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First published online 19 March 2009
doi: 10.1242/jcs.035238

Journal of Cell Science 122, 1134-1144 (2009)
Published by The Company of Biologists 2009
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Stability of the small {gamma}-tubulin complex requires HCA66, a protein of the centrosome and the nucleolus

Xavier Fant1, Nicole Gnadt1, Laurence Haren2 and Andreas Merdes1,2,*

1 Wellcome Trust Centre for Cell Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK
2 Centre National de la Recherche Scientifique–Pierre Fabre, 3 rue des Satellites, 31400 Toulouse, France


Figure 1
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  		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 (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 {gamma}-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.

 

Figure 2
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  		Fig. 2. Centrosome localization of HCA66 is regulated during the cell cycle. (A) Immunofluorescence of HCA66 (green) in U-2 OS cells, synchronized in G1 (top) or S phase (bottom). Cells were co-stained with {gamma}-tubulin (red). Insets on the right are magnifications of the centrosomal areas (top inset, {gamma}-tubulin; middle, HCA66; bottom, merge). Arrowheads indicate the position of the centrosome. Histogram showing percentage of cells with HCA66 localizing to the centrosome, in G1 and S phase. (B) Immunofluorescence analysis of HCA66 localization at different stages of mitosis. Arrowheads indicate areas of perichromatin staining. HCA66 is in green, {gamma}-tubulin is in red, DNA is in blue. (C) Top: interphase cell treated with 5 µm nocodazole to depolymerize microtubules, stained for HCA66 (red) and {alpha}-tubulin (green). Bottom: mitotic cell treated with 5 µm taxol to induce non-centrosomal microtubule asters, stained for HCA66 (red) and {alpha}-tubulin (green). Arrowheads indicate the localization of centrosome-associated HCA66. The centrosomal identity was verified with the centriole marker centrin (data not shown). (D) Representative images showing localization of HCA66 relative to the centrioles. Panel showing immunofluorescence of four centrioles stained for centrin (red) and HCA66 (green). Top: maximum intensity projections of a stack of optical sections. The selected areas are shown at the bottom at higher magnification, in deconvolved optical sections taken at 0.2 µm z-steps. Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Identification of centrosomal and nucleolar targeting sequences of HCA66. (A) U-2 OS cells, transfected with different GFP:HCA66 constructs as indicated on the left, stained with antibody against {gamma}-tubulin (red). The cell transfected with GFP:HCA661-149 is shown at two different exposures of the GFP channel. Numbers in the GFP panels indicate the exposure time relative to GFP:HCA66. Arrowheads indicate the positions of the centrosomes. (B) U-2 OS cells overexpressing GFP:HCA661-86 (green), co-stained with antibodies against centrin (white) and against {gamma}-tubulin, GCP2, Nedd1 or endogenous HCA66 (red). Positions of centrioles co-localizing with GFP:HCA661-86 are indicated by arrowheads. Relative exposure times are indicated, as in A. Centrosomal HCA66 (c) and nucleolar HCA66 (nl) are recorded in two separate focal planes, in the cell depicted in the first row. Histogram, left: the percentage of interphase cells with centrosome staining of {gamma}-tubulin, GCP2 or Nedd1 is indicated, following overexpression of GFP, GFP:HCA66 or GFP:HCA661-86. Graph, right: the intensity of {gamma}-tubulin immunofluorescence at the centrosome is shown as a function of fluorescence levels of overexpressed GFP:HCA661-86 at the centrosome. (C) U-2 OS cells transfected with GFP, GFP:HCA66 or GFP:HCA661-86 (top). Microtubules were depolymerized in the cold for 2 hours and allowed to regrow for 2 minutes, before being fixed and processed for immunofluorescence of {alpha}-tubulin (bottom). The corresponding histogram indicates the percentage of cells with re-grown microtubule asters after 2, 3.5 and 5 minutes. Scale bars: 10 µm.

 

Figure 4
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  		Fig. 4. HCA66 depletion leads to mitotic defects. (A) Immunoblots, probed with anti-HCA66, of U-2 OS cells treated with siRNA against HCA66 using two different oligonucleotides, HCA-2 and HCA-4, or control siRNA (C). {alpha}-Tubulin is shown as a loading control. (B) U-2 OS cells were transfected with HCA-4 oligonucleotides or control siRNA, and processed after 48 hours for immunofluorescence of HCA66 (green) and centrin to indicate the number of centrioles (insets show magnified areas of centrin staining). Mitotic cells are shown for both control and HCA66 siRNA. DNA is shown in blue. Histogram depicts the percentage of mitotic cells containing 0-1, 2, 3, 4, 5 or more centrioles per cells. Black columns, control cells; white columns, cells treated with HCA66 siRNA. (C) Control and HCA66-depleted U-2 OS cells treated with hydroxyurea (HU) to induce overduplication of centrioles. Graph depicts the percentage of cells containing ≤4 or >4 centrioles/cell. (D) Immunofluorescence of U-2 OS cells treated as in B. HCA66 is shown in green, {alpha}-tubulin in red and DNA in blue. One control cell in metaphase and three depleted cells in mitosis are shown. Histogram depicts the percentage of mitotic cells after control treatment or HCA66 siRNA, containing monopolar spindles (grey) or spindles with poorly separated poles (white). (E) Left panel: mitotic index of cells 48 hours after siRNA (control or HCA66). Mean from three experiments, error bars indicate s.d., ~500 cells were scored per condition. Right panel: frequency of different mitotic stages after 48 hours of HCA66 siRNA treatment (mean from three experiments, error bars indicate s.d., ~200 cells were scored per condition). (F) Percentage of cells with micronuclei after control and HCA66 siRNA for 48 hours (mean from three experiments, error bars indicate s.d., ~500 cells were scored per condition). (G) Growth curve of control-depleted cells and HCA66-depleted cells. Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. Depletion of HCA66 reduces pericentriolar localization of {gamma}-TuRC proteins. (A) U-2 OS cells were transfected with HCA66 siRNA or control siRNA, and processed after 48 hours for immunofluorescence of HCA66, {gamma}-tubulin, centrin, GCP2, GCP4, Nedd1, Aurora A and pericentrin, as indicated. Top panels left show interphase cells, the remaining panels show mitotic cells. Arrowheads indicate the positions of the centrosomes. (B) Histogram depicting the relative intensity of immunofluorescence at the mitotic centrosome, of proteins shown in A. Standard deviations are shown for both controls (black columns) and cells treated with HCA66 siRNA (white columns, n≥35). {gamma}-Tubulin immunofluorescence was measured both in mitosis and in interphase (n=85); in the graphs, the average immunofluorescence intensity in mitosis is defined as 100% and used as a reference for interphase. (C) The immunofluorescence intensity of polymerized microtubules is quantified in interphase and mitosis (n=25). The signal in mitotic control cells is set at 100%. Labelling as in B. Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. Depletion of HCA66 reduces {gamma}-TuSC protein levels. U-2 OS cells were transfected with `HCA-4' siRNA oligonucleotides against HCA66 or with control siRNA (C). Whole-cell lysates were analyzed by immunoblotting with anti-HCA66, anti-{gamma}-tubulin, anti-GCP2, anti-GCP3, anti-GCP4, anti-Aurora A, anti-Plk1, anti-Nedd1, anti-PCM-1 and anti-{alpha}-tubulin antibodies. Furthermore, immunoblot of cells treated with siRNA against {gamma}-tubulin, probed with antibodies against {gamma}-tubulin, GCP2, actin, CPAP and HCA66 are shown. Histogram indicating the signal intensities of western blots of HCA66, {gamma}-tubulin, GCP2 and GCP3 from HCA66-depleted cells. The mean values from four different experiments and standard deviations are shown. The percentages are expressed in relation to the respective control levels of each protein (=100%). The calculation of all values was performed after normalization over {alpha}-tubulin levels, to correct for uneven loading.

 

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© The Company of Biologists Ltd 2009