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First published online 27 November 2007
doi: 10.1242/jcs.020248

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Cep68 and Cep215 (Cdk5rap2) are required for centrosome cohesion

¹ Max-Planck-Institute for Biochemistry, Department of Cell Biology, Am Klopferspitz 18, D-82152 Martinsried, Germany
² Electron Microscopy Unit, Center for Plant Molecular Biology, University of Tübingen, Auf der Morgenstelle 5, D-72076 Tübingen, Germany

View larger version (32K): [in this window] [in a new window]   Fig. 1. Characterisation of anti-Cep68 and anti-Cep215 antibodies. (A) Primary structure of human Cep68L/S. Note that Cep68L comprises a 137 aa insertion compared with Cep68S (at residue 492) and both isoforms are predicted to form a C-terminal globular domain (at aa 618-757 in Cep68L and at aa 454-620 in Cep68S; grey). The horizontal line (antigen) denotes the region used for antibody production. The sequence data for Cep68 are available from Ensembl [accession no. ENST00000377990 (Cep68L) and ENST00000260569 (Cep68S)]. (B) Primary structure of human Cep215. Two isoforms with 1893 and 1814 residues, respectively, have been described; they are identical between residues 1 and 1396, followed by non-identical stretches of 259 (isoform 1) and 180 residues (isoform 2), and identical C-termini (starting at residue 1655 in isoform 1 and 1576 in isoform 2). Both isoforms are predicted to contain several coiled-coil regions (black, CC) and a microtubule association region (grey). The horizontal line (antigen) denotes the region used for antibody production. The sequence data for Cep215 cDNAs are available from NCBI [accession no. NM_018249 (isoform 1) and NM_001011649 (isoform 2)]. (C) Antibodies directed against Cep68 (R170) and corresponding pre-immune serum were tested on western blots of total cell lysates of HeLaS3, U2OS and 293T cells (left) and centrosome preparations from KE37 cells (centre). In the latter case, proteins show a retarded electrophoretic mobility because of the high sucrose concentration present in centrosome preparations. Arrowheads indicate Cep68L and -S. Right, western blot showing efficient depletion of Cep68 from U2OS cells by 48 hours siRNA, using oligonucleotide duplexes 262 or 263. (D) Antibodies directed against Cep215 (R174) and corresponding pre-immune serum were tested on western blots of total cell lysates of HeLaS3, U2OS and 293T cells (left) and centrosome preparations from KE37 cells (centre). In the latter case, proteins show a retarded electrophoretic mobility because of the high sucrose concentration present in centrosome preparations. Arrowheads indicate Cep215. Right, western blot showing efficient depletion of Cep215 from U2OS cells by 72 hour siRNA, using oligonucleotide duplexes 282 or 283. GL2 treatment is used for control and blotting for -tubulin illustrates equal loading.

View larger version (51K): [in this window] [in a new window]   Fig. 2. Distinct associations of Cep68 and Cep215 with centrosomes. Antibodies directed against Cep68 (A) or Cep215 (B) and corresponding pre-immune sera were used for immunofluorescence on U2OS cells. The antibodies (green) recognise the centrosome as indicated by colocalisation with -tubulin (red). Insets show enlarged areas to better visualise the centrosomes; note filamentous staining produced by anti-Cep68 antibody and compact staining produced by anti-Cep215 antibody. Pre-immune sera did not show any specific staining (top panels) and 48 hour siRNA treatment using duplex 262 targeting Cep68 or duplex 283 targeting Cep215 abolished Cep68 (A, bottom) and Cep215 signals (B, bottom), respectively. Note the centrosome splitting induced by siRNA-mediated depletion of either Cep68 or Cep215. Bars, 10 µm.

View larger version (85K): [in this window] [in a new window]   Fig. 3. Cell cycle analysis of centrosome localisation of Cep68 and Cep215. Asynchronously growing U2OS cells were co-stained with antibodies against -tubulin (central panels) and either Cep68 (A; upper panels) or Cep215 (B; upper panels). Lower panels show merged images including DNA staining by DAPI (Cep68 or Cep215 in green; -tubulin in red). Bars, 10 µm.

View larger version (99K): [in this window] [in a new window]   Fig. 4. High-resolution analysis of Cep68 and Cep215 localisation. (A,B) U2OS cells were co-stained with antibodies against -tubulin (middle columns) and either Cep68 (A; left column) or Cep215 (B; left column) and examined using deconvolution on a Deltavision instrument. Right columns show merged images with Cep68 or Cep215 in green and -tubulin in red. Bars, 2 µm. (C) U2OS cells were subjected to pre-embedding immuno-gold labelling EM. Cells were labelled with anti-Cep68 (top left) or anti-Cep215 (bottom left) antibodies, followed by nanogold-coupled secondary antibody. Controls (right) show centrioles stained with secondary antibody only. Note that Cep68 decorates striking fibres protruding away from the proximal ends of centrioles; proximal ends are identified by the proximity of fibres to nascent pro-centrioles (white arrow) and their distance to distal appendages (white arrowhead). Bars, 250 nm.

View larger version (85K): [in this window] [in a new window]   Fig. 5. Effect of Cep68 and Cep215 depletion on other cohesion proteins. U2OS cells were transfected for 48 hours with siRNA duplexes specific for Cep68 (A) or Cep215 (B) and then co-stained with the antibodies indicated (left columns) and antibodies against -tubulin (centre columns). Columns on the right show merged images with DAPI staining of DNA in blue, -tubulin in red and the various proteins in green. Bars, 10 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Mutual dependencies of cohesion-regulatory centrosomal proteins. U2OS cells were transfected for 48 hours with siRNA duplexes specific for rootletin (A), C-Nap1 (B) or pericentrin (C) and then co-stained with the antibodies indicated (left columns) and antibodies against -tubulin (centre columns). Columns on the right show merged images with DAPI staining of DNA in blue, -tubulin in red and the various proteins in green. Bars, 10 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 7. Recruitment of Cep68 but not Cep215 to rootletin fibres. Tagged constructs of rootletin, Cep68 and Cep215 were overexpressed in U2OS cells and filament formation as well as the recruitment of endogenous proteins to induced filaments was examined by immunofluorescence microscopy. Bars, 10 µm. (A) GFP-rootletin (left) was coexpressed with Myc-Cep68 or Myc-Cep215 (centre); merged images are shown on the right (GFP-rootletin in green; Myc in red). Note that Myc-Cep68 but not Myc-Cep215 colocalises with rootletin fibres. (B) Overexpressed Myc-Cep68 (centre) colocalises with endogenous rootletin at the centrosome (centre) but does not form polymers; merged image on the right shows Myc-Cep68 in green and rootletin in red. (C) Endogenous Cep68 (centre) colocalises with Myc-rootletin (left) overexpressed to low (top) and high (middle) levels, but endogenous Cep215 is not recruited to Myc-rootletin filaments (bottom); merged images on the right show Cep68 and Cep215 in green and Myc-rootletin in red.

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