1134 Research Article Stability of the small γ-tubulin complex requires HCA66, a protein of the centrosome and the nucleolus Xavier Fant1, Nicole Gnadt1, Laurence Haren2 and Andreas Merdes1,2,* 1Wellcome Trust Centre for Cell Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK 2Centre National de la Recherche Scientifique–Pierre Fabre, 3 rue des Satellites, 31400 Toulouse, France *Author for correspondence (e-mail: [email protected]) Accepted 16 December 2008 Journal of Cell Science 122, 1134-1144 Published by The Company of Biologists 2009 doi:10.1242/jcs.035238 Summary To investigate changes at the centrosome during the cell cycle, (γ-tubulin, GCP2, and GCP3) in HCA66-depleted cells. By we analyzed the composition of the pericentriolar material contrast, the levels of γ-tubulin ring complex proteins such as from unsynchronized and S-phase-arrested cells by gel GCP4 and GCP-WD/NEDD1 were unaffected. We propose that electrophoresis and mass spectrometry. We identified HCA66, HCA66 is a novel regulator of γ-tubulin function that plays a a protein that localizes to the centrosome from S-phase to mitosis role in stabilizing components of the γ-tubulin small complex, and to the nucleolus throughout interphase. Silencing of HCA66 which is in turn essential for assembling the larger γ-tubulin expression resulted in failure of centrosome duplication ring complex. and in the formation of monopolar spindles, reminiscent of the phenotype observed after γ-tubulin silencing. Immunofluorescence microscopy showed that proteins of the Supplementary material available online at γ-tubulin ring complex were absent from the centrosome in http://jcs.biologists.org/cgi/content/full/122/8/1134/DC1 these monopolar spindles. Immunoblotting revealed reduced protein levels of all components of the γ-tubulin small complex Key words: Centrosome, γ-Tubulin, Mitosis, Monopolar, Spindle Introduction phosphorylation-dependent manner. Recruitment of γ-tubulin in The centrosome constitutes a major microtubule-organizing centre mammalian cells may be further supported by a protein that in animal cells. Microtubules are nucleated and anchored at the associates with the γ-tubulin ring complex and that attaches to the surface of the centrosome, at the pericentriolar material. γ-Tubulin centrosome, termed GCP-WD or NEDD1 (Lüders et al., 2006; is a major component of the pericentriolar material and supports Haren et al., 2006). Journal of Cell Science microtubule nucleation. It is in dynamic exchange with a free To identify novel proteins that are responsible for cell cycle- cytoplasmic pool (Khodjakov and Rieder, 1999), and is found in dependent regulation of γ-tubulin, we compared the composition two major protein complexes (Oegema et al., 1999): a small ‘γ- of the pericentriolar material at different phases of the cell cycle. TuSC’ (γ-tubulin small complex), containing two molecules of γ- We characterized HCA66 as a protein of the nucleolus that tubulin associated with one molecule each of the γ-tubulin complex associates with the centrosome specifically from S-phase to mitosis. proteins GCP2 and GCP3; furthermore, a large ‘γ-TuRC’ (γ-tubulin HCA66 has initially been identified as an autoimmune antigen in ring complex), consisting of multiple γ-TuSCs and additional hepatocellular carcinomas, and has recently been found to bind to proteins, including GCP4, GCP5 and GCP6 (for a review, see the protein Apaf-1 of the apoptosis pathway (Wang et al., 2002; Raynaud-Messina and Merdes, 2007). The amount of γ-tubulin at Piddubnyak et al., 2007). In the present study, we demonstrate that the centrosome is regulated during the cell cycle and increases HCA66 is required for the stability of γ-TuSC proteins, and that sharply at the beginning of mitosis, when spindle formation requires silencing of HCA66 expression produces defects in centriole an increase in microtubule nucleation activity (Zheng et al., 1991; duplication and spindle microtubule assembly. Lajoie-Mazenc et al., 1994; Khodjakov and Rieder, 1999). After completion of mitosis, the amounts of centrosome-bound γ-tubulin Results are reduced to interphase levels. So far, the mechanisms that regulate HCA66 is a nucleolar protein that associates transiently with γ-tubulin-dependent activity are only partly understood. Several the centrosome kinases have been implicated in regulating the recruitment of γ- To study changes at the centrosome during the cell cycle, we tubulin to the centrosome, such as Aurora A and Plk1 (Lane and compared the protein composition of the pericentriolar material from Nigg, 1996; Hannak et al., 2001; Berdnik and Knoblich, 2002; unsynchronized Jurkat cells (66% in G1 phase, 25% in S phase, as Terada et al., 2003). Moreover, the cell cycle-dependent recruitment verified by flow cytometry), and from Jurkat cells arrested in S of γ-tubulin complexes depends on proteins of the pericentriolar phase by a double aphidicolin block (85% in S phase, 10% in G1). material such as pericentrin, ninein or ninein-like protein (Takahashi Centrosomes were isolated after lysis using a sucrose gradient, and et al., 2002; Casenghi et al., 2003; Chen et al., 2003; Zimmerman centrosome-containing fractions were pooled and extracted with 1 et al., 2004; Delgehyr et al., 2005). Whereas pericentrin recruits M potassium iodide to solubilize the pericentriolar material. The increased amounts of γ-tubulin complexes to the mitotic centrosome soluble pericentriolar material obtained from unsynchronized cells via GCP2 and GCP3, ninein and ninein-like protein anchor γ-tubulin (‘async’) or from S-phase-arrested cells (‘S’) was then compared preferably during interphase but are displaced during mitosis in a by gel electrophoresis (Fig. 1A). Bands with significantly increased HCA66 and γ-TuSC stability 1135 Fig. 1. HCA66 is a novel protein of the nucleolus and the centrosome. (A) Silver stained SDS-PAGE of pericentriolar material extracted from centrosomes of asynchronous Jurkat cells (async) or cells arrested in S phase (S). The arrowhead shows the band identified as HCA66. The sizes of the molecular weight markers (Mr) are indicated on the right. (B) Schematic representation of human HCA66, showing seven predicted HAT repeats (black boxes). Amino acid positions of the N and C termini (1 and 597), and the repeats are indicated. The region of HCA66 used for immunization of rabbits is indicated with a black line. (C) Immunoblots of whole cell lysates from HeLa and U-2 OS cells. Left: HeLa lysate probed with anti-HCA66 antibody (imm.) or the corresponding pre-immune serum (pre.). Right: lysates of regular U-2 OS cells or cells expressing GFP-HCA66, probed with anti-HCA66. Black arrowhead indicates HCA66, white arrowhead indicates GFP:HCA66. The positions of molecular weight markers (Mr) are indicated. (D) Immunoblots of U-2 OS cells after fractionation in buffer containing 50 mM Tris Journal of Cell Science (pH 6.8), 250 mM NaCl and the detergents Triton X-100 and deoxycholate (DOC) at increasing concentrations, as indicated. s, supernatant; p, pellet. (E) Immunofluorescence of U-2 OS cells stained with antibodies against endogenous HCA66, or transfected with GFP:HCA66. Cells were co-stained with antibodies against γ-tubulin or nucleophosmin (NPM). Nucleolar HCA66 is not in focus in the cell depicted in the second row. Arrowheads indicate the position of the centrosome. Scale bars: 10 μm. intensity in S phase were investigated by MALDI-tof mass were obtained in U-2 OS cells (Fig. 1C). Moreover, our antibody spectrometry. One of these bands (Fig. 1A) was identified as recognized a higher molecular weight band in lysates from U-2 OS hepatocellular carcinoma-associated antigen 66 (HCA66), a protein cells overexpressing GFP:HCA66, corresponding to the GFP-tagged of 597 amino acids. Database searches revealed highly homologous protein (Fig. 1C). Fractionation of cells with salt and detergent sequences from ESTs in mouse, Drosophila and budding yeast. revealed that HCA66 is largely insoluble. Most of HCA66 was found Alignment of HCA66 protein sequences (supplementary material in the pellet after extraction and centrifugation (Fig. 1D), and visual Fig. S1) showed that the N-terminal half of HCA66 (amino acids inspection revealed that all nuclei accumulated in these fractions. 1-202) is the most conserved region within the protein (30% identity Immunofluorescence experiments with our HCA66 antibody revealed + 31% conservative exchanges between budding yeast and human), a strong staining of the nucleolus, colocalizing with the marker suggesting an important role of this region for the function of the nucleophosmin (Fig. 1E). Consistently, proteomic analysis identified protein. Structure prediction software identified seven HAT repeats HCA66 as a nucleolar component (Andersen et al., 2005). Our in HCA66 (Fig. 1B). These are ‘half-a-tetratrico-peptide’ repeats immunofluorescence data further revealed one or two discrete dots with structural similarities to TPR and HEAT repeats; each repeat in the cytoplasm that colocalized with the centrosomal marker γ- is predicted to form two short amphipathic α-helices connected by tubulin (Fig. 1E). Expression of a GFP:HCA66 fusion construct a loop (Preker and Keller, 1998). HAT repeats in HCA66 were found confirmed the dual localization of HCA66 at the nucleolus and at the between amino acids 87-119, 121-153, 156-188, 304-335, 452-486, centrosome (Fig. 1E). We then tested by microscopy whether the 488-520 and 524-557. This type of repeats is thought to be involved association of HCA66 with the centrosome is cell cycle dependent, in protein-protein interactions. as indicated by our biochemical data on purified centrosomes (Fig. An antibody raised against a bacterially expressed fragment of 1A). We found that U-2 OS cells that were synchronized in S phase HCA66 recognized a single protein band of ~62 kDa on immunoblots and that were pulse labelled with bromo-deoxyuridine displayed of HeLa cell lysates (Fig. 1C). Equivalent immunoblotting results HCA66 localization at the centrosome in 91% (±5, n=250) of the 1136 Journal of Cell Science 122 (8) cells, whereas in cultures synchronized in G1 phase only 24% (±7, localization does not depend on polymerized microtubules (Fig.