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First published online 30 January 2007
doi: 10.1242/jcs.03366
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Research Article |
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
* Author for correspondence (e-mail: desdouets{at}cochin.inserm.fr)
Accepted 1 December 2006
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Summary |
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Key words: Polaris, Centrosome, Cilium, Intraflagellar transport, G1-S transition
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Introduction |
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; Nigg, 2002; Salisbury et al., 1999). A vertebrate centrosome comprises two barrel-shaped centrioles which are embedded within a protein-dense matrix known as the pericentriolar matrix (PCM). By nucleating and anchoring microtubules, centrosome influences most microtubule-dependent processes, including cell shape, polarity, adhesion and motility, as well as intracellular transport and positioning of organelles (Bornens, 2002; Fukasawa, 2002; Rieder et al., 2001). The centrosome also has a crucial role in cell division, mediating assembly and organization of the mitotic spindle that is required for correct chromosome segregation, and the positioning of the cleavage plane during cytokinesis (Doxsey, 2001; Piel et al., 2001). Consistent with this role, centriole duplication is coordinated with the cell cycle (Hinchcliffe and Sluder, 2001). Centrosomes are also the precursors of primary cilia, nonmotile sensory organelles found on most vertebrate cells (Badano et al., 2005; Quarmby and Parker, 2005). Typically, a single cilium projects from each cell, emerging from the elder centriole of the centrosome that has become a basal body (Sorokin, 1962). The presence of a cilium is associated with the establishment of polarity and differentiation of the cell (Wheatley et al., 1996). Thus, cilia are most abundant on non-proliferating G0 cells and entry into the cell cycle is preceded by ciliary resorption, whereas exit from mitosis is accompanied by ciliary assembly, a relationship that may reflect the use of the basal bodies and/or centrioles as mitotic spindle poles (Rieder et al., 1979).
Primary cilia assembly occurs by a process called intraflagellar transport (IFT) that was first described in Chlamydomonas (Kozminski et al., 1993; Rosenbaum and Witman, 2002). This process has recently become the focus of intense research interest owing to its association with human disease and developmental abnormalities, including polycystic kidney disease (PKD), hepatic and pancreatic defects, blindness and obesity, as well as skeletal patterning abnormalities and randomization of the left-right body axis (Hildebrandt and Otto, 2005). The first evidence that defective cilia are the proximal cause for PKD originated with the discovery that the gene mutated in the Oak Ridge polycystic kidney (Tg737orpk) mouse (Moyer et al., 1994) is homologous to a gene essential for ciliary assembly in Chlamydomonas, ift88 (Pazour et al., 2000) and in C. elegans, osm-5 (Haycraft et al., 2001; Qin et al., 2001). Whereas Tg737orpk mutants are viable, complete knockout of the mouse Tg737 gene (Tg737
2-3
-gal) is embryonic lethal (Murcia et al., 2000). Homozygous Tg7372-3-gal mutants die in early to mid-gestation and exhibit neural tube defects, random left-right body axis specification and a pronounced expansion of the anterior-posterior (AP) limb bud axis. In Tg737orpk and Tg7372-3-gal mutant mice, cilia are severely malformed or absent (Murcia et al., 2000; Pazour et al., 2000; Yoder et al., 2002a). In fact, in mammals, IFT88/polaris, the protein encoded by Tg737, is a core component of the intraflagellar transport machinery and is required for the formation of all cilia (Murcia et al., 2000; Pazour et al., 2000).
Growing evidence suggest a link between cilia, centrosome and proliferating disorders. Here, we show that the IFT protein IFT88/polaris, essential for cilia function, is also crucial for cell cycle progression. We demonstrated by immunofluorescence and biochemical analysis in proliferating cells, either ciliated or not, that IFT88/polaris is tightly associated with the centrosome throughout the cell cycle. Expression of a series of deletion constructs revealed the presence of two independent centrosome-targeting domains. Moreover, we tested whether IFT88/polaris participates in the regulation of cell cycle progression. Our results show that altered levels of IFT88/polaris concentration influence the G1-S cell cycle progression.
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; Pazour et al., 2000; Qin et al., 2001). To provide insight into its cellular distribution in proliferating ciliated cells (resorpted cilia) or in non ciliated cells, we generated polyclonal antiserum against the C-terminal region of the protein. As expected, in quiescent ciliated cells IFT88/polaris was localized to the basal body and along the axoneme (Fig. 1A). We investigated IFT88/polaris localization in proliferating cells either ciliated or not (ciliated cells, RPE-1 and CHO; non-ciliated cells, HeLa, 2A1.6) (shown for RPE-1 cells, Fig. 1B). Endogenous IFT88/polaris was found at one or two perinuclear focal points that colocalized with 
-tubulin in cells in G1 or G2 phase (Fig. 1B). This localization was confirmed biochemically (Fig. 1C). Low-speed Triton-X-100-soluble and -insoluble fractions of unsynchronized 2A1.6 cell lysates were prepared as described previously (Moudjou and Bornens, 1994) and submitted to western blot analysis together with centrosome sucrose-gradient preparations. IFT88/polaris was detected both in the Triton-insoluble fraction and, to a lesser extent, in the Triton-soluble fraction (Fig. 1C, left panel). Clearly, IFT88/polaris was as enriched in the centrosomal fraction as -tubulin (Fig. 1C, left panel). Exposure of the centrosomal fraction to various solubilizing treatments, including detergents and chaotropic agents, was done to assess the strength of this association. We found that IFT88/polaris, as well as -tubulin, can partly be solubilized in 2D buffer and about half of the protein can be solubilized in 8M urea (Fig. 1C, right panel). These results demonstrated a tight association of IFT88/polaris with the centrosome, which can only be disrupted in denaturing conditions.
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IFT88/polaris localizes to the centrosome throughout the cell cycle, independently of the microtubules network and the dynein motor
We next examined whether IFT88/polaris has a cell-cycle-dependent behaviour. For that, we used HeLa HC1 cells that constitutively expressed the centriole protein centrin1 fused to GFP (Piel et al., 2000). During G1, IFT88/polaris was present at the proximal ends of maternal centrioles (Fig. 2). In early S phase, centrioles begin to duplicate, and by G2/M, duplication is usually completed. At G2-M transition process, IFT88/polaris was still localized at the proximal ends of the mother and the daughter centrioles (Fig. 2; G2, prophase cells). By metaphase, when centrosomes become mature, IFT88/polaris staining was brighter at centrosomes than at any other cell cycle stage; by telophase IFT88/polaris staining reached its lowest levels (Fig. 2). We next asked how IFT88/polaris assembles at centrosomes, which serve as nucleation and organizing sites for cytoplasmic microtubules. In nocodazole-treated cells, IFT88/polaris remained centrosomal, as did the core centrosomal protein -tubulin (Fig. 3). We also investigated whether centrosomal assembly of IFT88/polaris depends on dynein-dynactin molecular motor by overexpressing a mutant of p150glued that antagonizes the function of the motor in vivo. We confirmed that overexpression of Dsred-p150217-548 in HeLa cells causes PCM1 mislocalization (Fig. 3). By contrast, as found for -tubulin, the centrosomal localization of IFT88/polaris was not affected in cells overexpressing Dsred-p150217-548 (Fig. 3). These data demonstrate that IFT88/polaris is assembled at the centrosome throughout the cell cycle in a microtubule- and dynein-independent manner.
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; Haycraft et al., 2001; Murcia et al., 2000; Pazour et al., 2000; Qin et al., 2001; Yoder et al., 2002b). To circumvent any phenotypes due to defects of cilia, all the following experiments have been performed in non-ciliated cells. To define sub-regions of IFT88/polaris involved in centrosomal targeting, we generated deletion mutant constructs fused to GFP either at the N-terminus or the C-terminus (mutants 1 to 7, see Materials and Methods for details) and expressed these truncated polypeptides into transiently transfected HeLa cells. As shown in Fig. 4A, the full-length sequence of IFT88/polaris contains ten copies of a tetratricopeptide repeat (TPR), with three copies located towards the N-terminus and seven repeats located closer to the C-terminus. Sequence analysis indicated also the presence of two putative coiled-coil domains. At low expression levels, exogenous full-length IFT88/polaris localized almost exclusively to the centrosome (Fig. 4B, left panel). At higher expression levels, multiple dots of various sizes were detected in cells (Fig. 4B, right panel) as previously described for the overexpression of other centrosomal proteins (Kim et al., 2004; Mayor et al., 2002); nevertheless, of several dots present, only one stained positive for -tubulin (Fig. 4B, right panel, Arrow). By analyzing the localization of truncated mutants, we demonstrated that mutants 2, 3, 6 and 7 were successfully recruited to the centrosome (Fig. 4C, example for 6). Like the full-length protein, these fusion proteins were identified as multiple dots with one that colocalized with -tubulin (Fig. 4C, example for 6) when expressed at high levels. By contrast, mutants 1 and 5 were distributed diffusely in the cytoplasm, like the GFP control protein, and colocalization with -tubulin was not observed (Fig. 4C, example for 5). Finally, distribution of mutant 4 was visible in the cytoplasm as multiple dots, but more irregular in shape and did not colocalize with -tubulin (Fig. 4C). From these experiments, we concluded that the two TPR domains were independently essential to target IFT88/polaris to the centrosome.
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Overexpression of IFT88/polaris prevents entry into S phase and induces apoptotic cell death
Since no late stages of mitosis were observed in transfected cells overexpressing IFT88/polaris, we sought to determine whether its ectopic expression caused changes in cell cycle progression. C-Nap1, a protein involved in centrosome cohesion, was used as control. Expression of GFP–C-Nap1 resulted in the formation of multiple dots without altering mitosis progression (Mayor et al., 2002). Nocodazole treated cells expressing only GFP or GFP–C-Nap1, showed a prominent accumulation of phases G2 and M (without treatment 16% of cells, with nocodazole 40%) (Fig. 5A). By contrast, the proportion of cells at G2/M was not significantly changed in cells expressing GFP-IFT88/polaris treated or not with nocodazole (19% or 21%, respectively, Fig. 5A). Thereafter, we determined whether its ectopic expression changed the progression into S phase. IFT88/polaris overexpression dramatically decreased the fraction of BrdU-positive cells at 36 hours post transfection (Fig. 5B, 4% compare with 25% and 32% for GFP and GFP–C-Nap1, respectively). The impairment of G1-S transition was confirmed by thymidine block experiments (Fig. 5C). In a final set of experiments, we studied the outcome of these cells. Morphological evaluation of cultures using Hoechst 33342 staining and fluorescent microscopy revealed a marked increase in the number of cells with nuclear condensation and fragmentation 48 hours after transfection (data not shown). Caspase 3 activity was also examined on transfected cells. As shown on Fig. 5D, only 4% of cells transfected with the GFP-control vector and 6% transfected with GFP–C-Nap1, expressed the activated form of caspase 3. By contrast, 40% of cells overexpressing IFT88/polaris were positive for cleaved caspase 3 at 48 hours post transfection (Fig. 5D). Taken together, the above results demonstrated that cells overexpressing IFT88/polaris arrested before S phase and then died by apoptosis.
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IFT88/polaris silencing promotes cell cycle progression to S, G2 and M phases
These results prompted us to analyze the growth characteristics of cells depleted from IFT88/polaris. Two non-overlapping IFT88/polaris small interference RNAs (siRNAs) (siPol1 and siPol2) were tested and produced similar results. Cells transfected with siPol1or siPol2 showed a specific reduction of about 60% of IFT88/polaris expression compared with control (scramble) transfected cells (Fig. 6A, left panel). We also confirmed by immunofluorescence staining that IFT88/polaris was greatly reduced at the centrosome in most cells (Fig. 6A, right panel). In contrast to cells overexpressing IFT88/polaris, reduction of the endogenous protein activated entry into S phase as demonstrated by BrdU labeling (from 21.1%±0.4 in control cells to 45.6%±2.3 in siPol1 cells at 48 hours, Fig. 6B). In fact, cells expressing reduced levels of IFT88/polaris protein exhibited an increased rate of proliferation (Fig. 6C). Furthermore, flow cytometric analysis for DNA content showed that inhibition of IFT88/polaris resulted in a significant reduction in cells in G1 (70.8%±2 with scramble siRNA, 50.3%±2.7 with siPol1) and a significant increase in cells in S phase (18.1%±3.6 with scramble siRNA, 31.1%±3.4 with siPol1) and in G2 and M phases (11.1%±1.9 with scramble siRNA, 18.6%±1 with siPol1) (Fig. 6D). In conclusion, IFT88/polaris silencing promoted cell-cycle progression to S and G2/M phases.
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Identification of IFT88/polaris downstream signaling partners involved in cell cycle regulation
To identify IFT88/polaris targets in cell-cycle regulation, we performed a yeast two-hybrid screening. The mouse full-length IFT88/polaris protein was used to screen a human fetal liver cDNA library. From the 3x106 yeast transformants obtained, we selected six clones that fulfilled our selection criteria. One of these clones contained the cDNA encoding amino acids 392 to 558 of Che-1 protein. Che-1 is known to interact with Rb protein and this binding affects the growth-suppressing function of Rb by relieving its inhibition of E2F1 transcriptional activity essential for G1-S transition (Bruno et al., 2002; Fanciulli et al., 2000). To test the specificity of this interaction in mammalian cells, we immunoprecipitated extracts from HEK293T cells that had been transfected with Myc-tagged full-length IFT88/polaris plasmid, with anti-Myc or anti-Che-1 antibodies. We demonstrated that Myc-tagged full-length IFT88/polaris could be coimmunoprecipitated by endogenous Che-1 (Fig. 7A, upper panel) and conversely, endogenous Che-1 could be coimmunoprecipitated with full-length IFT88/polaris (Fig. 7A, lower panel). More importantly, we observed interaction between endogenous IFT88/polaris and Che-1 (Fig. 7B). In order to understand the mechanism of action of IFT88/polaris at the G1-S transition, we analyzed whether the interaction between Che-1 and Rb is affected by IFT88/polaris overexpression. As shown in Fig. 7C, a reduced amount of Rb was immunoprecipitated by Che-1 in the cells overexpressing IFT88/polaris. Taken together, these results demonstrated that IFT88/polaris interacted with Che-1 and, thus, modulated its binding to Rb protein.
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). Here, we show that IFT88/polaris is tightly associated at the proximal end of centrioles throughout the cell cycle. This association is dependent on IFT88/polaris TPR domains and independent of the microtubule network and the dynein-dynactin motors. IFT88/polaris centrosomal localization is conserved in non-ciliated cells, suggesting an additional role beyond the classic IFT system.
A recent convergence of data has indicated an intimate regulatory relationship between ciliogenesis and cell cycle progression. Thus, members of protein families known for their mitotic functions play roles in ciliary assembly. For instance, a distant paralog of aurora kinase is essential for flagellar disassembly in Chlamydomonas (Pan et al., 2004). The centrosomal protein pericentrin, essential for correct spindle organization (Zimmerman et al., 2004), forms a complex with IFT proteins and is required for assembly of primary cilia (Jurczyk et al., 2004). However, proteins that are essential for cilia function appear to be indispensable for cell cycle progression. The FA2p protein, discovered in a genetic screen for Chlamydomonas mutants defective in deflagellation (Finst et al., 1998), is essential for G2-M transition (Mahjoub et al., 2002). Rootletin, a major structural component of the ciliary rootlet (Yang et al., 2002), regulates centrosome cohesion and separation during the cell cycle (Bahe et al., 2005; Yang et al., 2006). Indeed, the association between cilia and proliferative diseases has already raised the idea that cystoproteins provide a functional signal relay between the cilium, the centrosome and the cell cycle. Inversin, a protein of the axoneme, basal body and centrosome, binds to the anaphase-promoting-complex protein Apc2 (Morgan et al., 2002). Moreover, the Bardet-Bield syndrome protein BBS4, a protein of the basal body and the centrosome, has been shown to target pericentriolar material 1 (PCM1) to the pericentriolar region and, therefore, to control cell division (Kim et al., 2004). Here, we show for the first time that an IFT protein controls also cell cycle progression. Recently, another IFT component, IFT20 has also been shown to localize to the centrosome; nevertheless, its knockdown has no effect on cell cycle progression (Follit et al., 2006). In our case, alteration of the levels of IFT88/polaris in non-ciliated cells induces the following cell cycle defects: (1) overexpression of IFT88/polaris interferes with the G1-S cell cycle transition leading to a higher incidence of apoptotic cell death; (2) by contrast, inhibition of IFT88/polaris expression leads to cell-cycle progression to S and G2/M phases. Previous studies report in Tg737orpk mouse model that the pathology is associated with epithelial cell hyperproliferation (Richards et al., 1996; Zhang et al., 2005). On the basis of our findings, we propose that IFT88/polaris controls proliferation by regulating G1-S transition. Consistent with this idea, we demonstrate that IFT88/polaris interacts with Che-1, a key regulator for S phase entry. The role of Che-1 in cell proliferation is clearly linked to the growth-suppressing function of Rb (Bruno et al., 2002). Furthermore, we show that IFT88/polaris modulates Che-1 binding to Rb. Thus, we hypothesize that the overexpression of IFT88/polaris interferes with the G1-S cell cycle transition by titrating out Che-1, which is normally required at this crucial stage. It is tempting to speculate that, during cell cycle progression, IFT88/polaris, by interacting with Che-1, regulates its localization and/or its activity. It will be of interest to test this possibility and to identify other partners in order to better understand the molecular mechanisms by which IFT88/polaris controls G1-S transition.
The challenge is now to determine how dysfunction of primary cilia impacts upon processes such as cell cycle regulation. In vertebrates, primary cilia function as environmental sensors to transfer information from the extracellular space into the cell body (Davenport and Yoder, 2005). The ciliary membrane and cytoplasm are relatively isolated from cell body which offers the advantage of compartmentalization (Anderson, 1972), whereas IFT proteins allow for rapid transport of proteins between cilium and cell body (Kozminski et al., 1993). Cilia may provide information that help cells to remain in a differentiated state. Because cystoproteins change their subcellular localization according to the cell-cycle stage (Mollet et al., 2005; Morgan et al., 2002; Nurnberger et al., 2004; Sayer et al., 2006), cilia resorption may permit the redistribution of IFT proteins and/or ciliary proteins in the rest of the cell and, thus, promote cell-cycle progression. This model is fully compatible with our results on IFT88/polaris and suggests the existence of a mechanism that regulates proteins by shuttling them to the nucleus, the centrosome or to cilia.
Finally, we report a new cellular function for a component of the mammalian IFT by demonstrating that IFT88/polaris has a direct role in cell cycle progression. C. elegans, Trypasoma brucei and Chlamydomonas mutants for IFT88/polaris have not been shown to have defects in cell proliferation (Haycraft et al., 2001; Kohl et al., 2003; Pazour et al., 2000). This suggests that IFT88/polaris has others functions in higher eukaryotic organisms. In agreement with this idea, recent studies have demonstrated that IFT88/polaris is implicated in the regulation of the Sonic hedgehog signaling in mice (Liu et al., 2005); however, IFT mutations in Drosophila do not disrupt this pathway (Han et al., 2003). We can assume that polaris and, more generally, IFT proteins play roles in signaling pathways in vertebrates.
We are convinced that certain ciliary proteins are components of the cell cycle. Defining their role in this process will lead to a better understanding of the molecular mechanism involved in proliferative diseases such as PKD. Our findings may facilitate the understanding of the link between ciliary function and cellular response, and provide an entry point to define the dysfunctions associated with abnormal cilia formation.
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-mercaptoethanol, 5 mM sodium pyruvate, and penicillin and streptomycin (100 IU/ml and 100 µg/ml, respectively). All the cell lines were incubated at 37°C with 5% CO2 atmosphere.
Construction of expression vectors
Constructs encoding the different domains of IFT88/polaris were engineered by PCR using the IFT88/polaris encoding cDNA as a template (Tg737 cDNA 5.5). PCR fragments were then subcloned into the multicloning site of eukaryotic expression vectors: (1) pEGFP-N2 (Clontech), in fusion with GFP in C-terminal, (2) pGFP-C2, in fusion with GFP in N-terminal and (3) pcDNA3.1A (Invitrogen). All constructs were sequenced to verify intact reading frames. The series of deletion constructs used in this study is listed in Fig. 4A and were cloned in pEGFP-N2 (Clontech): 1 (amino acids 1-185), 2 (amino acids 1-430), 3 (amino acids 185-430), 4 (amino acids 294-441), 5 (amino acids 676-824), 6 (amino acids 330-824), 7 (amino acids 441-676).
Antibodies
Two antibodies against IFT88/polaris were generated according to the protocol of Agro-Bio (La Ferté St Aubin, France). The first antibody, IFT88/polaris-02078, was generated in rabbits by using a 21-residue-long peptide (NVHLAPETDEDDLYSGFNDYN) starting at position 3 of the mouse protein. For the anti-IFT88/polaris-740, a 1050-bp EcoRI digestion fragment of mouse IFT88/polaris (corresponding to amino acids 474-824) was cloned into the EcoRI site of the glutathione-S-transferase (GST) expression vector pGEX 4T1 (Pharmacia). Large-scale protein inductions were carried out, and the GST-IFT88/polaris fusion proteins were injected into rabbits for the production of polyclonal antibody. Commercial antibodies against -tubulin (GTU88, Sigma), 
-tubulin (B-5-1-2, Sigma), acetylated tubulin (6-11B-1, Sigma), actin (I-19, Santa Cruz), Myc (9E10, Santa Cruz), Rb (sc-50, Santa Cruz), Akt (Cell Signaling) and cleaved caspase 3 (Asp175, Cell Signaling) were also used according to the manufacturer's instructions. Secondary antibodies against rabbit and mouse IgG conjugated to Alexa Fluor-488 (Molecular Probes) or Texas Red (Jackson) were used as well.
Centrosome subfractionation
Soluble, insoluble and centrosome fractions were isolated from 2A1.6 lymphocytes as described previously (Moudjou and Bornens, 1994). Pelleted centrosomes were incubated for 1 hour at 4°C with extraction buffer (20 mM Tris-HCl pH 7.4, 2 mM EDTA) alone, with 1D buffer (extraction buffer containing 0.5% NP-40 and 0.5% deoxycholate), with 2D buffer (extraction buffer containing 0.5% NP-40, 0.5% deoxycholate and 0.1% SDS) or with 8 M urea. Proteins were then fractionated into pellet (P) and supernatant (S) by centrifugation at 10,000 g for 15 minutes. Proteins were detected by various antibodies and visualized by enhanced chemiluminescence (ECL, Amersham Biosciences).
Transfections, drug treatments, and FACS analysis
For transient transfection, HeLa cells were seeded onto glass coverslips (22x22 mm; 3x105 cells) and transfected with 1 µg of plasmid DNA using transfecting reagent exgen500 (Euromedex). At the indicated time point post transfection, cells were washed with PBS and fixed either with 4% paraformaldehyde (PFA) (15 minutes at 4°C) or methanol (6 minutes at –20°C). For synchronization experiments, cells were treated with either thymidine (2.5 mM, for 16 hours) or with nocodazole (165 nM, for 12 hours). For microtubule depolymerization and regrowth, HeLa cells were incubated in 25 µM nocodazole at 37°C for 1 hour. Following drug removal, cells were incubated for the indicated times to allow microtubule regrowth, and fixed and processed for immunofluorescence as described above. For FACS analysis, cells were collected by trypsinization and fixed in 70% ethanol-PBS overnight at 4°C. Cells were then incubated with RNAse (100 µg/ml) for 30 minutes at room temperature, stained with 15 µg/ml propidium iodide and analyzed on a Beckman Coulter flow cytometer using a gate on GFP-positive cells. The percentages of cells in G1 and S phases and at G2-M transition were determined using the software WincycleTM.
Immunofluorescence
Cells grown on glass coverslips were fixed either in 4% PFA for 15 minutes at 4°C and permeabilized for 2 minutes with ice cold methanol or fixed and permeabilized directly for 6 minutes with ice cold methanol. After blocking with 10% goat serum in PBS (30 minutes), cells were incubated for 1 hour with primary antibodies, washed in PBS containing 0.1% Tween 20 and incubated in secondary antibodies for 30 minutes, all at room temperature. Hoechst 33342 (0.2 µg/ml, Sigma) was included in the final wash to counterstain nuclei. Samples were mounted on slides in fluorescent mounting medium (DakoCytomation). The percentage of cells undergoing DNA synthesis was estimated by counting the number of BrdU-labeled cells using the BrdU Detection Kit II (Roche). Images were usually taken using a Nikon Statif Eclipse E600 microscope with 63x magnification, 1.4-0.7 NA PL-APO objectives, a DXM1200 cooled CCD camera (Nikon) and ACT-1 (Universal Imaging). For studies of colocalizations with centriolar markers, z-axis stacks were collected using a piezoelectric device mounted at the base of a 63x magnification, 1.4 NA PL-APO objective on a Leica DMRA2 microscope, a Coolsnap HQ camera controlled by Metamorph software (Universal Imaging) was used. The z-axis stacks were compiled as single 2D projections using ImageJ software. All images were imported into Adobe Photoshop v5.0 for contrast adjustment and figure assembly.
Knockdown of IFT88/polaris expression using siRNA
We synthesized 21-mer nucleotide siRNA duplexes (designed by oligoengine), comprised of a 19 nucleotide duplex corresponding to IFT88/polaris in HeLa cells (siPol1, position 667 from start codon: CCGAAGCACUUAACACUUA; siPol2, position 1650 from start codon: CUGAAACUUCACGCAAUCC) and dTdT overhangs at each 3'-end (Sigma-Proligo). A non-silencing siRNA-scramble was also used as control. For transfection, each duplex was mixed with oligofectamine (Invitrogen) according to the manufacturer's protocol and introduced (24 hours after plating) into cells cultured in 6-well plates. Analyses were performed 24 and 48 hours post transfection.
Yeast two hybrid screen
The entire coding sequence of mouse IFT88/polaris was subcloned into the bait plasmid pBTM116 to create pBTM116-Tg737. The Saccharomyces cerevisiae strain Y190, carrying Gal4-LacZ and Gal4-histidine fusion genes, was transformed with the bait construct and a human liver cDNA library (clontech) in the prey plasmid, pACT2. Transformants were first selected for histidine prototrophy and then assayed for induction of -galactosidase activity. Plasmids from positive clones were rescued into Escherichia coli and re-transformed into L40 containing either pBTM116-Tg737 or pBTM116. The sequences were compared with the database using the blast program.
Immunoprecipitation
For immunoprecipitation experiments cells were lysed at 4°C for 30 minutes in lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.5 mM EDTA, and 0.5 mM EGTA, protease inhibitor cocktail (Roche). After high-speed centrifugation, the lysates were immunoprecipitated by standard procedures using mouse anti-Myc monoclonal antibody clone 9E10 (Santa Cruz) or as described previously with Che-1 antibody (Bruno et al., 2002).
Data analysis and statistic
All values for statistical significance represent the mean ± standard deviation (s.d.). Mean values were compared using the Student's t-test. All comparisons of statistical significance have P values of P<0.05.
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