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Centrosomes direct cell polarity independently of microtubule assembly in C. elegans embryos

Abstract

Polarity establishment requires a symmetry-breaking event, resulting in an axis along which determinants are segregated. In Caenorhabditis elegans, oocytes are apolar and are triggered to polarize rapidly along one axis after fertilization. The establishment of this first polarity axis is revealed by the asymmetric distribution of PAR proteins and cortical activity in the one-celled embryo. Current evidence suggests that the centrosome–pronucleus complex contributed by the sperm is involved in defining the polarization axis1,2,3,4,5,6. Here we directly assess the contribution of the centrosome to polarity establishment by laser ablating the centrosome before and during polarization. We find that the centrosome is required to initiate polarity but not to maintain it. Initiation of polarity coincides with the proximity of the centrosome to the cortex and the assembly of pericentriolar material on the immature sperm centrosome. Depletion of microtubules or the microtubule nucleator γ-tubulin did not affect polarity establishment. These results demonstrate that the centrosome provides an initiating signal that polarizes C. elegans embryos and indicate that this signalling event might be independent of the role of the centrosome as a microtubule nucleator.

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Figure 1: The centrosome lies adjacent to the cortex at the time of polarity initiation.
Figure 2: Centrosome ablation prevents polarity establishment but not the propagation and maintenance of the posterior domain.
Figure 3: Polarity initiation requires a concomitant assembly of the pericentriolar material.
Figure 4: Polarity establishment and microtubule assembly are separable centrosomal functions.

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Acknowledgements

We thank: A. Desai, S. Eaton, C. Hoege, K. Oegema, N. Özlü, L. Pelletier, S. Quintin, A. Schlaitz, S. Schonegg, M. Srayko, J. Stear, A. Tudor-Constantinescu and M. van Breugel for comments on the manuscript; S. Grill for advice on laser ablation; A. Pozniakovsky for modified GFP vectors; and G. Seydoux for the gift of JH1380 worms. Some of the worm strains used in this study were obtained from the Caenorhabditis Genetics Center, funded by the NIH.

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Correspondence to Carrie R. Cowan.

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Supplementary information

Supplementary Figure 1

Effect of PCM depletion on contractile polarity establishment. (PDF 2199 kb)

Supplementary Figure 2

Analysis of centrosome-cortex juxtaposition in embryos depleted of g-tubulin, SPD-2, and SPD-5. (PDF 1794 kb)

Supplementary Figure 3

Centrosomal microtubules at the time of polarity initiation in control, spd-2(RNAi), and γ-tubulin(RNAi) embryos. (PDF 1956 kb)

Supplementary Figure 4

PAR-2 and ruffling polarity analysis following microtubule depletion by β-tubulin(RNAi) + 15 μm nocodazole. (PDF 1364 kb)

Supplementary Table 1 (DOC 19 kb)

Supplementary Movie 1

Centrosomes are adjacent to the cortex at the time of polarity initiation. Time lapse movie of a GFP::PAR-2; GFP::SPD-2 embryo prior to and during polarity establishment. The movie plays at roughly 120X actual speed. Embryo posterior is to the right. (MP4 694 kb)

Supplementary Movie 2

Centrosome ablation prevents polarity establishment. The GFP::SPD-2-labelled centrosome (indicated by the red circle) was ablated (red "x"); GFP::PAR-2 and cortical ruffling were used to assess polarity. The movie plays at roughly 120X actual speed. The embryo meiotic pole is to the left. (MP4 911 kb)

Supplementary Movie 3

Centrosome ablation prevents polarity establishment. The GFP::SPD-2-labelled centrosome (indicated by the red circle) was ablated (red "x"); GFP::PAR-2 was monitored to assess polarity. A failure in polar body extrusion is evident following centrosome ablation, although this defect was also observed occaisionally in control ablation embryos. The movie plays at roughly 120X actual speed. The embryo meiotic pole is to the left. (MP4 607 kb)

Supplementary Movie 4

Control ablation does not affect polarity establishment. An area of cytoplasm (indicated by the red circle) in the vicinity of the GFP::SPD-2-labelled centrosome was irradiated (red "x"), as for centrosome ablations. GFP::PAR-2 was used to assess polarity. The centrosomal SPD-2 is photobleached by the ablation but returns several frames later. The movie plays at roughly 120X actual speed. Embryo posterior is to the right. (MP4 547 kb)

Supplementary Movie 5

Centrosome ablation after polarity initiation does not affect the propagation of posterior polarity. The GFP::SPD-2-labelled centrosomes (indicated by the red circle) were ablated (red "x") during the expansion of the posterior domain (boundary indicated by green arrows). GFP::PAR-2 was used to monitor polarity pre- and post-ablation. The movie plays at roughly 120X actual speed. Embryo posterior is to the right. (MP4 664 kb)

Supplementary Movie 6

Centrosome ablation after polarity establishment does not affect the maintenance of posterior polarity. The GFP::SPD-2-labelled centrosomes (indicated by the red circles) were ablated (red "x") after the posterior domain had been formed (PAR-2 occupied half the embryo cortex). GFP::PAR-2 was used to monitor polarity pre- and post-ablation. The movie plays at roughly 120X actual speed. Embryo posterior is to the right. (MP4 724 kb)

Supplementary Movie 7

Cortical control ablation during polarity initiation does not affect the extension of posterior polarity. A small region of the cortex (indicated by the red circle) was ablated (red "x") during the formation of the posterior domain. GFP::PAR-2 was used to monitor polarity pre- and post-ablation; GFP::SPD-2 marks the centrosomes. Cortical damage, confirming the effectiveness of the ablation, can be seen later in the recording (indicated by a green arrow) The movie plays at roughly 120X actual speed. Embryo posterior is to the right. (MP4 735 kb)

Supplementary Figure and Movie Legends (DOC 32 kb)

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Cowan, C., Hyman, A. Centrosomes direct cell polarity independently of microtubule assembly in C. elegans embryos. Nature 431, 92–96 (2004). https://doi.org/10.1038/nature02825

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