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First published online 4 March 2003
doi: 10.1242/jcs.00341

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PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression

Angélique Augustin^1,*, Catherine Spenlehauer^1,*, Hélène Dumond¹, Josiane Ménissier-de Murcia¹, Matthieu Piel², Anne-Catherine Schmit³, Françoise Apiou⁴, Jean-Luc Vonesch⁵, Michael Kock⁶, Michel Bornens² and Gilbert de Murcia^1,

¹ Unité 9003 du CNRS, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, 67400 Illkirch, France
² Institut Curie, Section Recherche UMR 144 du CNRS, 26 Rue d'Ulm, F-75248 Paris, France
³ Institut de Biologie Moléculaire des Plantes, CNRS, 12 rue du General Zimmer, 67084, Strasbourg, France
⁴ Institut Curie, Section Recherche UMR 147 du CNRS, 26 Rue d'Ulm, F-75248 Paris, France
⁵ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 163, 67400 Illkirch, France
⁶ Pharmaceuticals Research, BASF AG, D-67056 Ludwigshafen, Germany

View larger version (58K): [in a new window]   Fig. 1A. (A) Sequence alignment of the five first members of the PARP family. Sequence alignment of amino acids 352 to 923 of human PARP-1 [hPARP-1, accession number P09874 (Cherney et al., 1987; Kurosaki et al., 1987; Uchida et al., 1987)], human PARP-2 [hPARP-2, AJ236912, (Ame et al., 1999)], human PARP-3 [hPARP-3, accession number NM_005485 (Johansson, 1999)], vault-particle-associated PARP [VPARP, accession number AF057160 (Kickhoefer et al., 1999)] and tankyrase [accession number AF082556 (Smith et al., 1998b)]. Cylinders and arrows schematically represent helices and ß-strands, respectively, as previously shown in the chicken PARP-1 structure (Ruf et al., 1996). (B) Schematic representation of the functional domains of hPARP-1 and hPARP-3. (C) Structure of the two possible versions of the human PARP-3 gene product by alternative splicing of the first exon. AS, acceptor site; DS, donor site. (D) PCR products loaded on 6% polyacrylamide gel. (E) Chromosomal mapping of hPARP-3: FISH of the hPARP-3 gene on a human lymphocyte chromosome spread (arrows). Chromosomes are counterstained with propidium iodide. (F,G) Chromosomal mapping of mouse PARP-3: FISH of mPARP-3 on a mouse fibroblasts chromosome spread. Chromosomes are counterstained with DAPI. The sequence data of hPARP-3 is available from GenBank/EMBL/DDBJ under accession number AY126341.

View larger version (24K): [in a new window]   Fig. 1D. (A) Sequence alignment of the five first members of the PARP family. Sequence alignment of amino acids 352 to 923 of human PARP-1 [hPARP-1, accession number P09874 (Cherney et al., 1987; Kurosaki et al., 1987; Uchida et al., 1987)], human PARP-2 [hPARP-2, AJ236912, (Ame et al., 1999)], human PARP-3 [hPARP-3, accession number NM_005485 (Johansson, 1999)], vault-particle-associated PARP [VPARP, accession number AF057160 (Kickhoefer et al., 1999)] and tankyrase [accession number AF082556 (Smith et al., 1998b)]. Cylinders and arrows schematically represent helices and ß-strands, respectively, as previously shown in the chicken PARP-1 structure (Ruf et al., 1996). (B) Schematic representation of the functional domains of hPARP-1 and hPARP-3. (C) Structure of the two possible versions of the human PARP-3 gene product by alternative splicing of the first exon. AS, acceptor site; DS, donor site. (D) PCR products loaded on 6% polyacrylamide gel. (E) Chromosomal mapping of hPARP-3: FISH of the hPARP-3 gene on a human lymphocyte chromosome spread (arrows). Chromosomes are counterstained with propidium iodide. (F,G) Chromosomal mapping of mouse PARP-3: FISH of mPARP-3 on a mouse fibroblasts chromosome spread. Chromosomes are counterstained with DAPI. The sequence data of hPARP-3 is available from GenBank/EMBL/DDBJ under accession number AY126341.

View larger version (43K): [in a new window]   Fig. 2. (A) Purification and characterization of recombinant hPARP-3 overexpressed in the Sf9/baculovirus system. Crude extract from infected Sf9 cells (lane a); Purified recombinant hPARP-3 (lanes b-e); DNA-binding activity of hPARP-3 detected by south-western blotting (lane c); autopoly(ADP-ribosyl)ation of purified hPARP-3 incubated with [-32P] NAD+ (lane d); inhibition of hPARP-3 autopoly(ADP-ribosyl)ation by 2 mM 3-Aminobenzamide (lane e). (B) Western blot detection of hPARP-3 in crude extracts from mouse lung (lane f), HeLa cells (lane g) or infected Sf9 cells (lane h) and purified recombinant hPARP-3 (lanes i,j) using two different anti hPARP-3 antibodies (see Materials and Methods).

View larger version (59K): [in a new window]   Fig. 3. hPARP-3 preferentially localizes to the daughter centriole throughout the cell cycle. (A) Confocal imaging of the subcellular distribution of hPARP-3 (red) at the centrosome marked with the anti-p34cdc2 antibody (green) in HeLa cells; d is a merge of a and b; (B) Colocalization of hPARP-3 immunostained with the polyclonal anti-hPARP-3 antibody 1650 (red) with HeLa HC1 cells stably expressing centrin (green) fused to GFP (Piel et al., 2000). h is a merge of e and f. (C) Merged images showing an asymmetric distribution of hPARP-3 (red) and centrin fused to GFP in S/G2 cells. hPARP-3 (red) was immunostained as in B. Strongly fluorescent dots of GFP-centrin (insets m and j) are attributed to the mother centrioles. (D) Immunofluorescence microscopy analysis using the 1650 polyclonal anti-hPARP-3 antibody (red) and the anti-acetylated -tubulin monoclonal antibody (green) showing the primary cilium of the mother centriole. (E) hPARP-3 subcellular distribution throughout the cell cycle. Colocalization of hPARP-3 with GFP-centrin in HeLa HC1 cells by confocal microscopy analysis using the polyclonal anti-hPARP-3 antibody 1650 (red). For all pictures, the magnifications are details of the area surrounding the arrowheads. Nuclei in c, g, i, p and s are stained with Hoechst or with DAPI in S phase. Bars, 10 µm.

View larger version (28K): [in a new window]   Fig. 4. hPARP-3 is detected in purified centrosomes. (A) Immunostaining of centrosome preparations from KE 37 cells. Immunolabelling was carried out using an anti-p34cdc2 antibody (green) and the polyclonal anti-hPARP-3 antibody 1650 (red). Bar, 2 µm. (B) Western blot of purified recombinant hPARP-3 (lane a), a sample of purified centrosomes (108) (lane b) and a KE 37 lysate containing a highly enriched centrosome preparation present in both the Triton-insoluble (lane c) and Triton-soluble fractions (lane d).

View larger version (70K): [in a new window]   Fig. 5. DNA damage induces centrosome amplification but does not relocate hPARP-3. HeLa HC1 cells expressing GFP-centrin (green) were immunostained with an anti-hPARP-3 (red) antibody 120 hours after treatment with 1 mM MNU (A-C) or with 1 mM hydrogen peroxide (D-F). Examples of monopolar and multipolar spindles are shown. Bars, 10 µm.

View larger version (30K): [in a new window]   Fig. 6. (A) hPARP-3 or N-ter hPARP-3 overexpression leads to G1/S cell cycle arrest. FACS analysis on undamaged (left column) and MNU-treated (right column) HeLa cells expressed by GST, GST N-ter hPARP-3 or GST-hPARP-3. (B) hPARP-3 (d-f) or its N-terminal domain (a-c) target the GFP fusion protein to the centrosome, which is immunostained with an anti -tubulin antibody (red). Bar, 10 µm.

View larger version (77K): [in a new window]   Fig. 7. hPARP-3 overexpression does not prevent centrosome amplification induced by hydroxyurea (HU) in CHO cells. Cells were transfected for 48 hours to express full-length or N-ter hPARP-3 as GFP-fusion proteins and cultured in the presence (C-F) or absence (A-B) of HU. Transfected cells were identified by fluorescence microscopy, and the number of centrosomes quantified using the antibody anti-glutamylated tubulin Gt335 (red). (C,D) Non-tranfected cells. (E-G) Transfected cells expressing GFP-hPARP-3. (H) Histogram showing the mean number of centrosomes±s.d. counted in non-transfected cells or in cells expressing GFP alone, GFP-hPARP-3 or GFP-N-ter hPARP-3 following HU treatment. The arrows point to centrosome amplification. Bars, 10 µm.

View larger version (37K): [in a new window]   Fig. 8. hPARP-3 interacts with hPARP-1 at the centrosome. (A) Extracts from HeLa cells expressing either GST (lane a), GST-hPARP-3 (lanes b and c) or GST-N-ter hPARP-3 (lane d) were submitted to GST pull-down experiments, and the interacting proteins were analyzed by western blotting. When indicated, a treatment with 2 mM 3AB was applied for 2 hours before harvesting cell extracts. The blot was firstly probed with the mouse anti-hPARP-1 antibody (EGT69) (asterisks in upper panel) and subsequently with a polyclonal anti-GST antibody to reveal the proper expression of the fusion proteins (lower panel). (B) Sample of purified centrosomes (107) separated on SDS-PAGE and analyzed by western blotting using successively antibodies against hPARP-1, hPARP-3 and -tubulin (lane e); purified recombinant hPARP-3 (50 ng) (lane f). (C) Subcellular localization of hPARP-1 in GFP-centrin expressing HeLa HC1 cells. hPARP-1 was detected using a monoclonal antibody (F1-23) followed by the anti-mouse fluor Alexa 568 conjugate (red). (D) Both hPARP-1 and hPARP-3, immunostained with their respective specific antibodies, are detected at the centrosome in HeLa cells. For all pictures, the magnifications are details of the area surrounding the arrowheads. Bars, 10 µm.