EC Accepts, published online ahead of print on 22 May 2009

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Eukaryotic Cell doi:10.1128/EC.00025-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Dictyostelium discoideum CenB is a bona fide centrin essential for nuclear architecture and centrosome stability

Sebastian Mana-Capelli, Ralph Gräf, and Denis A. Larochelle^*

Clark University, Department of Biology. 950 Main Street. Worcester, MA. 01610 US; Universität Potsdam, Institut für Biochemie und Biologie. ZELLBIOLOGIE. Karl-Liebknecht-Strasse 24-25, Haus 26, 14476 Potsdam-Golm Germany

^* To whom correspondence should be addressed. Email: dlarochelle{at}clarku.edu.

Centrins are a family of proteins within the calcium-binding EF-hand superfamily. In addition to the archetypical role at the MTOC, centrins have acquired multiple functionalities throughout the course of evolution. For example, centrins have been linked to different nuclear activities, including mRNA export and DNA repair. Dictyostelium discoideum centrin B is a divergent member of the centrin family. At the amino acid level, DdCenB shows 51 % identity with its closest relative and only paralog, DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a well supported monophyletic and divergent group within the centrin family of proteins. Interestingly, fluorescently-tagged versions of DdCenB were not found at the centrosome (in whole cells or isolated centrosomes). Instead, DdCenB localized to the nuclei of interphase cells. This localization disappeared as cells entered mitosis, although Dictyostelium cells undergo a closed mitosis in which the NE does not breakdown. DdCenB knockout cells exhibited aberrant nuclear architecture, characterized by enlarged and deformed nuclei, and loss of proper centrosome-nucleus anchoring (observed as NE protrusions). At the centrosome, loss of DdCenB resulted in defects in the organization and morphology of the MTOC, supernumerary centrosomes, and centrosome-related bodies. The multiple defects that loss of DdCenB generated at the centrosome can be explained by its atypical division cycle, transitioning into the NE as it divides at mitosis. On the basis of these findings, we propose that DdCenB is required at interphase to maintain proper nuclear architecture, and, before delocalizing from the nucleus, DdCenB is part of the centrosome duplication machinery.