An actin-dependent spindle position checkpoint ensures the asymmetric division in mouse oocytes

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.


Supplementary Figure 2. Effect of actin network depolymerization by cytochalasin D (a)
Cytochalasin D-treated (CD) and control oocytes were fixed at metaphase stage after isolation and stained with Alexa-488-phalloidin to label F-actin (green) and with Hoechst to label chromosomes (red). Oocytes were fixed after 1h and 2h of CD treatment (>3 independent experiments, n=20). Scale bar, 10 m. (b) The time of chromosome segregation was scored from GVBD to anaphase onset in CD (n= 10 CD-treated and n= 10 control oocytes, 2 independent experiments). Data are mean, with error bars displaying s.d. P values were calculated with Student's test (nonparametric test, two tailed). (c) Average of normalized Securin-GFP fluorescence in CD-treated oocytes. The line corresponds to the time of chromosome segregation (n= 11, 2 independent experiments). The chromosome position has been quantified by measuring the distance between the centre of mass of metaphase plate and the cortex. The plot shows the initial (at the drug washout) and final position (at anaphase onset) of chromosome in CD-treated (n=24), CD-treated washout (n=20) and control oocytes (n=20), 3 independent experiments. Data are mean, with error bars displaying s.d. P values were calculated with Student's test. (c) Control oocytes (control, n=32) and CD-treated oocytes (CD, n= 27) were fixed immediately after the drug washout and stained for Cyclin B (red), for F-actin (labelled by Alexa-488-phalloidin) and for DNA (cyan, with Hoechst 33258). Scale bar, 10 m. (d) The mean intensity of Cyclin B in the cytoplasm was measured in oocytes as shown in (c). Aggregation from 3 independent experiments, error bars display s.d. P values were calculated with Student's test (p=0.76; nonparametric test, two tailed).

Supplementary Figure 5. Spindle Position Checkpoint Assay II.
(a) Synchronized oocytes were transferred into a medium containing 0.5 g/ml of CD before or after spindle relocation. MG132 were at the late metaphase stage in order to prevent chromosome segregation during F-actin depolymerization for 2h. Next, oocytes were washout from CD/MG132 and transferred to the microscope for 4D live cells imaging to record the time of anaphase. (b) Time of chromosome segregation after the drug washout in hours. The time 0 correspond to the time of MG132/CD removal. (c) The spindle position was quantified by measuring the distance between the centre of metaphase plate and the cortex. The plot shows the initial (at the drug washout) and final distance (at anaphase onset) of chromosome from the cortex in m in oocytes treated by CD treated before (n=14) or after the spindle relocation (n=11, aggregation from 3 independent experiments). Data are mean, with error bars displaying s.d. P values were calculated with Student's test (nonparametric test, two tailed).

Supplementary Figure 6. The Spindle Position Checkpoint assay III (a)
We designed an assay to mechanically shift the spindle position by gentle centrifugation. This assay, named the SPC assay III included a step of depolymerization of the actin meshwork with CD at metaphase-I. Few minutes before the expected time of anaphase, culture medium was supplemented by MG132. Spindle position was shifted by a gentle centrifugation. We used chambers of Maro to centrifuge oocytes at 37°C at 1000 rpm during 1h. Immediately after centrifugation, the drugs were removed and we recorded the spindle displacement by confocal live imaging. (b) CD/MG132 treated oocytes (CD MG132, n= 13) and CD/MG132 centrifuged (CD MG132 CF, n=15) oocytes were fixed immediately after the drug washout and stained for Cyclin B. The mean intensity of cytoplasmic Cyclin B was measured in CD MG132 and CD MG132 CF oocytes (aggregation from 2 independent experiments, p = 0.103). Data are mean with error bars display s.d. P values were calculated with Student's test (nonparametric test, two tailed).

Supplementary Figure 7. Actin repolymerization in SPC assay. (a)
Oocyte expressing EGFP-UtrCH with F-actin in the spindle, in the left panel (spindle actin) and in the cytoplasm, in the right panel (cytoplasmic actin); from 3 min after CD washout. (b) The relative fluorescence intensity in the cytoplasm was quantified during actin network repolymerization, (c) F-actin length from branches to branches was measured after drug washout in CD-treated and non-treated oocytes. Data were mean from 9 oocytes (aggregation from 2 independent experiments). P values were calculated with Student's test (nonparametric test, two tailed). Scale bar, 5 m.

Supplementary Figure 8. Rab11a-mcherry labelled vesicles movement in SPC assay. (a)
Three-dimensional data sets of CD-treated and CD-treated washout live oocytes expressing Rab11a-mcherry were reconstructed in 3D using Imaris spot detection and tracked over 1 min. The colour bar shows the time scale for the time projection image. Scale bar: 3 m. (b) The mean of the speed was calculated in CD-treated, CD-treated washout and control oocytes (aggregation from 2 independent experiments). Data are mean, with error bars displaying s.d. P values were calculated with Student's test (nonparametric test, two tailed).