In most animals, female meiotic spindles are assembled in the absence of centrosomes. How microtubules (MTs) are organized into acentrosomal meiotic spindles is poorly understood. In Caenorhabditis elegans, assembly of female meiotic spindles requires MEI-1 and MEI-2, which constitute the microtubule-severing AAA+ ATPase Katanin. However, the role of MEI-2 is not known and whether MT severing is required for meiotic spindle assembly is unclear. Here, we show that the essential role of MEI-2 is to confer MT binding to Katanin, which in turn stimulates the ATPase activity of MEI-1, leading to MT severing. To test directly the contribution of MT severing to meiotic spindle assembly, we engineered Katanin variants that retained MT binding and MT bundling activities but that were inactive for MT severing. In vivo analysis of these variants showed disorganized microtubules that lacked focused spindle poles reminiscent of the Katanin loss-of-function phenotype, demonstrating that the MT-severing activity is essential for meiotic spindle assembly in C. elegans. Overall, our results reveal the essential role of MEI-2 and provide the first direct evidence supporting an essential role of MT severing in meiotic spindle assembly in C. elegans.
The authors declare no competing or financial interests.
N.J. and L.P. conceived the study; N.J., J.D. and L.P. designed the experiments; N.J., L.M., E.G. and L.P. performed the experiments. All the authors took part in the interpretation of results and preparation of the manuscript.
We acknowledge the ImagoSeine core facility of the Institut Jacques Monod, member of France-BioImaging (ANR-10-INBS-04) and certified IBiSA. L.M. is supported by a doctoral fellowship from the Ministère de l'Enseignement supérieur et de la Recherche. Work in the laboratory of L.P. is supported by the Agence Nationale de la Recherche [ANR-2012-BSV2-0001-01] and by the Fondation pour la Recherche Médicale [Equipe FRM DEQ20140329538]. Work in the J.D. laboratory is supported by an Emergence grant from the Mairie de Paris. E.G. is supported by an Association pour la Recherche sur le Cancer (ARC) post-doctoral fellowship.
Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.140830.supplemental
- Received June 8, 2016.
- Accepted August 12, 2016.