Securin degradation followed Cdc20 binding33. As observed in our previous examinations of by the onset of anaphase. A study, using a different mouse strain, SAC perturbations, chromosome segregation occurred before or reported that chromosome biorientation completes immediately at the onset of spindle relocation, resulting in the formation of a before the onset of anaphase. In the representative illustration of large polar-body or symmetric division, in Mad2DC-injected the experiment shown in this article, chromosomes are already oocytes compared with control oocytes (Supplementary positioned near the cortex during spindle formation and Fig. 12a–c). These results showed that SPC requires Mad2 chromosome biorientation. Future studies should investigate, in activity on kinetochores. mouse strains in which the spindle is often formed near the Previously, we showed that after chromosome biorientation, cortex, whether the delay in chromosome segregation after Mad2 bound 20–40% of kinetochores, which corresponded to the biorientation is systematically suppressed2. relocation of the spindle. To verify whether the delay in anaphase In the last two decades, numerous studies have identified onset is likely due to the enrichment of Mad2 on kinetochores, proteins involved in the regulation of actin network dynamics we imaged non-centrifuged oocytes expressing Mad2-EGFP and spindle relocation. One representative protein is Mos, the and H2B-mCherry after CD/MG132 washout. As expected, mutation of which inhibits mitogen-activated protein kinase Mad2-EGFP marked 10–30% of kinetochores, at the onset of activity and spindle relocation, but does not interfere with the spindle motion. We found that Mad2 is only delocalized from polar-body extrusion22,27,33. A recent study reported that, in kinetochores after cortical positioning, suggesting that the addition to these events, cortex softening is disturbed in mos À / À maintenance of SAC activity may be due to the spindle motion oocytes18,21. In contrast, Formin-2, an actin nucleator and (Supplementary Fig. 12d–f). Actin-filament repolymerization was contributor to asymmetric spindle positioning, plays an verified by imaging Rab11a-mCherry positive vesicle dynamic in essential role in cytoplasmic actin nucleation but does not reversine-treated and Mad2-inhibited oocytes. We found that the regulate cortical actin. Similar to other actin perturbations, the mean speed of vesicle movement was similar in all culture timing of polar-body extrusion is unaffected in Formin-2 and Mos conditions, indicating that the actin network was correctly mutant oocytes. These findings support our results, and suggest repolymerized (Supplementary Fig. 13a–c). All these results that both cortical and cytoplasmic actin are important for spindle reveal that SPC requires the SAC pathway via Mps1, Mad2 and position sensing. Cdc20. Here, we showed that the regulation of chromosome segrega- tion in vertebrate oocytes is more complex than a ‘biological clock’ and the asymmetric spindle positioning combined. For the Discussion first time, we reveal the existence of a SPC, based on the activity Our data showed that the metaphase I spindle moves to the of the SAC, alongside a dynamic actin network. In addition to the cortex in three phases. In phase I, after bipolarization of the mechanical model involving actin and the SAC pathway, other spindle, it moves slowly toward the closest cortex. Next, in phase factors may be involved in the regulation of the SPC and remain II, the speed of displacement increases exponentially until it to be discovered. reaches a maximum, with the spindle in a specific spatial position. This increase in speed is correlated with the enrichment of actin filaments on the spindle pole during relocation. Finally, in phase Methods Culture and micromanipulation of oocytes. Ovaries were collected from III, the spindle finishes its journey by significantly decreasing its 8-week-old FVB or CAGH2BEGFP mice that were maintained according to the speed until it becomes stationary. Subsequently, chromosome guidelines of EMBL laboratory animal resources, 44–48 h after priming with 5 IU segregation occurs. In the SPC assay, we shifted the spindle from pregnant mare serum gonadotropin. Oocytes were isolated by puncturing the its initial position to a random position in the cytoplasm by gentle ovaries with needles in M2 medium supplemented with 100 nM of IBMX (3-isobutyl-1-methylxanthine). To induce resumption of meiosis, oocytes were centrifugation. We observed that, depending on its initial distance incubated in IBMX-free medium. In some experiments, the medium was from the cortex, the spindle started its motion in phases I, II or supplemented with low concentration of CD (0.5 mgmlÀ 1) and MG132 (4 mM) to III. These results suggest that the increase in velocity during prevent spindle relocation and chromosome segregation. To inhibit the activity of relocation depends on the initial distance of the spindle from the Mps1, oocytes were treated with reversine (an inhibitor of Msp1, 1.5 mM). In cortex. The number of F-actin filaments attached to the spindle control, medium was supplemented corresponding amounts of DMSO (dimethyl sulfoxide). Microinjection of in vitro-transcribed RNAs to the oocytes was pole and the number of actin branches connecting the spindle to performed with 5 pl EB3-mCherry, 5 pl mCherry-H2B, 5 pl EGFP-MAP4, 1 pl the cortex define the speed of relocation. This is supported by a Mad2-EGFP, Rab11a-mCherry, Cdc20R132A and Mad2DC of 1–2 mg ml À 1 mRNAs. recent elegant study combining in vitro manipulation and mathematical modelling, showing a link between the spindle position, the F-actin density and the pulling force28. Immunofluorescence. Oocytes were fixed for 30–60 min at 37 °Cin100mM We proposed that the spindle position via the dynamic actin HEPES (pH7; titrated with KOH), 50 mM EGTA (pH7; titrated with KOH), 10 mM MgSO4, 2% formaldehyde (methanol free) and 0.2% Triton X-100. Oocytes were network determines the timing of chromosome segregation. In left in PBS (phosphate-buffered saline) with 0.1% Triton X-100 overnight at 4 °C. our previous study, we showed that chromosome biorientation F-actin staining was carried out in PBS with 0.1% Triton X-100 and 3% bovine occurs in four phases. During the first three phases, chromosomes serum albumin with Alexa-Fluor-488–Phalloidin (Molecular Probes, A12379; 1:100). DNA was stained with 5 mg ml À 1 Hoechst 33342 or DAPI (Molecular and kinetochores move to the surface of the microtubule ball, Probes). As primary antibodies, we used mouse Cyclin B (Abcam, ab2949; 1:100). undergo congression to yield the prometaphase belt, and become As secondary antibodies, we used Alexa-555-labelled goat anti-mouse (Life bioriented with the barrel-shaped bipolar spindle formation1,2. technologies, A21422; 1:500).

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Confocal microscopy. Images were acquired with a customized Zeiss LSM780 References or Meta 510 confocal microscope, using a 40x C-Apochromat 1.2 NA water 1. Kitajima, T. S., Ohsugi, M. & Ellenberg, J. Complete kinetochore tracking immersion objective lens. We recorded spindle and chromosomes dynamics by reveals error-prone homologous chromosome biorientation in mammalian time-lapse (4D) imaging. For 10 cells in parallel, we imaged 25 z-confocal sections oocytes. Cell 146, 568–581 (2011). (every 3 mm) every 7 min for 5–10 h, which covers spindle migration to anaphase 2. Lane, S. I. & Jones, K. T. Non-canonical function of spindle assembly onset of meiosis I. checkpoint proteins after APC activation reduces aneuploidy in mouse oocytes. For spindle tracking with MAP4-EGFP and EB3-mCherry, we imaged 25 Nat. Commun. 5, 3444 (2014). z-confocal sections (every 3 mm) of 512  512 pixel xy images, in total covering at 3. Musacchio, A. Spindle assembly checkpoint: the third decade. Philos. Trans. R. least 70 mm  70 mm  75 mm volume, at time interval of 7 min for 10 h from early Soc. Lond. B. Biol. Sci. 366, 3595–3604 (2011). metaphase stage or after drug washout. 4. Gui, L. & Homer, H. Spindle assembly checkpoint signalling is uncoupled from m For Mad2-EGFP, we imaged 17 z-confocal sections (every 2.0 m) of 512  512 chromosomal position in mouse oocytes. Development 139, 1941–1946 (2012). pixel xy images, in total covering a 37.5 mm  37.5 mm  34.0 mm volume, at time 5. Jones, K. T. & Lane, S. I. Molecular causes of aneuploidy in mammalian eggs. interval of 10 min from 1 to 6 h after drugs washout, covering the spindle Development 140, 3719–3730 (2013). migration. 6. Nagaoka, S. I., Hodges, C. A., Albertini, D. F. & Hunt, P. A. Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors. SPC assay I. Oocytes expressing H2B-EGFP were collected at GV stage. After Curr. Biol. 21, 651–657 (2011). scoring GVBD, oocytes were transferred in M2 medium supplemented with 7. Hunt, P. & Hassold, T. Female meiosis: coming unglued with age. Curr. Biol. 0.5 mg.ml À 1 of CD. At the time of anaphase onset (7 h after GVBD), oocytes were 20, R699–R702 (2010). transferred CD-free medium into the microscope to score the location and the time 8. Rieder, C. L., Cole, R. W., Khodjakov, A. & Sluder, G. The checkpoint delaying of chromosome segregation in non-treated (DMSO), CD-treated and CD-treated anaphase in response to chromosome monoorientation is mediated by an washout oocytes (Supplementary Fig. 4). inhibitory signal produced by unattached kinetochores. J. Cell Biol. 130, 941–948 (1995). SPC assay II. To induce maturation, fully-grown oocytes were incubated in 9. Tsurumi, C., Hoffmann, S., Geley, S., Graeser, R. & Polanski, Z. The spindle IBMX-free medium. After scoring GVBD, synchronized oocytes were transferred assembly checkpoint is not essential for CSF arrest of mouse oocytes. J. Cell in medium supplemented with low dose of CD (0.5 mg.ml À 1) at metaphase I stage Biol. 167, 1037–1050 (2004). before or after spindle relocation. In addition, all oocytes were treated with low 10. Homer, H. A. et al. Mad2 prevents aneuploidy and premature proteolysis of dose of MG132 (4 mM, an inhibitor of the proteasome) to prevent chromosome cyclin B and securin during meiosis I in mouse oocytes. Genes Dev. 19, 202–207 segregation by inhibiting Securin/Cyclin B degradation. MG132 was added at the (2005). late metaphase I to avoid any damage on the spindle steady-state. After drugs 11. Homer, H., Gui, L. & Carroll, J. A spindle assembly checkpoint protein washout, oocytes were immediately transferred into the microscope to score the functions in prophase I arrest and prometaphase progression. Science 326, position and the time of chromosome segregation. The time of anaphase has been 991–994 (2009). calculated between the drug washout and the onset of chromosome segregation 12. McGuinness, B. E. et al. Regulation of APC/C activity in oocytes by a (Supplementary Fig. 5). Bub1-dependent spindle assembly checkpoint. Curr. Biol. 19, 369–380 (2009). 13. Wassmann, K., Liberal, V. & Benezra, R. Mad2 phosphorylation regulates its SPC assay III. Oocytes expressing H2B-EGFP were collected at GV stage, association with Mad1 and the APC/C. EMBO J. 22, 797–806 (2003). microinjected with 5 pl of EB3-mCherry and transferred immediately in IBMX-free 14. Hached, K. et al. Mps1 at kinetochores is essential for female mouse meiosis I. medium. Two hours after GVBD, oocytes were treated with CD (0.5 mgmlÀ 1). Development 138, 2261–2271 (2011). Seven hours after GVBD, oocytes were either treated with MG132 (4 mM) or 15. Kolano, A., Brunet, S., Silk, A. D., Cleveland, D. W. & Verlhac, M.-H. MG132 combined with reversine (1.5 mM). Oocytes were transferred in Maro Error-prone mammalian female meiosis from silencing the spindle assembly chamber positioned in 50 ml conical tube; and centrifuged for 1 h at 1,000 r.p.m. at checkpoint without normal interkinetochore tension. Proc. Natl Acad. Sci. USA 37 °C. After the centrifugation, oocytes were then released into MG132- and 109, E1858–E1867 (2012). cytochalasin-D-free medium and transferred into the microscope for 4D live 16. Hoffmann, S., Maro, B., Kubiak, J. Z. & Polanski, Z. A single bivalent efficiently imaging (Supplementary Fig. 6). inhibits cyclin B1 degradation and polar body extrusion in mouse oocytes indicating robust SAC during female meiosis I. PloS One 6, e27143 (2011). 17. Kubiak, J. Z., Ciemerych, M. A., Hupalowska, A., Sikora-Polaczek, M. & Spindle position and orientation tracking To measure the kinetics of spindle . Polanski, Z. On the transition from the meiotic to mitotic cell cycle during early relocation, we injected oocytes with mRNA encoding EB3-mCherry to label mouse development. Int. J. Dev. Biol. 52, 201–217 (2008). microtubules. We then imaged the entire oocyte volume during spindle relocation using an autofocussing macro (25z sections; spacing: 3 mm, time interval: 18. Schuh, M. & Ellenberg, J. A new model for asymmetric spindle positioning in from 7 min, maximum total duration: 10 h; parameters vary slightly in some mouse oocytes. Curr. Biol. 18, 1986–1992 (2008). experiments). Drifts during image acquisition were corrected with the ‘3D drift 19. Azoury, J. et al. Spindle positioning in mouse oocytes relies on a dynamic correction’ function of Fiji. The cytoplasmic background has been enhanced using meshwork of actin filaments. Curr. Biol. 18, 1514–1519 (2008). filtering (Gaussian blur) and Image Calculator in Fiji (http://rsb.info.nih.gov/ij/). 20. Yi, K. et al. Sequential actin-based pushing forces drive meiosis I chromosome migration and symmetry breaking in oocytes. J. Cell Biol. 200, 567–576 (2013). 21. Dumont, J. et al. Formin-2 is required for spindle migration and for the late Rab11a-mCherry vesicle tracking. Three-dimensional data sets of vesicles were steps of cytokinesis in mouse oocytes. Dev. Biol. 301, 254–265 (2007). acquired using Zeiss’s Multi Time Series macro (13 sections; spacing: 1.5 mm; time 22. Verlhac, M. H., Lefebvre, C., Guillaud, P., Rassinier, P. & Maro, B. Asymmetric B interval: 6.3 s; total duration: 1 min). For peak enhancement of vesicle signals division in mouse oocytes: with or without Mos. Curr. Biol. 10, 1303–1306 and background subtraction, vesicle images processed by a 10-pixel Gaussian blur (2000). were subtracted from the same images processed by a 2-pixel Gaussian blur. Drifts 23. Maro, B., Johnson, M. H., Webb, M. & Flach, G. Mechanism of polar body during image acquisition were corrected with the ‘Correct 3D drift’ function of Fiji formation in the mouse oocyte: an interaction between the chromosomes, the using the transmitted light. Vesicles were tracked using Imaris 3D spot tracking cytoskeleton and the plasma membrane. J. Embryol. Exp. Morphol. 92, 11–32 and the mean speed was calculated over 1 min. Average, s.d., and statistical significance based on Student’s t-test were calculated in SigmaPlot software. (1986). 24. Fraschini, R., Venturetti, M., Chiroli, E. & Piatti, S. The spindle position checkpoint: how to deal with spindle misalignment during asymmetric cell Measurement of the cytoplasmic actin network density and actin-filament division in budding yeast. Biochem. Soc. Trans. 36, 416–420 (2008). length. To measure the average filament length between two nodes, we recorded 25. Schuh, M. & Ellenberg, J. Self-organization of MTOCs replaces centrosome time-lapse imaging of oocytes expressing EGFP-UtrCH (time interval: 1 min) few function during acentrosomal spindle assembly in live mouse oocytes. Cell 130, minutes after the drug washout. The filament length between two nodes or between 484–498 (2007). a node and the end of the filament in the cytoplasm was measured using Fiji. To 26. Almonacid, M. et al. Active diffusion positions the nucleus in mouse oocytes. measure the actin-filament density, the cytoplasmic background has been sub- Nat. Cell Biol. 17, 470–479 (2015). tracted using filtering by Fiji software after the CD washout. 27. Chaigne, A. et al. A soft cortex is essential for asymmetric spindle positioning in mouse oocytes. Nat. Cell Biol. 15, 958–966 (2013). Statistics. Average (mean), s.d. and statistical significance based on Student’s 28. Chaigne, A. et al. A narrow window of cortical tension guides asymmetric t-test (always two tailed) were calculated in Excel and SigmaPlot. The sample size spindle positioning in the mouse oocyte. Nat. Commun. 6, 6027 (2015). was chosen on the basis of Student’s t-test. The end of all boxes plot defines the 29. Herbert, M. et al. Homologue disjunction in mouse oocytes requires proteolysis 25th and 75th percentiles, with a line at the median and error bars defining the of securin and cyclin B1. Nat. Cell Biol. 5, 1023–1025 (2003). 10th and 90th percentiles. The 2D and the 3D plots were generated by the plotting 30. Madgwick, S. et al. Maintenance of sister chromatid attachment in mouse eggs software Python (xy) (http://code.google.com/p/pythonxy/) or SigmaPlot software. through maturation-promoting factor activity. Dev. Biol. 275, 68–81 (2004).

8 NATURE COMMUNICATIONS | 6:7784 | DOI: 10.1038/ncomms8784 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8784 RETRACTED ARTICLE

31. Rattani, A. et al. Dependency of the spindle assembly checkpoint on Cdk1 Acknowledgements renders the anaphase transition irreversible. Curr. Biol. 24, 630–637 (2014). We gratefully acknowledge Kim Nasmyth, Andrea Musacchio and Melina Schuh, D 32. Nabti, I., Marangos, P., Bormann, J., Kudo, N. R. & Carroll, J. Dual-mode respectively, for Cdc20R132A cDNA, Mad2 C, Rab11a-mCherry cDNA, for discussions regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and advices. We would like to thank Marvin Albert, Kota Miura and the members of and fidelity. J. Cell Biol. 204, 891–900 (2014). Ellenberg lab for discussions; the members of EMBL Laboratory Animal Resources, 33. Verlhac, M. H. et al. Mos activates MAP kinase in mouse oocytes through two especially, Yvonne Peterson, Silke Brohn, Angela Burro, Andrea Genthner, Marika opposite pathways. EMBO J. 19, 6065–6074 (2000). Krudwig and Klaus Schmitt for excellent support. This work was supported by Marie 34. Holubcova´, Z., Howard, G. & Schuh, M. Vesicles modulate an actin network for Curie Long-Term Fellowships (FP7; A.M.) and by the German Research Council: asymmetric spindle positioning. Nat. Cell Biol. 15, 937–947 (2013). Deutsche Forschungsgemeinschaft (SPP 1384; J.E.). 35. Pfender, S., Kuznetsov, V., Pleiser, S., Kerkhoff, E. & Schuh, M. Spire-type actin nucleators cooperate with Formin-2 to drive asymmetric oocyte division. Curr. Biol. 21, 955–960 (2011). Author contributions 36. Kotani, T., Yasuda, K., Ota, R. & Yamashita, M. Cyclin B1 mRNA translation is A.M. designed experiments with the help of J.E. A.M. conducted experiments, and temporally controlled through formation and disassembly of RNA granules. analysed data with the help of M.E. and A.Z.P. J.M.H. and S.H. wrote the matlab code for J. Cell Biol. 202, 1041–1055 (2013). the spindle position and orientation tracking. A.M. wrote the manuscript. J.E. conceived 37. Burkel, B. M., von Dassow, G. & Bement, W. M. Versatile fluorescent probes the project. for actin filaments based on the actin-binding domain of utrophin. Cell Motil. Cytoskeleton 64, 822–832 (2007). 38. Schuh, M. An actin-dependent mechanism for long-range vesicle transport. Additional information Nat. Cell Biol. 13, 1431–1436 (2011). Supplementary Information accompanies this paper at http://www.nature.com/ 39. Overlack, K., Krenn, V. & Musacchio, A. When Mad met Bub. EMBO Rep. 15, naturecommunications 326–328 (2014). 40. Uchida, K. S. K. et al. Kinetochore stretching inactivates the spindle assembly Competing financial interests: The authors declare no competing financial checkpoint. J. Cell Biol. 184, 383–390 (2009). interests. 41. Reis, A. et al. Prometaphase APCcdh1 activity prevents non-disjunction in Reprints and permission information is available online at http://npg.nature.com/ mammalian oocytes. Nat. Cell Biol. 9, 1192–1198 (2007). reprintsandpermissions/ 42. Jin, F. et al. Cdc20 is critical for meiosis I and fertility of female mice. PLoS Genet. 6, e1001147 (2010). How to cite this article: Metchat, A. et al. An actin-dependent spindle position 43. Santaguida, S., Tighe, A., D’Alise, A. M., Taylor, S. S. & Musacchio, A. checkpoint ensures the asymmetric division in mouse oocytes. Nat. Commun. 6:7784 Dissecting the role of MPS1 in chromosome biorientation and the spindle doi: 10.1038/ncomms8784 (2015). checkpoint through the small molecule inhibitor reversine. J. Cell Biol. 190, 73–87 (2010). 44. Santaguida, S., Vernieri, C., Villa, F., Ciliberto, A. & Musacchio, A. Evidence This work is licensed under a Creative Commons Attribution 4.0 that Aurora B is implicated in spindle checkpoint signalling independently of International License. The images or other third party material in this error correction. EMBO J. 30, 1508–1519 (2011). article are included in the article’s Creative Commons license, unless indicated otherwise 45. Yamagishi, Y., Yang, C.-H., Tanno, Y. & Watanabe, Y. MPS1/Mph1 in the credit line; if the material is not included under the Creative Commons license, phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC users will need to obtain permission from the license holder to reproduce the material. components. Nat. Cell Biol. 14, 746–752 (2012). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

NATURE COMMUNICATIONS | 6:7784 | DOI: 10.1038/ncomms8784 | www.nature.com/naturecommunications 9 & 2015 Macmillan Publishers Limited. All rights reserved. DOI: 10.1038/ncomms9124 Retraction: An actin-dependent spindle position checkpoint ensures the asymmetric division in mouse oocytes

Aı¨cha Metchat, Manuel Eguren, Julius M. Hossain, Antonio Z. Politi, Se´bastien Huet & Jan Ellenberg

Nature Communications 6:7784 doi:10.1038/ncomms8784 (2015); Published 15 Jul 2015; Updated 29 Jul 2015

This paper has been retracted as it was submitted by the corresponding author A.M. without the knowledge or consent of the other researchers listed on the paper or their institution, utilising incorrect contact details. With the exception of the corresponding author, all other researchers have requested that the paper be retracted.

NATURE COMMUNICATIONS | 6:8124 | DOI: 10.1038/ncomms9124 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved.