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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Gillies, T. E. & Cabernard, C. Cell division orientation in animals. Curr. Biol 21, R599–R609 (2011).
Moore, J. K. & Cooper, J. A. Coordinating mitosis with cell polarity: molecular motors at the cell cortex. Semin. Cell. Dev. Biol. 21, 283–289 (2010).
Thery, M. & Bornens, M. Cell shape and cell division. Curr. Opin. Cell. Biol. 18, 648–657 (2006).
Carreno, S. et al. Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells. J. Cell. Biol. 180, 739–746 (2008).
Kunda, P., Pelling, A. E., Liu, T. & Baum, B. Moesin controls cortical rigidity, cell rounding, and spindle morphogenesis during mitosis. Curr. Biol. 18, 91–101 (2008).
Kunda, P. & Baum, B. The actin cytoskeleton in spindle assembly and positioning. Trends Cell. Biol. 19, 174–179 (2009).
Verlhac, M. H., Lefebvre, C., Guillaud, P., Rassinier, P. & Maro, B. Asymmetric division in mouse oocytes: with or without Mos. Curr. Biol. 10, 1303–1306 (2000).
FitzHarris, G., Marangos, P. & Carroll, J. Changes in endoplasmic reticulum structure during mouse oocyte maturation are controlled by the cytoskeleton and cytoplasmic dynein. Dev. Biol. 305, 133–144 (2007).
Halet, G. & Carroll, J. Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes. Dev. Cell. 12, 309–317 (2007).
Brunet, S. & Verlhac, M. H. Positioning to get out of meiosis: the asymmetry of division. Hum. Reprod. Update. 1, 68–75 (2011).
Szollosi, D., Calarco, P. & Donahue, R. P. Absence of centrioles in the first and second meiotic spindles of mouse oocytes. J. Cell. Sci. 11, 521–541 (1972).
Leader, B. et al. Formin-2, polyploidy, hypofertility and positioning of the meiotic spindle in mouse oocytes. Nat. Cell. Biol. 4, 921–928 (2002).
Dumont, J. et al. Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes. Dev. Biol. 301, 254–265 (2007).
Azoury, J. et al. Spindle positioning in mouse oocytes relies on a dynamic meshwork of actin filaments. Curr. Biol. 18, 1514–1519 (2008).
Schuh, M. & Ellenberg, J. A new model for asymmetric spindle positioning in mouse oocytes. Curr. Biol. 18, 1986–1992 (2008).
Li, H., Guo, F., Rubinstein, B. & Li, R. Actin-driven chromosomal motility leads to symmetry breaking in mammalian meiotic oocytes. Nat. Cell. Biol. 10, 1301–1308 (2008).
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).
Larson, S. M. et al. Cortical mechanics and meiosis II completion in mammalian oocytes are mediated by myosin-II and Ezrin-Radixin-Moesin (ERM) proteins. Mol. Biol. Cell 21, 3182–3192 (2010).
Burkel, B. M., von Dassow, G. & Bement, W. M. Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin. Cell. Motil. Cytoskeleton. 64, 822–832 (2007).
Sun, S. C. et al. Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes. PLoS One 6, e18392 (2011).
Yi, K. et al. Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes. Nat. Cell. Biol. 13, 1252–1258 (2011).
Nolen, B. J. et al. Characterization of two classes of small molecule inhibitors of Arp2/3 complex. Nature 460, 1031–1034 (2009).
Verlhac, M. H., de Pennart, H., Maro, B., Cobb, M. H. & Clarke, H. J. MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes. Dev. Biol. 158, 330–340 (1993).
Verlhac, M. H. et al. Mos is required for MAP kinase activation and is involved in microtubule organization during meiotic maturation in the mouse. Development 122, 815–822 (1996).
Verlhac, M. H. et al. Mos activates MAP kinase in mouse oocytes through two opposite pathways. EMBO J. 19, 6065–6074 (2000).
Phillips, K. P. et al. Inhibition of MEK or cdc2 kinase parthenogenetically activates mouse eggs and yields the same phenotypes as Mos(-/-) parthenogenotes. Dev. Biol. 247, 210–223 (2002).
Tong, C. et al. Effects of MEK inhibitor U0126 on meiotic progression in mouse oocytes: microtuble organization, asymmetric division and metaphase II arrest. Cell. Res. 13, 375–383 (2003).
Azoury, J., Lee, K. W., Georget, V., Hikal, P. & Verlhac, M. H. Symmetry breaking in mouse oocytes requires transient F-actin meshwork destabilization. Development 138, 2903–2908 (2011).
Mendoza, M. C. et al. ERK-MAPK drives lamellipodia protrusion by activating the WAVE2 regulatory complex. Mol. Cell. 41, 661–671 (2011).
Nakanishi, O., Suetsugu, S., Yamazaki, D. & Takenawa, T. Effect of WAVE2 phosphorylation on activation of the Arp2/3 complex. J. Biochem. 141, 319–325 (2007).
Sun, S. C. et al. WAVE2 regulates meiotic spindle stability, peripheral positioning and polar body emission in mouse oocytes. Cell. Cycle 10, 1853–1860 (2011).
Fink, J. et al. External forces control mitotic spindle positioning. Nat. Cell. Biol. 13, 771–778 (2011).
Lecuit, T., Lenne, P. F. & Munro, E. Force generation, transmission, and integration during cell and tissue morphogenesis. Annu. Rev. Cell. Dev. Biol. 27, 157–184 (2011).
Nizak, C. et al. Recombinant antibodies against subcellular fractions used to track endogenous Golgi protein dynamics in vivo. Traffic 4, 739–753 (2003).
Klemke, R. L. et al. Regulation of cell motility by mitogen-activated protein kinase. J. Cell. Biol. 137, 481–492 (1997).
Nguyen, D. H. et al. Myosin light chain kinase functions downstream of Ras/ERK to promote migration of uro kinase-type plasminogen activator-stimulated cells in an integrin-selective manner. J. Cell. Biol. 146, 149–64 (1999).
Deng, M., Williams, C. J. & Schultz, R. M. Role of MAP kinase and myosin light chain kinase in chromosome-induced development of mouse egg polarity. Dev. Biol. 278, 358–366 (2005).
Saitoh, M., Ishikiwa, T., Matsushima, S., Naka, M. & Hidaka, H. Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase. J. Biol. Chem. 262, 7796–7801 (1987).
Simerly, C., Nowak, G., de Lanerolle, P. & Schatten, G. Differential expression and functions of cortical myosin IIA and IIB isotypes during meiotic maturation, fertilization, and mitosis in mouse oocytes and embryos. Mol. Biol. Cell. 9, 2509–2525 (1998).
Pasternak, C. & Elson, E. L. Lymphocyte mechanical response triggered by cross-linking surface receptors. J. Cell. Biol. 100, 860–872 (1985).
Pasternak, C., Spudich, J. A. & Elson, E. L. Capping of surface receptors and concomitant cortical tension are generated by conventional myosin. Nature 341, 549–551 (1989).
Dai, J., Ting-Beall, H. P., Hochmuth, R. M., Sheetz, M. P. & Titus, M. A. Myosin I contributes to the generation of resting cortical tension. Biophys. J. 77, 1168–1176 (1999).
Rosenblatt, J., Cramer, L. P., Baum, B. & McGee, K. M. Myosin II-dependent cortical movement is required for centrosome separation and positioning during mitotic spindle assembly. Cell 117, 361–372 (2004).
Sedzinski, J. et al. Polar actomyosin contractility destabilizes the position of the cytokinetic furrow. Nature 476, 462–466 (2011).
Canman, J. C. & Bement, W. M. Microtubules suppress actomyosin-based cortical flow in Xenopus oocytes. J. Cell. Sci. 110, 1907–1917 (1997).
Dumont, J. et al. A centriole- and RanGTP-independent spindle assembly pathway in meiosis I of vertebrate oocytes. J. Cell. Biol. 176, 295–305 (2007).
Breuer, M. et al. HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells. J. Cell. Biol. 191, 1251–1260 (2010).
Mori, M. et al. Intracellular transport by an anchored homogeneously contracting F-actin meshwork. Curr. Biol. 21, 606–611 (2011).
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).
Colledge, W. H., Carlton, M. B., Udy, G. B. & Evans, M. J. Disruption of c-mos causes parthenogenetic development of unfertilized mouse eggs. Nature 370, 65–68 (1994).
Hashimoto, N. et al. Parthenogenetic activation of oocytes in c-mos-deficient mice. Nature. 370, 68–71 (1994).
Guck, J. et al. Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. Biophysical. J. 88, 3689–3698 (2005).
Suresh, S. et al. Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria. Acta. Biomaterialia 1, 15–30 (2005).
Hou, H. et al. Deformability study of breast cancer cells using microfluidics. Biomedical. Microdev. 11, 557–564 (2009).
Verlhac, M. H., Kubiak, J. Z., Clarke, H. J. & Maro, B. Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development 120, 1017–1025 (1994).
Reis, A., Chang, H. Y., Levasseur, M. & Jones, K. T. APCcdh1 activity in mouse oocytes prevents entry into the first meiotic division. Nat. Cell. Biol. 8, 539–540 (2006).
Tsurumi, C., Hoffmann, S., Geley, S., Graeser, R. & Polanski, Z. The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes. J. Cell. Biol. 167, 1037–1050 (2004).
Terret, M. E. et al. DOC1R: a MAP kinase substrate that control microtubule organization of metaphase II mouse oocytes. Development 130, 5169–5177 (2003).
Evans, E. & Yeung, A. Apparent viscosity and cortical tension of blood granulocytes determined by micropipet aspiration. Biophys. J. 56, 151–160 (1989).
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.
The authors declare no competing financial interests.
Integrated supplementary information
(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.
(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 Information (PDF 2109 kb)
Supplementary Information (XLSX 38 kb)
Frames were 1 h apart. The movie starts at NEBD and stops at NEBD+7 h. One Z plane is shown. (AVI 2758 kb)
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)
Note that the plasma membrane does not detach from the cytoplasm, as cytoplasmic particles can be seen flowing into the micropipette. (AVI 4504 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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