A soft cortex is essential for asymmetric spindle positioning in mouse oocytes

Abstract

At mitosis onset, cortical tension increases and cells round up, ensuring correct spindle morphogenesis and orientation. Thus, cortical tension sets up the geometric requirements of cell division. On the contrary, cortical tension decreases during meiotic divisions in mouse oocytes, a puzzling observation because oocytes are round cells, stable in shape, that actively position their spindles. We investigated the pathway leading to reduction in cortical tension and its significance for spindle positioning. We document a previously uncharacterized Arp2/3-dependent thickening of the cortical F-actin essential for first meiotic spindle migration to the cortex. Using micropipette aspiration, we show that cortical tension decreases during meiosis I, resulting from myosin-II exclusion from the cortex, and that cortical F-actin thickening promotes cortical plasticity. These events soften and relax the cortex. They are triggered by the Mos–MAPK pathway and coordinated temporally. Artificial cortex stiffening and theoretical modelling demonstrate that a soft cortex is essential for meiotic spindle positioning.

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Figure 1: Cortical F-actin thickening appears during meiosis.
Figure 2: Cortical and cytoplasmic F-actin define two distinct compartments.
Figure 3: The Arp2/3 complex nucleates the subcortex.
Figure 4: Mos–MAPK triggers nucleation of the Arp2/3-dependent subcortex.
Figure 5: The cortex softens in meiosis I owing to myosin-II exclusion from the cortex that decreases cortical tension and to the the subcortex that promotes cortical plasticity.
Figure 6: Mos–MAPK triggers myosin-II exclusion from the cortex and nucleation of the subcortex resulting in cortex softening, required for spindle migration in meiosis I.

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Acknowledgements

We thank T. Pollard (Yale University, USA) for providing the CK666 and F. Perez (Curie Institute, France) for providing SF9-expressing plasmids. We thank J. Teillon for helping set up the photoactivation experiments. We thank C. Klein for his help with the analysis of the photoactivation data. We also thank A. Roux, M. Piel, A. Echard, A. Gautreau, E. Derivery and R. Li for helpful discussions. This work was supported by grants from the Ligue Nationale Contre le Cancer (EL2009-EL2012/LNCC/MHV) and from the Agence Nationale pour la Recherche (ANR-08-BLAN-0136-01 to MHV and ANR-08-BLAN-0012-12 to CS). A. Chaigne is a recipient of a fellowship from the Ecole Normale Supérieure (ENS) Paris. C. Campillo acknowledges financial support from the Association pour la Recherche contre le Cancer (ARC). N. S. Gov wishes to thank the Mayent-Rothschild Foundation for the Visiting Professor grant at the Curie Institute.

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Contributions

A.C., M-H.V. and M-E.T. designed the experiments, interpreted the results and wrote the manuscript. A.C., C.U-D. and M.E.T. carried out most of the experiments. J.A. performed the preliminary observations that started the project. M.A. assisted in the photoactivation experiments. I.Q. assisted in genotyping the animals. A.C. and C.C. carried out the micropipette experiments. C.C., P.N. and C.S. designed the micropipette experiments and interpreted the results. N.S.G. and R.V. designed the physical model. M.E.T. and M.H.V. conceived and supervised the project.

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Correspondence to Marie-Hélène Verlhac or Marie-Emilie Terret.

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Integrated supplementary information

Supplementary Figure 1 Spatial analysis of PA-GFP dynamics in mouse oocytes.

(a) Profiles of PA-GFP fluorescence decay after photoactivation over time in the subcortex and cytoplasm of NEBD+6 h oocytes. Mean of n = 6 wt oocytes photoactivated in the subcortex and n = 5 wt oocytes photoactivated in the cytoplasm, assessed over 2 independent experiments. Standard errors are plotted on each curve. See methods section for photoactivation analysis. The curves are are not statistically different using a t test for each timepoint. (b) Profiles of PA-GFP fluorescence decay in regions adjacent to the photoactivated subcortical region of NEBD+6 h oocytes over time. Mean of n = 6 wt oocytes, assessed over 2 independent experiments. Standard errors are plotted on each curve. Statistical differences were assessed for each timepoint using a t test. All curves are not statistically different from each other. (c) Profiles of PA-GFP fluorescence decay in regions adjacent to the photoactivated cytoplasmic region of NEBD+6 h oocytes over time. Mean of n = 5 wt oocytes, assessed over 2 independent experiments. Standard errors are plotted on each curve. Statistical differences were assessed for each timepoint using a t test. All curves are not statistically different from each other.

Supplementary Figure 2 U0126 mimics mos-/- phenotype on the cortical actin thickening and Myosin-II cortical localization.

(a) Bar graph showing measures of the subcortex in μ at NEBD+3 h and NEBD+7h in wt, mos-/- or U0126 treated oocytes expressing GFP-UtrCH. Mean of n = 28 wt oocytes, n = 32 mos-/- oocytes and n = 34 U0126 treated oocytes, assessed over 8 independent experiments. Standard deviation is plotted on each bar. The statistical significance of differences was assessed with a t test with Welch correction, p value <0.0001. (b) Bar graph showing the ratio between the average intensities of cortical and cytoplasmic Myosin-II at different stages in wt, mos-/- or U0126 treated oocytes expressing SF9-GFP. Mean of n = 16 wt oocytes, n = 18 mos-/- oocytes and n = 17 U0126 treated oocytes, assessed over 3 independent experiments. For each oocyte, 6 measurements were taken in the cortex and in the cytoplasm. Standard error is plotted on each bar. The statistical significance of differences presented in Supplementary Table S1 was assessed with a t test with Welch correction.

Supplementary Figure 3 The cortical F-actin thickening is essential for spindle migration in meiosis I.

(a) Bar graph showing the percentage of symmetric, asymmetric and absence of division in DMSO and CK666 treated oocytes. Mean of n = 155 DMSO treated oocytes and n = 134 CK666 treated oocytes, assessed over 4 independent experiments. Standard deviation is plotted on each bar. The statistical significance of differences was assessed with a t test, p values = 0.0007 for symmetric division and 0.0041 for asymmetric division. (b) Time-lapse spinning disk confocal microscopy of Histone-RFP expressing oocytes followed during meiotic maturation. The upper panel corresponds to a DMSO treated oocyte while the lower panel shows a CK666 treated one. The red dotted circle highlights the initial position of chromosomes. A projection of 3 Z planes is shown. Times from NEBD are indicated in hours. Scale bar, 10 μm.

Supplementary Figure 4 ConA effects on meiosis I division, actin meshworks organization and Myosin-II localization.

(a) Graph representing the kinetics of division in untreated, SuccConA treated and ConA treated oocytes. For all kinetics, the percentage of division is calculated only from the population of oocytes that actually undergo a division. (b) Bar graph showing the percentage of oocytes undergoing blebbing and polar body extrusion (PB1) in untreated, SuccConA treated and ConA treated conditions at NEBD+7 h. Mean of n = 74 untreated oocytes, n = 90 SuccConA treated oocytes and n = 87 ConA treated oocytes, assessed over 4 independent experiments. Standard deviation is plotted on each bar. The statistical significance of differences was assessed with a t test with welch correction, p values = 0.0951 for untreated versus SuccConA and 0.0009 for SuccConA versus ConA. (c) Time-lapse spinning disk confocal microscopy of untreated, SuccConA treated and ConA treated oocytes observed every 20 min starting from NEBD+7 h until NEBD+9 h. The red asterisks mark the first polar bodies. Scale bar, 10 μm. (d) Confocal spinning disk images of NEBD+7 h oocytes expressing GFP-UtrCH treated or not with ConA. The left panels correspond to unsaturated images, the right panels to saturated images. One Z plane is shown. On the right are magnified regions of these oocytes outlined by the orange squares. Due to culture conditions on agarose coated glass slides, the stainings appear blurry. Scale bar, 10 μm. (e) Time-lapse spinning disk confocal microscopy of NEBD+7 h oocytes expressing a Myosin-II intrabody (black) treated or not with ConA. One Z plane is shown. Scale bar, 10 μm.

Supplementary Figure 5 Theoretical model of spindle motion.

(a) The spindle moves in one dimension along its long axis, which we call axis (0,x), 0 being the center of the oocyte and x the position of the center of gravity of the spindle at a given time. L represents the distance between the closest spindle pole and the cortex before the spindle starts migrating. The origin (x = 0) is at the cell center. The spindle is shown with a finite size, while in the model only its center is described, not its spatial extent. (b) Velocity of the spindle along its long axis as a function of cortex stiffness (k). From top to bottom k = 0,01; 0,023; 0,034; 0,65; 10 nN μm (using L = 17 μm, = 0,05 nN, l0 = 6 μm). The velocity is plotted normalized by the value of the maximal velocity vmax, defined when the spindle reaches the cortex for k = kL = 0,023 nN/μm. The curves for k = 0,65 nN/μm and 10 nN/μm are indistinguishable.

Supplementary Figure 6 Spindle positioning in mouse oocytes is the result of an imbalance of forces favored by a soft cortex.

A schematic showing the relationship between Mos expression, cortical Myosin-II enrichment, cortical thickening, cortical mechanics and spindle positioning during meiotic maturation in wt and mos-/- oocytes. Chromosomes are shown in red, microtubules in green and actin in violet.

Supplementary Figure 7 Full scans of the blots from Fig. 4e-f.

Supplementary Table 1 Statistical analysis of Fig. 5e and Supplementary Fig. S2b. NS: not statistically different.

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Supplementary Table 1

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Time-lapse movie showing a WT oocyte expressing GFP–UtrCH (grey) and histone–RFP (red).

Frames were 1 h apart. The movie starts at NEBD and stops at NEBD+7 h. One Z plane is shown. (AVI 2758 kb)

Time-lapse movie showing a WT oocyte expressing GFP–UtrCH.

Frames were 5 s apart. The movie starts at NEBD+7 h and lasts 5 min. One Z plane is shown. (AVI 202 kb)

Pseudocolored time-lapse movies showing WT NEBD+6 h oocytes expressing PA–GFP–UtrCH (upper cells) and PA–GFP (lower cells) photoactivated in the subcortex (cells on the left) or in the cytoplasm (cells on the right).

Black, lowest intensity; white, highest intensity. Frames were 100 milliseconds apart. The movies start at NEBD+6 h and last 25 seconds. One Z plane is shown. (AVI 4595 kb)

13-second movie showing micropipette aspiration of a WT NEBD+1h mouse oocyte.

Note that the plasma membrane does not detach from the cytoplasm, as cytoplasmic particles can be seen flowing into the micropipette. (AVI 4504 kb)

31-second movies showing the relaxation after suction of a NEBD+6 h mos-/- oocyte (left cell, elastic behaviour) and a NEBD+6 h WT oocyte (right cell, plastic behaviour). (AVI 9449 kb)

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Chaigne, A., Campillo, C., Gov, N. et al. A soft cortex is essential for asymmetric spindle positioning in mouse oocytes. Nat Cell Biol 15, 958–966 (2013). https://doi.org/10.1038/ncb2799

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