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Active diffusion positions the nucleus in mouse oocytes

Abstract

In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function.

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Figure 1: A cytoplasmic actin meshwork nucleated by formin 2 involved in nucleus positioning.
Figure 2: Microtubules and myosin II are not involved in nucleus positioning.
Figure 3: Actin vesicle dynamics.
Figure 4: The cytoplasmic actin meshwork generates global cytoplasmic activity.
Figure 5: Cytoplasmic activity and nucleus movement require myosin Vb.
Figure 6: A model for nucleus positioning relying on a pressure gradient and cytoplasm fluidization.

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Acknowledgements

We thank J-Y. Tinevez (Institut Pasteur, Paris) for sharing the resources for TrackMate and MSD analysis, J. Unruh (Stowers Institute for Medical Research, Kansas City) for sharing the resources for STICS analysis, M. Schuh (MRC Cambridge) for providing the MyoVa and Vb tail plasmids and M-E. Terret for critical reading of the manuscript. M. Almonacid is a recipient of post-doctoral fellowship from the Ligue Nationale contre le Cancer. This work was supported by a grant from the Ligue Nationale Contre le Cancer (EL/2012/LNCC/MHV). This work has received support from the Fondation Bettencourt Schueller, and support under the program ‘Investissements d’Avenir’ launched by the French Government and implemented by the ANR, with the references: ANR-10-LABX-54 MEMO LIFE, ANR-11-IDEX-0001-02 PSL* Research University. W.W.A. is a recipient of post-doctoral fellowships from La Fondation Pierre-Gilles de Gennes and Marie Curie Actions. M.B. is a recipient of an Axa PhD fellowship. T.B. was supported by the French Agence Nationale de la Recherche (ANR-11-JSV5-0002). N.S.G. gratefully acknowledges financial support from the ISF (grant number 580/12).

Author information

Authors and Affiliations

Authors

Contributions

M.A. and M-H.V. conceived and supervised the project. M.A. performed all experiments. M.A. and M-H.V. analysed all experiments. M.A. and W.W.A. performed the optical trapping experiments. M.A., W.W.A., T.B. and M-H.V. analysed optical trapping experiments. M.B. designed the software for cytoplasmic activity analysis. P.M. assisted in image analysis. N.S.G. and R.V. designed the mathematical model. M.A. and M-H.V. wrote the manuscript, which was seen and corrected by all the authors.

Corresponding author

Correspondence to Marie-Hélène Verlhac.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 2 Formin 2-GFP expression as a function of the position of the nucleus 3 h after injection.

The red circle points out the population of oocytes with delayed nuclei. Expression levels are quantified by the integrated fluorescence intensity of Formin 2-GFP over an entire oocyte normalized by the oocyte surface (in μm2). Correlation analysis: correlation coefficient rs = −0.1484, p-value = 0.4256, indicative of the non-correlation of the data. Non-linear regression fitting to a straight line: low correlation coefficient r2 = 0.0007 makes the data incompatible with a linear model. n = 31 oocytes. Data are aggregated from two independent experiments.

Supplementary Figure 3 Microtubules are not involved in nucleus maintenance at the centre.

Nucleus remains central in wt oocytes after Nocodazole and Cytochalasin D treatment. Left: time-lapse movie of a wt oocyte injected with Rango (merged in green) and treated with Nocodazole and Cytochalasin D. Right: mean velocities of nucleus centroid (in μm min−1) as a function of time. n = 13 oocytes. Time is in minute, scale bar is 15 μm and error bars display SEM. Data are aggregated from two independent experiments.

Supplementary Figure 4 Microtubules and Myosin II are not involved in nucleus positioning.

a: Percentage of migrating versus non-migrating nuclei in Fmn2−/− oocytes injected with Formin 2, untreated, on treatment with Blebbistatin or Taxol. n = 23 oocytes for Fmn2−/− +Fmn2;n = 22 oocytes for Blebbistatin, n = 18 oocytes for Taxol. b: Microtubule (MTs) network organization in untreated oocytes (left) and in oocytes treated with Taxol (right). Oocytes express the marker of microtubule (+) ends EB3-GFP. Time-projections from one movie (frame interval: 357 ms, 480 frames, movie duration: 2 min 51 s), single medial plane. Please note the absence of EB3 comets in the oocyte treated with Taxol, indicative of impaired microtubule network dynamics. Untreated: n = 8 oocytes, treated: n = 7 oocytes. c: Nucleus repositioning following Formin 2 injection into Fmn2−/− oocytes on 100 μM Blebbistatin treatment. Left panel: time-lapse movie of an Fmn2−/− oocyte injected with cRNAs coding for Formin 2 and the nuclear probe Rango (merged in green). Right panel: mean velocities of nucleus centroid (in μm min−1) as a function of time. Black arrow points out the end of nucleus movement, where the velocity stabilizes. n = 10 oocytes. Error bars display SEM. d: Nucleus repositioning on Taxol treatment. Left panel: time-lapse movie of an Fmn2−/− oocyte injected with cRNAs coding for Formin 2 and the nuclear probe Rango (merged in green). Right panel: mean velocities of nucleus centroid (in μm min−1) as a function of time. n = 13 oocytes. Error bars display SEM. Data are aggregated from two independent experiments for Fmn2−/− +Fmn2, Blebbistatin and Taxol in a, c and d. Data shown represent one out of one experiment in b.

Supplementary Figure 5 Actin distribution and mechanical properties of the cytoplasm.

a: No differences in actin density are detected across the cytoplasm of Fmn2−/− oocytes injected with Formin 2. Actin densities are represented as the ratio of GFP-UtrCH integrated fluorescence intensities of cytoplasmic regions near the cortex, in the middle and near the nucleus normalized by the region area over integrated fluorescence intensity of the entire oocyte normalized by the oocyte surface. P-value for one-way ANOVA = 0.48. n = 8 oocytes. Error bars display SEM. b: No density gradient of actin vesicles is observed in Fmn2−/− oocytes injected with Formin 2. Actin vesicles density as a function of the distance from the cortex. Densities are represented as a ratio with the highest density observed in the area 10–15 μm away from the cortex (relative density of 1). n = 8 oocytes. c: Direct measurements by optical trapping of mechanical properties in different regions of the cytoplasm in wt oocytes. Elastic (G′) and viscous (G′′) moduli are measured in Pa at 10 Hz. Mean and SD for elastic moduli are 4.6 ± 2.2 near cortex, 4.6 ± 2.3 in the middle, 4.3 ± 1.8 near nucleus. Mean and SD for viscous moduli are 4.9 ± 2.8 near cortex, 4.3 ± 1.7 in the middle, 4.0 ± 1.5 near nucleus. P-values for Kolmogorov–Smirnov test for equality of distributions are 0.83 for cortex-middle, 0.98 for cortex-nucleus, 0.91 for middle-nucleus (elastic moduli); 0.98 for cortex-middle, 0.98 for cortex-nucleus, 0.72 for middle-nucleus (viscous moduli). n = 32 vesicles measured in 11 oocytes Data are aggregated from five independent experiments in a and b and from three independent experiments in c.

Supplementary Table 1 Statistics source data.

Supplementary information

Supplementary Information

Supplementary Information (PDF 297 kb)

Supplementary Video 1

Nucleus repositioning upon Formin 2 injection into Fmn2−/− oocytes. Time-lapse movie of an Fmn2−/− oocyte injected with cRNAs coding for Formin 2 together with the nuclear probe Rango (in green). Frames are taken every 20 min. Movie duration is 360 min. (AVI 639 kb)

Supplementary Video 2

Nucleus stays off-centred in Fmn2−/− oocytes. Time-lapse movie of a Fmn2−/− oocyte injected with cRNA coding for the nuclear probe Rango (green). Frames are taken every 20 min. Movie duration is 360 min. (AVI 621 kb)

Supplementary Video 3

Nucleus keeps its central position in wt oocytes treated with 1 μg ml−1 Cytochalasin D. Time-lapse movie of a wt oocyte injected with cRNA coding for the nuclear probe Rango (green). Frames are taken every 20 min. Movie duration is 300 min. (AVI 504 kb)

Supplementary Video 4

Nucleus repositioning following Formin 2 injection into Fmn2−/− oocytes upon 1 μM Nocodazole treatment. Time-lapse movie of an Fmn2−/− oocyte injected with cRNAs coding for Formin 2 together with the nuclear probe Rango (green). Frames are taken every 20 min. Movie duration is 300 min. (AVI 595 kb)

Supplementary Video 5

Nucleus stays off-centred in Fmn2−/− oocytes injected with Formin 2 upon 2 μM Taxol treatment. Time-lapse movie of a Fmn2−/− oocyte injected with cRNAs coding for Formin 2 together with the nuclear probe Rango (green). Frames are taken every 20 min. Movie duration is 300 min. (AVI 476 kb)

Supplementary Video 6

Nucleus repositioning following Formin 2 injection into Fmn2−/− oocytes upon 30 μM ML-7 treatment. Time-lapse movie of an Fmn2−/− oocyte injected with cRNAs coding for Formin 2 and the nuclear probe Rango (green). Frames are taken every 20 min. Movie duration is 300 min. (AVI 618 kb)

Supplementary Video 7

Nucleus repositioning following Formin 2 injection into Fmn2−/− oocytes upon 100 μM Blebbistatin treatment. Time-lapse movie of an Fmn2−/− oocyte injected with cRNAs coding for Formin 2 and the nuclear probe Rango (green). Frames are taken every 20 min. Movie duration is 300 min. (AVI 453 kb)

Supplementary Video 8

Nucleus repositioning in oocytes expressing Ezrin-mCherry-VCA. Time-lapse movie of an Fmn2−/− oocyte injected with cRNAs coding for Formin 2 and Ezrin-mCherry-VCA. Frames are taken every 1 min. Movie duration is 300 min. (AVI 10220 kb)

Supplementary Video 9

Dynamics of the F-actin cytoplasmic meshwork during nucleus movement in an Fmn2−/− oocyte injected with Formin 2. Time-lapse movie of an Fmn2−/− oocyte injected with cRNAs coding for Formin 2 and the F-actin probe GFP-UtrCH. Frame interval: 557 ms, 480 frames, movie duration: 4 min 27s, single medial plane. This movie is used in Figures 3a and 3c for mapping vesicles directions and tracks, respectively. (AVI 25813 kb)

Supplementary Video 10

Dynamics of the F-actin cytoplasmic meshwork during nucleus movement in an Fmn2−/− oocyte injected with Formin 2. Time-lapse movie of an Fmn2−/− oocyte injected with cRNAs coding for Formin 2 and the F-actin probe GFP-UtrCH. Frame interval: 556 ms, 480 frames, movie duration: 4 min 27s, single medial plane. (AVI 26781 kb)

Supplementary Video 11

Cytoplasmic activity in a wt oocyte. Transmitted light movie. Frames are taken every 1 min. Movie duration is 49 min. This movie is used for STICS analysis in Fig. 4a, top panel and Fig. 5c, left panel. (AVI 1543 kb)

Supplementary Video 12

Cytoplasmic activity in an Fmn2−/− oocyte. Transmitted light movie. Frames are taken every 1 min. Movie duration is 50 min. This movie is used for STICS analysis in Fig. 4a, middle panel. (AVI 1496 kb)

Supplementary Video 13

Cytoplasmic activity in an Fmn2−/− oocyte injected with cRNAs coding for Formin 2. Transmitted light movie. Frames are taken every 1 min. Movie duration is 50 min. This movie is used for STICS analysis in Fig. 4a, bottom panel. (AVI 1336 kb)

Supplementary Video 14

Cytoplasmic activity in a wt oocyte expressing the MyoVb tail. Transmitted light movie. Frames are taken every 1 min. Movie duration is 50 min. This movie is used for STICS analysis in Fig. 5c, right panel. (AVI 1346 kb)

Supplementary Video 15

Nucleus repositioning upon Formin 2 injection into Fmn2−/− oocytes. Time-lapse movie of an Fmn2−/− oocyte injected with cRNAs coding for Formin 2. Frames are taken every 1 min. Movie duration is 349 min. (AVI 8799 kb)

Supplementary Video 16

Arrest of nucleus movement is concomitant with the drop in cytoplasmic activity in oocytes expressing MyoVb tail. Time-lapse movie of an Fmn2−/− oocyte injected with Formin 2 together with MyoVb tail. Frames are taken every 1 min. Movie duration is 349 min. (AVI 27471 kb)

Supplementary Video 17

Cytoplasm of a wt oocyte injected with 0.1 μm latex fluorescent beads, 5 h after injection. Frame interval: 555 ms, 480 frames, movie duration: 4 min 27 s, single medial plane. The first frame of this movie is displayed in Fig. 6c, left panel. (AVI 25252 kb)

Supplementary Video 18

Cytoplasm of a wt oocyte injected with 0.1 μm latex fluorescent beads, 18 h after injection. Frame interval: 556 ms, 480 frames, movie duration: 4 min 27 s, single medial plane. The first frame of this movie is displayed in Fig. 6c, right panel. (AVI 15189 kb)

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Almonacid, M., Ahmed, W., Bussonnier, M. et al. Active diffusion positions the nucleus in mouse oocytes. Nat Cell Biol 17, 470–479 (2015). https://doi.org/10.1038/ncb3131

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