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First published online 30 January 2007
doi: 10.1242/jcs.03366

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The intraflagellar transport component IFT88/polaris is a centrosomal protein regulating G1-S transition in non-ciliated cells

¹ Institut Cochin, Département Génétique et Développement, Faculté de médecine R. Descartes, UM 3, Paris, F-75014 France
² INSERM, U567, Faculté de médecine R. Descartes, UM 3, Paris, F-75014 France
³ CNRS, UMR8104, Faculté de médecine R. Descartes, UM 3, Paris, F-75014 France
⁴ Université Paris 5, Faculté de médecine R. Descartes, UM 3, Paris, F-75014 France
⁵ INSERM U785, Université Paris XI,CHB Paul Brousse, Villejuif Cedex, F-94804 France
⁶ Laboratoire de Biologie du Cycle Cellulaire et de la Motilité, UMR144 Institut Curie/CNRS, 12 rue Lhomond, Paris cedex 05, F-75248 France

View larger version (65K): [in this window] [in a new window]   Fig. 1. IFT88/polaris localizes to basal bodies and axonemes in quiescent ciliated cells and to centrosomes in proliferating cells. (A) RPE-1 cell immunostained with an antibody against acetylated-tubulin (6-11B-1, green) to label the axoneme of the cilium, and with anti-IFT88/polaris (740, red) to stain IFT88/polaris which was localized to the axoneme and the basal body. Insets, higher magnifications of cilium. Ciliary assembly was induced by culturing RPE-1 cells in medium supplemented with 0.25% serum for 48 hours. Bar, 10 µm. (B) Confocal immunofluorescence microscopy analysis, using anti-IFT88/polaris 740 (green) and anti--tubulin (GTU88, red), were performed on exponentially growing RPE-1 cells. Bars, 5 µm. Nuclei in A and B are stained with Hoechst 33342 dye. (C) Left panel, western blot analysis of Triton-X-100-soluble (S) and -insoluble (I) protein fractions from 2A1.6 cells, and of a highly enriched centrosomal fraction (CTR). Right panel, biochemical extraction of centrosome-associated IFT88/polaris. Supernatant (S) and pellet (P) of centrosomal fractions obtained under different extraction conditions (see Materials and Methods) were immunoblotted with anti-IFT88/polaris (02078), anti--tubulin (GTU88) and anti-Akt antibodies. Detection of Akt, a well-characterized cytoplasmic protein, was used as a negative control to verify the purity of the centrosomal fraction.

View larger version (49K): [in this window] [in a new window]   Fig. 2. IFT88/polaris is localized to the centrosome throughout the cell cycle. Immunofluorescence images of endogenous IFT88/polaris in HeLa cells at different stages of cell cycle that stably express GFP-centrin1. Right panel, merged images of IFT88/polaris (red), centrin1 (green), and nuclei (blue) from cells in G1, G2, prophase, metaphase, anaphase and telophase. Insets, higher magnifications of centrioles stained for GFP-centrin1 (left) and IFT88/polaris (anti-IFT88/polaris 740, middle) in cells at G1 and G2. Bars, 5 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 3. Centrosomal assembly of IFT88/polaris is not dependent on polymerized microtubules or the dynein-dynactin molecular motor. (A) Hela cells were either mock-treated (no drug) or treated with nocodazole for 1 hour and allowed to recover for 0 or 5 minutes. Cells were stained with -tubulin antibody (methanol fixation, Tub2.1, green) to follow depolymerization (0 minutes) or polymerization (5 minutes after recovery) of microtubules, or co-stained with anti-IFT88/polaris 740 (PFA 4% fixation, green) and anti--tubulin (PFA 4% fixation, red) antibodies. IFT88/polaris and -tubulin localizations were analyzed on cells treated or not with the nocodazole (n=200). Bars, 10 µm. (B) HeLa cells were transiently transfected with DsRed-p150217-548, which inhibits dynein-dynactin motor function, and co-stained with antibodies against PCM1, –tubulin or IFT88/polaris (green). The left panel shows untransfected cells with expected PCM1 localisation (greens staining, arrow) and transfected DsRed-p150217-548 cells with PCM1 mislocalization (scattered staining). Middle and right panels show that, after expression of plasmid DsRedp150217-548, centrosomal localization in transfected cells is unchanged for -tubulin and IFT88/polaris (arrows). Bars, 10 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 4. IFT88/polaris is targeted to the centrosome by its TRP domains. (A) Deletion constructs. Numbers indicate the positions of amino acids, black boxes designate the predicted coiled-coil domain of IFT88/polaris, grey boxes designate the tetratricopeptide repeat domains (TPR). Results of the immunolocalization of GFP-tagged polypeptides are summarized on the right. (B) HeLa cells were transiently transfected with GFP-IFT88/polaris full-length and, 24 hours later, processed for immunofluorescence microscopy (GFP staining). Depending on the expression level, ectopic IFT88/polaris expression resulted in the formation of a single dot (left panel) or multiple dots (right panel). The arrowhead points to the dot containing the centrosome (-tubulin labeling, data not shown). Bars, 10 µm. (C) Phenotypes observed after ectopic expression of GFP-tagged truncated IFT88/polaris polypeptides (green). Transfected cells were stained with -tubulin (red) 24 hours post transfection. Although polypeptides 1, 4 and 5 produced proteins that were not found at the centrosome (see 4 and 5), other polypeptides containing TRP domains (2, 3, 6 and 7) produced proteins that were recruited to the centrosome (see 6). Depending on expression levels, ectopic expression of mutants 2, 3, 6 and 7 resulted in the formation of a single dot (6 first panel) or of multiple dots (6, second panel). Bars, 10 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Overexpression of IFT88/polaris induces an arrest in the cell cycle before S phase and an apoptotic death. (A) HeLa cells were transfected for 36 hours to express either GFP-IFT88/polaris or GFP–C-Nap1 and these cells were treated or not with nocodazole for 12 hours. Cell-cycle-phase distribution of GFP-positive cells was assessed using propidium iodide. Proportions were estimated by triplicate measurements using FACS analysis and the software WincycleTM. (B) HeLa cells were transfected for 36 hours to express GFP-IFT88/polaris, GFP–C-Nap1 or GFP. BrdU staining was analyzed on GFP-positive cells after labeling with anti-BrdU monoclonal antibody (n=150). (C) HeLa cells were transfected for 36 hours to express GFP-IFT88/polaris, GFP–C-Nap1. These cells were synchronized by thymidine block for 12 hours and released for 6 hours. Cell-cycle-phase distribution of GFP-positive cells was assessed using iodide propidium and FACS analysis. (D) Caspase-3-like activity was measured 48 hours after transfection in GFP-positive cells (n=150). Data are from three independent experiments and presented as the mean ± s.d.

View larger version (30K): [in this window] [in a new window]   Fig. 6. IFT88/polaris downregulation causes cells proliferation. (A) Two non-overlapping IFT88/polaris siRNAs (siPol1 and siPol2) were tested and were found to produce similar results. Data obtained with siPol1 are illustrated in this figure. HeLa cells were transfected with scramble or IFT88/polaris siRNA (siPol1). Cell lysates (48 hours post transfection) were subject to western blot (left panel) using either the anti-IFT88/polaris antibody 740 (top) or an anti-actin antibody (I-19, bottom). HeLa cells transfected with scramble or IFT88/polaris siRNA (siPol1) were also immunostained (right panel) with anti-IFT88/polaris antibody 740. (B) HeLa cells were transfected with scramble or siPol1, treated with BrdU during 4 hours before the indicated time and BrdU-positive cells were analyzed (n=300). (C) Growth curves of cells transiently transfected with scramble or siPol1. (D) FACS analysis, 48 hours post tranfection. The histogram indicates the percentage of cells at a given cell cycle phase. Data are from three independent experiments and presented as the mean ± s.d.

View larger version (20K): [in this window] [in a new window]   Fig. 7. IFT88/polaris interacts with Che-1 and inhibits its interaction with Rb protein. (A) HEK293T cells were transfected with control plasmid (pcDNA3.1A) or Myc-tagged full-length IFT88/polaris (pcDNA3.1A-Tg737). Lysates (500 µg) from transfected cells were immunoprecipitated with anti-Myc antibody or anti-Che-1 antibody and analyzed by western blot using specific anti-IFT88/polaris (740) or Che-1 antibodies. (B) Lysates (1 mg) from non transfected HEK293T cells were immunoprecipitated with anti-Che-1 antibody and analyzed by western blot using a specific anti-IFT88/polaris (740) antibody. (C) HEK293T cells were transfected with control plasmid or Myc-tagged full-length IFT88/polaris. Lysates (500 µg) from transfected cells were immunoprecipitated with anti-Che-1 antibody and analyzed by western blot using specific anti-Rb, anti-IFT88/polaris (740) or Che-1 antibodies. I.P., immunoprecipitation; I.B, immunoblot.