Their functional relevance, however, remains largely unexplored

quent phosphorylation of at least one key substrate for bipolar spindle assembly, the microtubule depolymerizing kinesin MCAK. Thus, the two Xenopus Sgo proteins have non-overlapping functions in chromosome segregation. Our results further suggest that this functional specificity could rely on the association of XSgo1 and XSgo2 with different MedChemExpress ATL-962 regulatory subunits of the PP2A complex. The EMBO Journal 31, 14671479. doi:10.1038/ emboj.2012.4; Published online 24 January 2012 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19828691 Subject Categories: cell & tissue architecture; cell cycle Keywords: Aurora B; chromosome segregation; cohesion; mitosis; phosphatase Corresponding author. Molecular Oncology Programme, Spanish National Cancer Research Centre, Melchor Fernandez Almagro 3, Madrid 28029, Spain. Tel.: 34 917328000 ext 3470; Fax: 34 917328033; E-mail: [email protected] 5 Present address: Molecular and Cellular Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA Received: 13 July 2011; accepted: 22 December 2011; published online: 24 January 2012; corrected: 21 March 2012 & 2012 European Molecular Biology Organization XSgo2 regulates the spindle assembly pathway mediated by CPC T Rivera et al promoting Aurora B activation, but not its localization. Thus, we show for the first time that an Sgo protein can modulate CPC activity. We also provide evidence that suggests that association of XSgo1 and XSgo2 with different regulatory subunits of the PP2A holoenzyme could mediate their specific functions in the chromosome segregation process. Results Identification of Xenopus Sgo2 A BLAST search for Sgo2 orthologues in X. laevis identified a cDNA encoding a polypeptide of 127 amino acids with significant homology to the C-terminal region of Sgo proteins. By means of the RACE technique, we obtained a 3-kb long full-length cDNA that encodes a protein of 1029 amino acids showing weak but significant homology to human and mouse Sgo2 and which contains the N-terminal coiled-coil and C-terminal basic regions characteristic of the Sgo protein family. An antibody raised against this protein recognizes a main band of the expected size in the egg extracts, 130 kDa, as well as an unspecific band of 200 kDa. Only the former is immunodepleted by the antibody to o5% of the endogenous levels and addition of the mRNA encoding full-length XSgo2 to the depleted extracts restores the presence of the 130-kDa protein. By immunofluorescence, we observed that XSgo2 distributes all over the chromatin in interphase nuclei assembled in the egg extracts, same as XSgo1, although there is no colocalization between the two proteins. In mitosis, XSgo2 accumulates at centromeres, labelled by XSgo1 and this accumulation depends on Bub1 and Aurora B mitotic kinases, but not on XSgo1. Thus, the regulation of Sgo2 targeting is conserved between human and Xenopus. Proper sister chromatid cohesion in the absence of XSgo2 The contribution of Sgo2 to sister chromatid cohesion in mitosis is unclear. We found that, unlike XSgo1, depletion of XSgo2 has no impact on centromeric cohesion, assayed by measuring the distances between sister centromeres in replicated chromosomes assembled in the egg extracts. Similarly, the accumulation of cohesin at centromeres observed in metaphase chromosomes is not perturbed in the absence of XSgo2. Thus, we decided to test whether depletion of Sgo proteins affects spindle assembly around sperm chromatin. In mock-depleted or XSgo1-depleted extracts, most sperm nuclei directed the fo