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Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex

2015; Elsevier BV; Volume: 25; Issue: 13 Linguagem: Inglês

10.1016/j.cub.2015.05.033

1879-0445

Valeria Palumbo, Claudia Pellacani, Kate J. Heesom, Kacper B. Rogala, Charlotte M. Deane, Violaine Mottier-Pavie, Maurizio Gatti, Silvia Bonaccorsi, James G. Wakefield,

Photosynthetic Processes and Mechanisms

Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers [1Weisenberg R.C. Microtubule formation in vitro in solutions containing low calcium concentrations.Science. 1972; 177: 1104-1105Crossref PubMed Scopus (879) Google Scholar, 2Walczak C.E. Heald R. Mechanisms of mitotic spindle assembly and function.Int. Rev. Cytol. 2008; 265: 111-158Crossref PubMed Scopus (284) Google Scholar]. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions [3Hartl F.U. Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded protein.Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2782) Google Scholar, 4Westermann S. Weber K. Post-translational modifications regulate microtubule function.Nat. Rev. Mol. Cell Biol. 2003; 4: 938-947Crossref PubMed Scopus (570) Google Scholar]. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies [5Mottier-Pavie V. Cenci G. Vernì F. Gatti M. Bonaccorsi S. Phenotypic analysis of misato function reveals roles of noncentrosomal microtubules in Drosophila spindle formation.J. Cell Sci. 2011; 124: 706-717Crossref PubMed Scopus (16) Google Scholar], but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the Tubulin chaperone complex, TCP-1/CCT, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.