ABSTRACT
Centrosomes are major microtubule-organizing centers composed of centrioles surrounded by an extensive proteinacious layer called the pericentriolar material (PCM). In Caenorhabditis elegans embryos, the mitotic PCM expands by Polo-like kinase 1 (PLK-1) phosphorylation-accelerated assembly of SPD-5 molecules into supramolecular scaffolds. However, how PLK-1 phosphorylation regulates SPD-5 assembly is not known. We found that a mutant version of SPD-5 that is insensitive to PLK-1 phosphorylation (SPD-54A) could localize to PCM but was unable to rescue the reduction in PCM size and density when wild-type SPD-5 levels were decreased. In vitro, purified SPD-54A self-assembled into functional supramolecular scaffolds over long time scales, suggesting that phosphorylation only controls the rate of SPD-5 scaffold assembly. Furthermore, the SPD-5 scaffold, once assembled, remained intact and supported microtubule nucleation in the absence of PLK-1 activity in vivo. We conclude that PLK-1 is required for rapid assembly of the PCM scaffold but not for scaffold maintenance or function. Based on this idea, we developed a theoretical model that adequately predicted PCM growth rates in different mutant conditions in vivo. We propose that PLK-1 phosphorylation-dependent conversion of SPD-5 into an assembly-competent form underlies PCM formation in vivo and that the rate of this conversion determines final PCM size and density.
Footnotes
Competing interests
The authors declare no competing or financial interests.
Author contributions
O.W., J.B.W., and D.Z. designed the experiments and wrote the manuscript. O.W. performed the in vivo experiments and quantifications of centrosome size and density as well as protein levels from western blots and stainings. D.Z. developed and tested the model with help from F.J. J.B.W. performed the in vitro experiments and in vivo analysis of centrosome size in the plk-1as embryos. Y.L.W. and K.O. created the plk-1as gfp::spd-5 strain. A.S. immunostained embryos. A.A.H. assisted with experimental design.
Funding
This project was funded by the Max-Planck-Gesellschaft (Max Planck Society) and the European Commission's Seventh Framework Programme [grant number FP7-HEALTH-2009-241548/MitoSys] and a Max Planck Research Network in Synthetic Biology (MaxSynBio) grant to A.A.H. and F.J. J.B.W. was supported by an EMBO fellowship [no. ALTF 759-2012] and MaxSynBio.
Supplementary information
Supplementary information available online at http://bio.biologists.org/lookup/doi/10.1242/bio.020990.supplemental
- Received July 29, 2016.
- Accepted August 25, 2016.
- © 2016. Published by The Company of Biologists Ltd
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