npg Cep57 maintains spindle pole integrity Cell Research (2012) 22:1390-1401. 1390 © 2012 IBCB, SIBS, CAS All rights reserved 1001-0602/12 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr Cep57, a NEDD1-binding pericentriolar material component, is essential for spindle pole integrity Qixi Wu1, *, Runsheng He1, *, Haining Zhou1, Albert CH Yu2, Bo Zhang1, Junlin Teng1, Jianguo Chen1, 3 1The State Key Laboratory of Biomembrane and Membrane Bioengineering and The Key Laboratory of Cell Proliferation and Dif- ferentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, China; 2Department of Neurobi- ology, Neuroscience Research Institute, School of Basic Medical Sciences, Peking University, Beijing 100191, China; 3The Center for Theoretical Biology, Peking University, Beijing 100871, China Formation of a bipolar spindle is indispensable for faithful chromosome segregation and cell division. Spindle in- tegrity is largely dependent on the centrosome and the microtubule network. Centrosome protein Cep57 can bundle microtubules in mammalian cells. Its related protein (Cep57R) in Xenopus was characterized as a stabilization factor for microtubule-kinetochore attachment. Here we show that Cep57 is a pericentriolar material (PCM) component. Its interaction with NEDD1 is necessary for the centrosome localization of Cep57. Depletion of Cep57 leads to unaligned chromosomes and a multipolar spindle, which is induced by PCM fragmentation. In the absence of Cep57, cen- trosome microtubule array assembly activity is weakened, and the spindle length and microtubule density decrease. As a spindle microtubule-binding protein, Cep57 is also responsible for the proper organization of the spindle micro- tubule and localization of spindle pole focusing proteins. Collectively, these results suggest that Cep57, as a NEDD1- binding centrosome component, could function as a spindle pole- and microtubule-stabilizing factor for establishing robust spindle architecture. Keywords: Cep57; centrosome; spindle; cell cycle Cell Research (2012) 22:1390-1401. doi:10.1038/cr.2012.61; published online 17 April 2012 Introduction the spindle microtubule-based tensions during mitosis. The loss of centriole cohesion and PCM factors, such The centrosome, comprising two centrioles and a sur- as astrin [4], Kizuna [5] and tastin [6], leads to centriole rounding pericentriolar material (PCM), is the primary disengagement and PCM fragmentation. The collapse of microtubule-organizing center (MTOC) in animal cells. the centrosome yields new MTOCs and multiple spindle After duplication of the centrosome in interphase, sub- poles. Thus, abnormalities in number, structure and com- sequent mitosis centrosome maturation and separation position of the centrosome frequently result in abnormal is required for orchestrating the bipolar spindle microtu- spindle formation. bule arrays [1]. At the onset of mitosis, the centrosomes The spindle microtubule architecture is also governed generate a large number of microtubules, which form the by microtubule-associated proteins (MAPs), including mechanical structure of the spindle [2, 3]. Thus, the cen- motor [7] and non-motor MAPs [8] at the spindle poles trosome must form a strong structure that can withstand and kinetochores and along spindle microtubules, which modulate spindle microtubule organization spatially and temporally to determine spindle morphology. The spindle pole proteins NuMA [9] and TPX2 [10] focus the spindle *These two authors contributed equally to this work. microtubule minus end, while microtubule-dependent Correspondence: Junlin Tenga, Jianguo Chenb aTel: +86-10-62767044; Fax: +86-10-62755786 motor proteins Eg5 [11], MCAK [12] and the dynein E-mail: [email protected] complex [9] generate motor force in the microtubule and bTel: +86-10-62755786; Fax: +86-10-62755786 transport mitotic proteins to ensure spindle assembly. E-mail: [email protected] Defects in spindle MAPs often disrupt the balance of Received 28 December 2011; revised 1 February 2012; accepted 6 March 2012; published online 17 April 2012 spindle microtubule-mediated forces, which in turn affect Cell Research | Vol 22 No 9 | September 2012 Qixi Wu et al. npg 1391 the overall spindle assembly. by the Cep57 antibody, whereas other representative Cep57, originally named translokin, was shown to centrosome components, pericentrin, centrin and ninein be involved in cytoplasmic FGF-2 intracellular traf- were not precipitated (Figure 1D). Among the tested ficking [13, 14] and was identified as a centrosome γ-TuRC components, NEDD1 appeared to have the component by a proteomics screen [15]. There are two strongest binding affinity to Cep57. Immunostaining of Cep57 family members in vertebrate cells, Cep57 and HeLa cells showed that Cep57 colocalized extensively Cep57-related protein (Cep57R) [16]. Xenopus Cep57 with NEDD1 at the centrosome (Figure 2A). To further (xCep57), a Cep57R homologous protein, is localized to investigate their interaction at the centrosome, we per- the kinetochore and centrosome and required for stable formed fluorescence resonance energy transfer (FRET) microtubule-kinetochore attachment and centrosome- analyses in HeLa cells probed by Cep57 (Alexa Fluor microtubule anchorage [17]. Mammalian Cep57 is a 488) and NEDD1 (Alexa Fluor 568) using an acceptor multidomain centrosome protein with an unconventional photobleaching assay. Compared with the control cells N-terminal centrosome-localization domain and a C- probed by Cep57 and centrin-2, the fluorescence intensi- terminal microtubule-binding domain, which is not local- ty increased after NEDD1 bleaching (Figure 2A and 2B), ized to the kinetochore. Exogenously expressed Cep57 suggesting that the NEDD1-Cep57 FRET signal was not can bind, bundle and stabilize microtubules [16]. Thus, the consequence of experimental error. The presence of the two Cep57 family members may have different func- FRET confirmed the specific interaction between Cep57 tions during mitosis. Genetic analysis showed that Cep57 and NEDD1 (Figure 2B). mutations can cause mosaic variegated aneuploidy (MVA) We then sought to find the region of NEDD1 that en- syndrome [18]. The precise localization of Cep57 at the abled the interaction with Cep57. NEDD1 consists of centrosome and its role during mitosis in mammalian cells remain elusive. Here, we show that Cep57 acts as a PCM component through binding to NEDD1 and is required for spindle microtubule organization and main- tenance of spindle pole integrity. Results Cep57 is a PCM component and is associated with NEDD1 We generated polyclonal mouse and rabbit antibod- ies against recombinant mouse Cep57. Immunoblotting of HeLa cell lysates with the affinity-purified antibody recognized a specific band with the expected molecular weight (Supplementary information, Figure S1A). Im- munostaining of HeLa cells showed that Cep57 localized within the PCM proteins pericentrin and γ-tubulin (Figure 1A and Supplementary information, Figure S1B), and localized adjacent to centrin-2, a marker of the centrioles (Figure 1B). The centrosome localization of Cep57 was irrespective of cell-cycle status (Supplementary informa- tion, Figure S1B and S1C) and intact microtubule net- work (Supplementary information, Figure S1D). Immu- Figure 1 Cep57 is a PCM component. (A, B) Immunostaining of noelectron microscopy showed that Cep57 was localized Cep57 (green, A; red, B), pericentrin (red, A) or centrin-2 (green, to the electron-dense area around centrioles (Figure 1C, B) in HeLa cells. Nuclear DNA was stained with DAPI (blue) arrowheads and Supplementary information, Figure S1E in all figures of this paper. Boxed regions, close-up of the cen- and S1F), but not on the centriolar microtubule wall. The trosome. Bar, 5 µm. (C) Immunoelectron microscopy images of above data suggested that Cep57 is a PCM component HeLa cell centrosome. Arrowheads indicate 5 nm gold particles. rather than a centriole protein. Bar, 100 nm. (D) Cep57 is associated with γ-tubulin ring com- plex components. Asynchronized (A) or metaphase-arrested To identify the centrosome-binding partner of Cep57, (M) HeLa cell lysates were immunoprecipitated with anti-Cep57 we performed immunoprecipitation (IP) experiments antibody or non-specific rabbit IgG. The inputs (left) and im- with the antibody against Cep57. The γ-TuRC compo- munoprecipitants (right) were immunoblotted with the indicated nents γ-tubulin, GCP2 and NEDD1 were precipitated antibodies. www.cell-research.com | Cell Research npg Cep57 maintains spindle pole integrity 1392 Figure 2 Cep57 binds to the N-terminus of NEDD1. (A, B) Immunostaining of Cep57 (Alexa Fluro 488) and centrin-2 or NEDD1 (Alexa Fluro 568) in HeLa cells, which were subjected to acceptor photobleaching. Representative images show the pre- and post-photobleaching states of the centrosome. Insets show the FRET intensities encoded by using the scale bar on the right. Bar, 1 µm. The graph shows the FRET efficiencies. n = 10. Error bars represent mean ± SEM. (C) Lysates of 293T cells expressing Cep57-GFP were incubated with glutathione-agarose beads coated with GST or GST-NEDD1 fusion protein. The inputs and the proteins bound to the beads were immunoblotted with anti-GFP antibody. (D) Co-IP with anti-Myc and anti- GFP antibodies were performed in lysates of 293T cells coexpressing Cep57-GFP and Myc vector or Myc-NEDD1 constructs, followed by immunoblotting with the indicated antibodies. FL, Myc-NEDD1 full-length. 1-350, Myc-NEDD1 (1-350). 341-end, Myc-NEDD1