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First published online September 12, 2003
doi: 10.1242/10.1242/jcs.00743

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Characterisation of PGs1, a subunit of a protein complex co-purifying with tubulin polyglutamylase

Catherine Regnard2,*, Didier Fesquet1, Carsten Janke1, Dominique Boucher2, Elisabeth Desbruyères2, Annette Koulakoff3, Christine Insina1, Pierre Travo1 and Bernard Eddé1,{ddagger}

1 Centre de Recherches de Biochimie Macromoléculaire, CNRS, 34293 Montpellier, France
2 Laboratoire de Biochimie Cellulaire, CNRS, Université Paris 6, 75252 Paris, France
3 INSERM U114, Collège de France, 75005 Paris, France



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  		Fig. 1. Screening of anti-TPG hybridomas. Of the 330 hybridomas tested, 15 are presented here. TPG activity was measured in both the supernatant (top) and bead fraction (bottom) of each IP. Incorporation of [3H]Glu into {alpha}-tubulin (light-grey bars) and ß-tubulin (dark-grey bars) was measured separately for the supernatants and together for the pellets (black bars). The control (Ctl) is an IP performed with the culture medium alone. Numbering refers to hybridoma names.

 


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  		Fig. 2. Immunoprecipitation of brain TPG and identification of the PGs1 subunit. (A) Silver staining of Fraction IV (lane 1) and the IP fraction obtained with mAb206 (lane 2). In addition to the light (LC) and heavy (HC) antibody chains (arrowheads), three protein species (arrows) are found at high levels in the IP fraction. (B) Immunoprecipitation was performed with mAb206 and analysed by western blotting with L83. Comparable amounts of fraction IV (lane 1), unbound fraction (lane 2) and IP fraction (lane 3) were loaded on the gel. (C) L83 immunoprecipitates TPG activity. S1-S3 correspond to the IP supernatants obtained after the first, second and third cycle of IP, and B1-B3 to the bead fractions. Similar proportions of each fraction were tested for TPG activity (upper panel) and analysed by western blotting with L83 (lower panel). The position of p32 and of the antibody light chain are indicated by arrows. (D) Enrichment of PGs1 during TPG purification. Brain fractions I (initial supernatant) and II-IV [20x, 160x and 400x purified fractions, respectively, as described by Regnard et al. (Regnard et al., 1998Go)] were analysed by western blotting with L83. Equal amounts of proteins were loaded in each lane. (E) PGs1 mRNA expression profile. Total RNA from various mouse tissues were analysed by northern blotting and probed with a 32P-labelled PGs1 cDNA ORF.

 


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  		Fig. 3. Confocal images of PGs1 localisation in mouse neural cells in primary culture. (A-C) Mouse brain neurones were cultured for 2 (A), 8 (B) and 13 (C) days, then fixed with paraformaldehyde and double stained with L83 (left panels, green) and GT335 (middle panels, red). Right panels present the corresponding merged images. (D,E), Mouse brain neural cells fixed with paraformaldehyde and double stained with L83 (left panels in D and E, green) and GT335 (middle panel in D, red) or anti-{gamma}-tubulin mAb GTU-88 (middle panel in E, red). Right panels show the corresponding merged images. Staining of PGs1 was observed at the base of primary cilia (arrowheads) as well as of multiple cilia emerging from ependymal cells (arrows). Note also some PGs1 staining along the cilia. Scale bars: 20 µm.

 


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  		Fig. 4. PGs1 is localised at the centrosome in 1009 EC cells. Exponentially growing 1009 EC cells were fixed with methanol and double stained with L83 (left panels) and anti-polyglutamylated tubulin (GT335, right panel in A) or anti-{gamma}-tubulin (GTU-88, right panel in B), or anti-AKAP450 (CTR-453, right panel in C). Scale bars: 10 µm.

 


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  		Fig. 5. PGs1-GFP localises to the centrosome. (A,B) 1009 cells were transfected with a PGs1-GFP construct and analysed 24 hours after transfection. Cells were fixed with paraformaldehyde and stained with L83 (right panel in A) or CTR-453 (right panel in B). Left panels show PGs1-GFP fluorescence. (C,D), PGs1-GFP fluorescence of stable transfected 1009 cells, fixed with paraformaldehyde (C) or unfixed (D). Scale bars: 10 µm. (E) Western blotting of 1009 (lane 1) and stable transfected 1009/PGs1-GFP (lane 2) cell extracts with L83. A single band at 60x103 was detected in the latter but not in the former. Note that the endogenous PGs1 is not detected.

 


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  		Fig. 6. PGs1 co-localises with PKA. (A,B) Exponentially growing 1009 EC cells were fixed with methanol and double stained with L83 (left panels) and anti-RII{alpha} (right panel in A) or anti-RIIß (right panel in B). (C,D) 1009 cells were transfected with a PGs1-GFP construct and analysed 24 hours after transfection. Cells were fixed with paraformaldehyde and stained with anti-RII{alpha} (right panel in C) or anti-RIIß (right panel in D). Left panels show PGs1-GFP fluorescence. Scale bars: 10 µm.

 


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  		Fig. 7. 3D reconstruction of PGs1 at the centrosome. 1009 EC cells were fixed with methanol and double stained with L83 (red) and GT335 (A, green) or GTU-88 (B, green) or CTR-453 (C, green) or DM1A (D, green). Confocal images were analysed by deconvolution. Projections of all planes are shown in left panels. 3D reconstructions are shown in the middle and right panels in two different orientations. In D (right), the green colour was made semi-transparent to better visualise the red staining. The arrowheads in D (left), indicate PGs1 dots associated with MTs.

 


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  		Fig. 8. Localisation of PGs1 is dependent on the integrity of the MT network. 1009 cells were incubated with (B) or without (A) 10 µM nocodazole for 45 minutes. After washing off nocodazole, MTs were allowed to regrow for 3 minutes (C) or 6 minutes (D). (E) 1009 cells were incubated with 5 µM taxol for 45 minutes. Methanol-fixed cells were double stained with L83 (left panels in A-E) and DM1A (right panels in A-E). Scale bars: 10 µm. (F-H) Confocal green fluorescence images of living 1009/PGs1-GFP cells before (F) and after incubation with 10 µM nocodazole for 45 minutes (G) and after 10 minutes of regrowth (H).

 


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  		Fig. 9. PGs1 disappears from centrosomes at mitosis. (A,B) Fixed 1009/PGs1-GFP cells immunolabelled with CTR-453 (middle panels). Upper panels show the green fluorescence of PGs1-GFP and lower panels DAPI staining of DNA. The intensity of labelled cells in interphase (I), metaphase (M) and anaphase (A) is compared. (C) Time-lapse video microscopy of 1009/PGs1-GFP cells. Phase contrast (left panels) and green fluorescence (right panels) images were taken at 10 minutes intervals. Arrows indicate a cell that goes through mitosis during the time course of the experiment.

 




© The Company of Biologists Ltd 2003