RESEARCH ARTICLE Cytoskeleton, Month 2010 000:000–000 (doi: 10.1002/cm.20433) C V

2010 Wiley-Liss, Inc.

Basal Body Duplication in Paramecium: The Key Role of Bld10 in Assembly and Stability of the Cartwheel Maria Jerka-Dziadosz,1 Delphine Gogendeau,2,3 Catherine Klotz,2,3 Jean Cohen,2,3 Janine Beisson,2,3 and France Koll2,3* 1

Department of Cell Biology, M. Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland CNRS, Centre de Ge´ne´tique Mole´culaire, FRE 3144, 91198 Gif-sur-Yvette, France 3 Universite´ Paris-Sud, 91405 Orsay cedex, France 2

Received 6 November 2009; Revised 28 December 2009; Accepted 5 January 2010 Monitoring Editor: Ritsu Kamiya

Basal bodies which nucleate cilia and flagella, and centrioles which organize centrosomes share the same architecture characterized by the ninefold symmetry of their microtubular shaft. Among the conserved proteins involved in the biogenesis of the canonical 9-triplet centriolar structures, Sas-6 and Bld10 proteins have been shown to play central roles in the early steps of assembly and in establishment/stabilization of the ninefold symmetry. Using fluorescent tagged proteins and RNAi to study the localization and function of these two proteins in Paramecium, we focused on the early effects of their depletion, the consequences of their overexpression and their functional interdependence. We find that both genes are essential and their depletion affects cartwheel assembly and hence basal body duplication. We also show that, contrary to Sas6p, Bld10p is not directly responsible for the establishment of the ninefold symmetry, but is required not only for new basal body assembly and stability but also for Sas6p maintenance at mature basal bodies. Finally, ultrastructural analysis of cells overexpressing either protein revealed two types of early assembly intermediates, hub-like structures and generative discs, suggesting a conserved scaffolding process. V 2010 Wiley-Liss, Inc. C

Key Words:

cartwheel, basal-body, centriole, SAS-6, Bld10p/Cep135, ninefold symmetry, pre-assembly stages

Additional Supporting Information may be found in the online version of this article. Delphine Gogendeau’s present address is CNRS, Institut Curie, UMR 144, F-75005 Paris. *Address correspondence to: France Koll, CNRS, Centre de Ge´ne´tique Mole´culaire, Avenue de la Terrasse, Gif-sur-Yvette 91198, France. E-mail: [email protected] Published online in Wiley InterScience (www.interscience. wiley.com).

Introduction

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tudies on centriole duplication in human cells, Caenorhabditis and Drosophila and basal body biogenesis in protists such as Chlamydomonas, Tetrahymena, or Paramecium led to the discovery of conserved proteins involved in centriolar structure duplication among which are delta, epsilon and gamma tubulins, centrins, SAS-4/CPAP/CENPJ, SAS-6, Cep135/Bld10 [reviewed in Bettencourt-Dias and Glover, 2007]. SAS-6 and Bld10/Cep135 are coiled coil proteins, the first one discovered in Caenorhabditis [Dammermann et al., 2004; Leidel et al., 2005] and the second one in Chlamydomonas [Matsuura et al., 2004] and human cells [Ohta et al., 2002] respectively. These proteins are of particular interest as they have been shown in Chlamydomonas to be specific constituents of the cartwheel [Matsuura et al., 2004; Hiraki et al., 2007; Nakazawa et al., 2007]. The cartwheel constitutes a scaffolding structure for the ninefold symmetry of the centriole or basal body formed of a stack of discs, each comprising a central hub from which emanate nine spokes, as particularly well illustrated in Chlamydomonas [O’Toole et al., 2003; Geimer and Melkonian, 2004]. While a cartwheel is present in all pro-centrioles or pro-basal bodies, developing by either the canonical/ templated or the de novo pathway [Anderson and Brenner, 1971; Tamm and Tamm, 1980] it will either disappear from the mature centriole as in mammalian cells [Vorobjev and Chentsov, 1982; Alvey, 1986] or be stably retained in the mature basal body as in Chlamydomonas [Cavalier-Smith, 1974] and other protists [Dippell, 1968; Allen, 1969]. Interestingly, in C. elegans which harbors a simplified centriole of smaller diameter, devoid of cartwheel and organized around a central tube, with only nine microtubule singlets instead of the canonical nine triplet organization, both Bld10p/Cep135 and cartwheel are absent. Conversely, in both Chlamydomonas and human cells, SAS-6 and Bld10/Cep135 were shown to be partly interdependent [Kleylein-Sohn et al., 2007; Nakazawa et al., 2007] for basal body and centriole biogenesis. To gain further insight into their functional relationships, it seemed of 1

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interest to investigate the roles of both SAS-6 and Bld10p/ Cep135 in the ciliate Paramecium, a study previously carried out in parallel in Chlamydomonas [Hiraki et al., 2007; Nakazawa et al., 2007]. Paramecium provides a particularly suitable model to analyze the biogenesis of ciliary basal bodies with a permanent cartwheel, but also presents specific experimental advantages: easy detection and precise analysis of defective basal body duplication [Beisson et al., in press] simple and efficient RNAi procedures [Galvani and Sperling, 2002]. By the localization of GFP-PtSas6p and GFP-PtBld10p proteins and RNAi experiments, we show that both proteins are required for basal body assembly, are constituents of the cartwheel where they localize at distinct sites and that PtSas6p is essential for establishment of the ninefold symmetry. This functional analysis extends to ciliates and ciliary basal bodies the conclusions of previous studies on Chlamydomonas [Hiraki et al., 2007; Nakazawa et al., 2007], mammalian cells [Kleylein-Sohn et al., 2007], and Drosophila [Rodrigues-Martins et al., 2007; Blachon et al., 2009]. Furthermore, our observations provide two new insights into basal body assembly and stability. We demonstrate for the first time that, in addition to its role in basal body assembly PtBld10p is also required for PtSas6p maintenance at mature basal bodies and therefore plays a pivotal role in cartwheel stability. In addition, ultrastructural analysis of cells overexpressing GFP-PtSas6p or GFP-PtBld10p revealed several pre-assembly structures: an electron dense generative disc as first described by Dippell [Dippell, 1968] and observed in Chlamydomonas [Cavalier-Smith, 1974] as well as a ‘‘hub’’ reminiscent of the central tube of C. elegans [Pelletier et al., 2006] and of the hub with some spokes devoid of microtubules. Such stages have been not clearly identified previously except in certain ‘‘de novo’’ assembly process [Tamm and Tamm, 1980]. These observations, which suggest a conserved sequence of assembly steps for the centriolar structure, will be discussed in relation with the functional and morphological data obtained in the other models.

Materials and Methods Culture Conditions Two strains of Paramecium tetraurelia were used: the wild type stockd4-2 and the mutant nd7-1 which carries a recessive mutation at the ND7 locus preventing trichocyst exocytosis [Skouri and Cohen, 1997]. Cells were grown at 27! C in a wheat grass infusion, BHB (L’arbre de vie) or WGP (Pines International), bacterised with K. pneumoniae and supplemented with 0.8 lg/ml b-sitosterol [Sonneborn, 1970]. Gene Identification By BLAST search at ParameciumDB [Arnaiz et al., 2007] we identified four Paramecium genes encoding 594 amino acid proteins homologous to SAS-6: PtSAS6a, PtSAS6b, PtSAS6c, and PtSAS6d (GSPATG00011824001, GSPATG00008149001, GSPATG00005603001, and GSPATG00024268001). These

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genes could be separated in two subfamilies (PtSAS6A and PtSAS6B), each containing two sequences resulting from the last whole genome duplication [Aury et al., 2006]. Two genes encoding 1295 amino acid proteins homologous to the Bld10 protein of Chlamydomonas [Matsuura et al., 2004] were identified. The two genes, PtBLD10a (GSPATG0000943800) and PtBLD10b (GSPATG00019908001), result from the last whole genome duplication. Phylogenetic Analysis Phylogenetic analyses were performed at http://www.phylogeny.fr/ using the « a` la carte mode » [Dereeper et al., 2008]. Alignments were performed with Clustal and the phylogenetic reconstruction was done using PHYLIP (neighborjoining method) with the JTT model for amino acid substitution. The bootstrap values were calculated generating 1000 replicates and the tree reconstructed by using Treedyn. Gene Cloning The coding region of Paramecium SAS6a, SAS6c, and BLD10 genes were amplified from genomic DNA by PCR using specific primers into which a linker containing KpnI restriction sites was added. These fragments were cloned into the KpnI restriction site at the 30 end of the GFP synthetic gene that was designed by Meyer and Cohen and which had been introduced into the pPXV vector, the recombinant gene being under the control of the Paramecium calmodulin regulators. After cloning, the genes were entirely sequenced. The synthetic gene encoding the mRFPmars protein [Fischer et al., 2004] was amplified by PCR and cloned at the KpnI restriction site located at the Nterminal part of the PtSAS6 gene integrated into the pPXV vector. For gene silencing, only a part of each gene was amplified by PCR and cloned into the L4440 [Timmons and Fire, 1998] vector or into the Litmus 28 vector (NEB) wherein the EcoR1 site was replaced by a Srf1 site. The PCR fragments were cloned into this Srf1 site according to the protocol of the pPCRscriptTM Cloning Kit (Stratagene). These vectors allow synthesis of double-stranded RNA from two T7 promoters. The constructs were introduced by transformation into the HT115 E. coli strain. Paramecium Transformation nd7-1 cells unable to discharge trychocysts were transformed by micro-injection into their macronucleus [Gilley et al., 1988] of DNA containing a mixture of the plasmids of interest (5 lg/ll) and of plasmid DNA directing the expression of the ND7 gene [Skouri and Cohen, 1997]. Transformants were screened for their ability to discharge their trychocysts and further analyzed. Gene Silencing Gene silencing was performed by the feeding method [Galvani and Sperling, 2002]. Wild type cells were fed with double-stranded-RNA-expressing bacteria and transferred daily

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into fresh medium. Control cells were fed with bacteria carrying the complete coding region of the ND7 gene. To inactivate PtSAS6, cells were fed with two different strains producing dsRNA homologous to the 1336 bp 50 -terminal part of PtSAS6c and to the 1340 bp 50 terminal part of PtSAS6b respectively. Feeding with a single strain is expected to inactivate only one subfamily, whereas mixture of the two types of bacteria is expected to inactivate all four PtSAS6 genes. Indeed each produced dsRNA was 87% identical to the gene of the same subfamily with a total of seven segments of perfect identity over " 23 bp [Garnier et al., 2004]. To inactivate PtBLD10, we used bacteria producing dsRNA homologous to the 683 bp fragment extending from base 6 to 689 of PtBLD10a. Fluorescence Microscopy Immuno-staining of cells was carried out as previously described [Beisson et al., 2001]. The monoclonal anti-tubulin ID5 (1:100) [Wehland and Weber, 1987], generously provided by J. Wehland, and the polyclonal anti-GFP antibody (1:100, Interchim), were used with the appropriate secondary antibodies from Jackson ImmunoResearch labs (West Grove) at a dilution of 1:500. Cells were observed under a Zeiss Axioskop 2-plus fluorescence microscope equipped with a Roper Coolsnap-CF intensifying camera with GFP filters. Images were processed with Metamorph software (Universal Imaging). Electron Microscopy For TEM, cells were fixed in 1.5% glutaraldehyde and 1% osmic acid in cacodylate buffer (Sigma) for 1 h on ice, washed three times in the buffer, then in H2O. Both random samples from growing cultures and groups of five to ten individual dividing cells were embedded into 2% Agarose (Miles-Seravac). Agar blocks were then dehydrated in graded series of EtOH or Acetone and embedded in Durcupan (Sigma). Thin sectioned samples were contrasted in uranyl acetate and lead citrate and photographed on Electron Microscopes JEM 1200 EX, or registered digitally on EM JEM1400 equipped with Morada 11 Mpx and Olympus iTEM software. Measurements of basal bodies were made using the ImageJ (NIH) software [Abramoff et al., 2004]. For Immuno Electron Microscopy, samples of ca 20 cells were incubated in 100 ll of 1% Triton in PHEM buffer [Schliwa and van Blerkom, 1981] for 3 min, and fixed for 10 min in 4% paraformaldehyde in PHEM buffer. After 2 # 10 min washes in TBST* buffer [Keryer et al., 1990] containing 3% BSA, cells were incubated for 1 h with a polyclonal anti-GFP antibody 1/50 in TBST*/BSA, washed and incubated in 1/50 IgM GAR (Sigma) for 1 h. After two washes in TBST*BSA and two washes in PBS, cells were fixed in 2% glutaraldehyde in PBS in 4! C overnight. Cells were then embedded in agar blocks, dehydrated in graded series of EtOH or acetone and embedded in Durcupan (Sigma). Sections were contrasted with uranyl acetate and lead citrate and observed in TEM.

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Results Four PtSAS6 and Two PtBLD10/CEP135 Genes in Paramecium The four SAS6 genes identified in Paramecium (PtSAS6a, PtSAS6b, PtSAS6c, and PtSAS6d) correspond to two subfamilies (PtSAS6A and PtSAS6B) coding for proteins which share 97% similarity within a subfamily and 93% between subfamilies. The four Sas6 proteins (Supporting Information Fig. S1A) contain the conserved PISA domain [Leidel et al., 2005]. In Tetrahymena, two Sas6 proteins sharing only 57% similarity have been reported to have distinct functions [Culver et al., 2009]. When separately aligned with these two Tetrahymena Sas6ps, all four Paramecium sequences turned out to be closer to TtSas6bp (Supporting Information Fig. S1B). The two PtBLD10 genes, issued from the last Paramecium whole genome duplication, share 87% identity. RNA-seq data (Meyer, Duret and Saudemont personal communication) indicate that the four PtSAS6 and the two PtBLD10 genes are all transcribed. Hereafter we will refer to PtSAS6 as SAS6 and PtBLD10 as BLD10. GFP-Sas6p and GFP-Bld10p Localize at Basal Bodies, at Distinct Sites of the Cartwheel The localization of Sas6p and Bld10p was examined on transformant cell lines expressing GFP-Sas6ap, GFP-Sas6cp, or GFP-Bld10ap. In the three cases, and at all stages of the cell cycle, the GFP signal localized exclusively at the basal body as demonstrated by double labeling with an anti-GFP and the monoclonal anti-tubulin ID5 (Supporting Information Fig. S2A). The specificity of the GFP-labeling was ascertained by direct visualization of the GFP signal on fixed transformant cells (Supporting Information Fig. S2B) and by the disappearance of the fluorescence upon silencing of the corresponding gene in the transformants (not shown). The co-localization of the two proteins at basal bodies was further demonstrated by observing transformants expressing both RFP-Sas6p and GFPBld10p (Supporting Information Fig. S2C). In agreement with all previous observations on Chlamydomonas [Hiraki et al., 2007; Nakazawa et al., 2007] immuno-electron microscopy observations demonstrate that the two proteins localize at distinct sites within basal bodies, GFP-Sas6p at the hub and GFP-Bld10p at the spokes of the cartwheel (Supporting Information Fig. S3). These localizations are further supported by observations in human cells [Kleylein-Sohn et al., 2007]. It can be noted that in Paramecium, GFP-Sas6p is detected at the site of new basal body assembly before assembly of the microtubular shaft (Fig. 5A and Supporting Information Fig. S3B).That GFP-Bld10p is recruited at the new basal body sites before microtubule assembly can be also visualized in immunofluorescence at the whole cell level (Supporting Information Fig. S4). Sas6p and Bld10p are Essential in Early Steps of Basal Body Assembly To ascertain the function of SAS6 and BLD10 in Paramecium, gene silencing experiments were carried out by the

Bld10p Stabilizes SAS-6p at the Cartwheel

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Fig. 1. Basal body duplication is inhibited in SAS6 and BLD10 silenced cells. BLD10 (A) and SAS6 (C) silenced cells were fixed by their first division after transfer into the feeding medium and labeled with the ID5 antibody. In the middle panel (B), a control cell at the same mid-division stage displays the characteristic longitudinal groups of four basal bodies issued from parental groups of two. Inhibition of basal body duplication is attested by the many single dots and by a significantly reduced density of basal bodies in the silenced cells. Bar: 10 lm. Insets: enlargement X4. D and E: These sections through the same SAS6 cell silenced for the only B subfamily shows one unit (D) with two old basal bodies (obb) and a newly formed basal body (nbb) not yet parallel to the old bbs, and another unit (E) where no structure is visible at the site (arrow) of development of the new basal body. Evidence that activation of basal body duplication has taken place in E as in D is provided by the fact that the anterior old basal body of the pair has developed its own ciliary rootlet (cr).

feeding technique [Galvani and Sperling, 2002]. Figure 1 shows the defects induced by the depletion of Sas6p and Bld10p on cells undergoing their first division under silencing conditions and labeled by the ID5 antibody. The well mapped spatio-temporal pattern of basal body duplication during division [Iftode et al., 1989] allow detection of any defect in their localisation or duplication. The numerous unduplicated basal bodies (single dots on Figs. 1A and 1C) in Sas6p or Bld10p depleted cells demonstrate that depletion of either protein blocks new basal body assembly. In the case of the SAS6 genes, the effects of inactivation of each subfamily separately and both simultaneously were studied. The terminal phenotype was the same in all three conditions. However, upon inactivation of a single subfamily (Figs. 1D and 1E), the phenotype was established more slowly, suggesting that the two SAS6 subfamilies concur in fulfilling the same function. At the ultrastructural level (Fig. 2), the polarity landmarks (ciliary rootlet or microtubule ribbons) which flank each preexisting basal body unambiguously mark old versus neoformed basal bodies. In cells undergoing their first, second or third division under SAS6 inactivation conditions, in all the observed neo-formed basal bodies (n ¼ 116, from three different samples), the cartwheel was either absent (40%) or abnormal. Although defective basal bodies appear devoid of the central hub, they still harbor spokes connected to the microtubules (Figs. 2F and 2G). Longitudinal sections show a significant (ca. 20%) length reduction after two to three divisions (Fig. 2D, Table I). In addition, transverse sections of 41 basal bodies showed three basal bodies containing eight regularly arranged microtubule triplets instead of nine (Figs.

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2E and 2F) and a few basal bodies in which microtubule triplets were mis-positioned (Fig. 2C). In cells undergoing their first division under BLD10 silencing conditions, partly distinct defects (Figs. 2H–2N) were observed. Although weaker than under Sas6p depletion, some length reduction of basal bodies (12%) was also observed (Table I). In 70% of either transverse or cross-sections (n ¼ 48), the cartwheel appears disturbed or absent: the hub is present, sometimes in duplicate (Fig. 2I), but the spokes are missing or abnormal (Fig. 2N); in other cases, tiers of the cartwheel are not properly attached to the peripheral triplets and many abnormal nascent basal bodies have misplaced microtubules (Fig. 2K). The surprising occurence of two hubs may result of an unbalanced level of Sas6p, leading to erratic number/positioning of hubs in the absence of constraining interactions with Bld10p. After 48 h of inactivation, about 38% of new basal bodies (n ¼ 36) appeared as incomplete rings with empty triplet sites or mis-localized microtubules (Figs. 2J and 2L). All these observations point to a role of Bld10p in proper microtubule arrangement or localisation at the cartwheel spokes. Finally, rare anomalies in axoneme structure—e.g., missing outer doublets—were recorded (Fig. 2M), which most likely reflected defects in the corresponding basal body rather than a direct role of Bld10p in axoneme structure, as recently documented in Drosophila [Mottier-Pavie and Megraw, 2009]. Despite some overlap between the phenotypes observed under depletion of either Sas6p or Bld10p, distinct ultrastructural defects are nevertheless observed, marked length reduction in the case of Sas6p, disordered microtubular shaft in the case of Bld10p.

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Fig. 2. Ultrastructural abnormalities of basal bodies in SAS6 and BLD10 silenced cells. A and B: In control cells, a transversal (A) and longitudinal (B) section of basal bodies reveal the normal ultrastructure of the cartwheel (cw), showing in cross-section the central hub (hb) and radial spokes (sp) and the nine microtubular triplets. The ciliary rootlet (cr) points to the right and anterior of the basal body and the microtubule ribbons (mr) mark the left site of the basal body. C–G: Sas6p depleted cells (RNAi SAS6) fixed 18 or 72 h after feeding exhibit diverse basal body defects: displaced triplets (C) in the anterior, i.e., newly formed basal body (nbb), shorter microtubule barrel (D), missing triplets arranged with eightfold symmetry (E–F), absence of structured cartwheel in the neo-formed anterior basal body (G). H–N: Bld10p depleted cells (RNAi BLD10) exhibit different basal body defects. H: A longitudinal section through a mature basal body shows absence of a cartwheel and unequal length of microtubule triplets (arrow). I: Abnormal hub and spoke organization. J: A triplet is missing (star) in a basal body displaying a ninefold symmetrical pattern. K: Probasal body at orientation perpendicular to the mother basal body with irregular number and arrangement of microtubular triplets indicated by arrows. L: Three missing triplets (star). M: Cross-section of a ciliary axoneme with missing doublets (arrow). N: Disorganized hub and spokes (arrow). Bar: 100 nm.

Bld10p is Required for Stable Integration of Sas6p at the Basal Bodies In order to ascertain the functional relationships between Sas6p and Bld10p, we performed cross-inactivation experiments and thus examined the effect of inactivation of the SAS6 genes in GFP-Bld10p expressing cells and of inactivation of the BLD10 genes in GFP-Sas6p expressing cells (Fig. 3). After two to three divisions under SAS6 silencing conditions, all basal bodies retained the GFP-Bld10 labeling (Fig. 3A), suggesting that the integration or the stability of

Bld10p in basal bodies is not affected by depletion of Sas6p. In contrast, in cells expressing GFP-Sas6p and silenced for BLD10, the GFP signal totally disappeared from all basal bodies within two divisions (Fig. 3B). Since in such cells preexisting and neo-formed basal bodies coexist, this result demonstrates that Bld10p is not only necessary to stabilize Sas6p in the cartwheel during assembly of neo-formed basal bodies, but is also required for its maintenance in the preexisting ones. This observation indicates that in Paramecium, Sas6p is a dynamic component of the centriolar structure as in other

Table I. Basal Body Dimensions are Affected Under SAS6 and BLD10 Inactivation

Length (nm) Diameter (nm)

ND7

Bld10

SAS6 24 h

SAS6 72 h

391 6 8.3 (n ¼ 29) 122.1 6 1.9 (n ¼ 36)

342 6 7.1 (n ¼ 28) 116.7 6 2.4 (n ¼ 51)

310 6 12 (n ¼ 23) 107 6 1.4 (n ¼ 38)

308 6 6 (n ¼ 28) 105.3 6 1.7 (n ¼ 41)

Basal body dimensions were measured on scanned negatives using image J software. The length was measured on longitudinal sections of basal bodies and the width on transverse sections. The mean length of basal bodies in all experimental samples (SAS6 and BLD10 RNAi) are significantly shorter (P < 0.001) than in the control sample (ND7 RNAi), but no significant difference is observed in the two Sas6p depleted samples after 24 h and 72 h, respectively under RNAi conditions. As for the diameter of basal bodies, it is significantly smaller in Sas6p depleted cells than in the controls, with no significant difference between the two samples. The smaller diameter of basal bodies in Sas6p depleted cells reflects the presence the 8-triplets basal bodies (Figs. 2E and 2F).

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Bld10p Stabilizes SAS-6p at the Cartwheel

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Fig. 3. Bld10p is required for maintenance of Sas6p at the basal bodies. Transformants expressing GFP-Bld10p were silenced for SAS6 (A) and transformants expressing GFP-Sas6p were silenced for BLD10 (B). As a control, transformants expressing GFP-Sas6p were silenced for PtCEN2 (C). The cells were imunolabelled with 1D5 (red) and anti-GFP antibodies (green). While silencing of SAS6 does not affect the localization of GFP-Bld10p (A), silencing of BLD10 in GFP-Sas6p expressing cells leads to the loss of the GFP signal from all basal bodies (B). In contrast, in transformants expressing GFP-Sas6p, depletion of PtCen2p, which is not required for basal body assembly but contributes to define the budding site of the new basal body [Ruiz et al., 2005] does not affect GFP-Sas6p maintenance at basal bodies (C). In panels A and B, the control cell is on the left and the silenced cell on the right. In panel C, only the silenced cell is shown as the control is identical to that in panel B. Bar: 10 lm; On the lower part of each panel, enlargements of the double labeling X6.

organisms [Kleylein-Sohn et al., 2007; Strnad et al., 2007; Dammermann et al., 2008; Pearson et al., 2009]. In order to ascertain whether the observed disappearance of Sas6p upon BLD10 silencing was the result of a specific interaction with Bld10p or reflected an intrinsic instability of the fusion between Sas6p and GFP, we examined the effect of the silencing of another basal body component, centrin2p encoded by CEN2 [Ruiz et al., 2005]. Under inactivation of CEN2 (Fig. 3C), the GFP-Sas6p signal was retained in all basal bodies indicating that neither the recruitment of Sas6p into new basal bodies nor its stability depend on PtCen2p, and therefore that the instability of Sas6p observed after BLD10 inactivation is specific. In Chlamydomonas [Nakazawa et al., 2007] and in mammalian cells [Kleylein-Sohn et al., 2007], absence/depletion of Bld10p/Cep135 was observed to prevent Sas6p recruitment. Our observations provide the first experimental evidence that Sas6p requires Bld10p to be maintained at mature basal bodies. This result leads us to consider that inactivation or mutation of BLD10 may have a dominant negative effect on SAS6. Overexpression of GFP-Sas6p or GFP-Bld10p Reveals Basal Body Assembly Intermediates Transformation in Paramecium is achieved by microinjection of the tagged gene at high copy number into the macronucleus of cells physiologically expressing their wild-type endogenous homologous gene thus leading to a variable level of overexpression [Skouri and Cohen, 1997; Ruiz et al., 1998]. In most clones transformed by GFP-Sas6p or GFP-Bld10p, this overexpression had no apparent physiological effect as cell cycle and morphology were normal. In some clones, aggregates of the GFP-tagged protein were observed (Supporting Infor-

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mation Fig. S5), without other phenotypic effect, except for limited patches of basal body overduplication (Supporting Information Fig. S6). In both GFP-Sas6p and GFP-Bld10p expressing cells however, some basal bodies displayed anomalies: additional stacks of cartwheel structures (Fig. 4D), or cartwheel tiers protruding from the basal body (Fig. 4C) in GFPSas6p expressing cells, or shortened/defective microtubule walls at the proximal end (Figs. 4A and 4C) or missing triplets in the case of GFP-Bld10p (Fig. 4F). These defects resemble those described in Chlamydomonas both in bld12 (CrSAS-6 gene) [Nakazawa et al., 2007] and bld10 mutants [Hiraki et al., 2007]. Among the 10 transformant clones studied, all had a normal phenotype, as judged by growth rate and cell morphology, except for one which presented growth and morphology defects resembling those observed under BLD10 silencing. In this clone, striking anomalies were observed in basal bodies, such as accumulation of dense material (Fig. 4G) or ectopic development of an abortive basal body (Fig. 4H). Phenotypically normal transformants revealed a number of very early steps of basal body assembly (Fig. 5) not often detected in control cells, where they are presumably less abundant and possibly more transient. On EM transverse sections of GFP-Sas6p and GFP-Bld10p transformants fixed at early stages of division, two distinct types of circular structures are observed: either a disc of variable electron density (Figs. 5A, 5C, 5D, and 5F) resembling the ‘‘generative disc’’ first described in Paramecium [Dippell, 1968], or a ‘‘pre-cartwheel’’ with a central hub and thin radial prolongations similar to short spokes (Figs. 5A, 5B, and 5E). Particularly striking is the presence of two ‘‘hubs’’ in a GFP-Bld10 transformant (Fig. 5E). These structures develop precisely at the sites where both GFP-Sas6p and GFP-Bld10p are detected by immunogold labeling (Figs. 5A and 5D). As shown in

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Fig. 4. Over-expression of GFP-Sas6p and GFP-Bld10p induces basal body abnormalities. A: The longitudinal sections (A and E) show that some of the microtubular triplets are shorter (arrows), a situation likely to account for the missing triplets observed in cross-sections (B and F, stars). C: The remnants of the cartwheel structure seem to be evacuated from the basal body of a GFP-Sas6p expressing cell. D: Longitudinal section though a basal body of GFP-Sas6p expressing cell showing an increased number of cartwheel stacks (seven instead of the normal number of three to five) in a young, as yet unciliated basal body. E–F: Both the longitudinal and the cross-section show the instability of the microtubular shaft. G: In the GFP-Bld10p tansformant, which showed an abnormal phenotype, the most proximal part of the basal body is surrounded by fibro-granular material (arrows). H: Longitudinal section through a basal body displaying an abortive basal body at an abnormal location at the base of a newly formed basal body. Bar 100 nm.

Fig. 5G, the identification of this site and the orientation of the structures are unambiguous as ciliary rootlet, transverse microtubule ribbon and parasomal sac provide landmarks for both antero-posterior and proximo-distal polarities of the old basal body, while the longitudinal section of the cilium gives a measure of the distance of the observed hub or disc from the old basal body: the more axial the section trough the cilium (Figs. 5B and 5C), the closer to the basal body is the observed structure. ‘‘Hubs’’ devoid of dense surrounding material (Figs. 5B and 5E) are found closer to the old basal body than discs (Figs. 5C and 5F). Whether this observation reflects a spatial or temporal sequence remains to be ascertained. Finally, it can be pointed out that the clearest images are those of hubs; in contrast, when the section cuts through the disc, whatever is assembled is blurred and the idenfication of a thin structure like the hub becomes difficult. Nevertheless, faint hubs with radial prolongations can be discerned within discs (Figs. 5B, 5E, and 5F), an observation which also supports the idea that the hub and spokes, and then of the pre-cartwheel, do not develop after the generative disc.

Discussion The cartwheel, within the proximal part of centrioles and basal bodies, is generally the first recognizable structure of the developing centriole or basal body [reviews in Azimzadeh and Bornens, 2007; Strnad and Go¨nczy, 2008]. However the functions of this elaborate structure, with its central hub and

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nine radial spokes, still is a riddle. It is dispensable for the generation of a ninefold symmetrical organization since there is no cartwheel in the C. elegans centriole. A cartwheel is also dispensable to maintain the ninefold symetrical organisation as it disappears in the mature centriole or basal body in mammalian cells [Alvey, 1986]; Lemullois et al., 1988]. However, key proteins required to initiate assembly of basal bodies or centrioles, SAS-6 and Cep135/Bld10 localize at the cartwheel. While SAS-6 is conserved and required for assembly of both centrioles [Kleylein-Sohn et al., 2007] and basal bodies [Nakazawa et al., 2007; Vladar and Stearns, 2007; Culver et al., 2009], Bld10p/Cep135 [Ohta et al., 2002; Matsuura et al., 2004] is less conserved and its absence in T. brucei excludes a direct role in assembly of the cartwheel, since newly formed basal bodies in Trypanosoma have a cartwheel [Lacomble et al., 2009]. Furthermore, recent studies reveal new functions for Bld10/Cep135, in centrosome splitting through an interaction with C-NAP1 [Kim et al., 2008] and in Drosophila in assembly of motile cilia [Mottier-Pavie and Megraw, 2009]. In order to gain further insight into the role of BLD10 in cartwheel and basal body assembly, and examine its relationship with SAS-6, we have characterized both types of genes and investigated their function in Paramecium, where all basal bodies display a permanent cartwheel and nucleate motile cilia. By RNAi experiments, immuno-fluorescence and immuno-electron microscopy of GFP-tagged proteins, we showed that both Sas6p and Bld10p were required for basal body assembly and permanently detected

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Fig. 5. Early steps of basal body assembly in GFP-Sas6p and GFP-Bld10p transformants. Sections through dividing cells expressing GFP-Sas6p (A–C) and GFP-Bld10p (D–F) show different aspects or stages of pre-basal bodies in dividing cells. In all images, the anterior of the cell is toward the viewer showing a transverse section of the cell anterior to an old basal body which can be localized and oriented after the sections through cilium (cil), ciliary rootlet (cr), transverse microtubular ribbon (mr) and/or parasomal sac (ps) (Compare scheme on Fig. 5G). Immunogold labeling of early dividing cells of GFP-Sas6p (A) and GFP-Bld10p (D) reveal localization of gold grains near the hub (A) or outside of the hub in the dense material surrounding it (D, arrowheads). In sections B and E a circular structure (hb arrows), ca. 50 nm in diameter, is present closest to the old bb. Note in E the presence of two such structures. On more anterior sections (C and F), a disc filled with dark fibro-granular material is visible (gd). In GFP-Bld10p (F arrow) a centrally located hub-like structure in the disc is visible. The diameter of the disc or clump corresponds to that of pre-basal bodies with microtubule triplets already assembled in WT cells. Bar: 100 nm. G: Schematic representation of the planes of sections in top view (upper drawing) and side view (lower drawing). A-P and R-L indicate the anterior-posterior and right-left axes of the cell. In the left panel, the plane of section runs anterior of the old basal body (obb). Depending othe anglewith respect to the R-L axis it will cut through some or all of the following organelles: parasomal sac (ps), striated ciliary rootlet (cr), developing daughter basal body, transverse microtubular ribbon (mr). On the lower panel, depending on the angle with respect to the longitudinal axis of the basal body þcilium, the section will cut through different parts of the developing baal body, of old one and its cilium. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

at the cartwheel, with Sas6p centrally localized at the hub, and Bld10p at the tip of the spokes, in agreement with previous studies on diverse organisms [Matsuura et al., 2004; Leidel et al., 2005; Hiraki et al., 2007; Kleylein-Sohn et al., 2007; Nakazawa et al., 2007; Rodrigues-Martins et al., 2007; Culver et al., 2009; Mottier-Pavie and Megraw, 2009]. Our functional analyses and ultrastructural observations permitted us to gain further insight into three aspects of basal body assembly: the respective functions of Sas6p and Bld10p, their functional interdependence, the visualization of pre-cartwheel stages of basal body assembly. In conclusion, we will discuss to what extent our observations fit in a conserved assembly pathway of the centriolar structure. Respective Functions of Sas6p and Bld10p Beyond a similar blockage of basal body duplication by the first division under silencing of either SAS6 or BLD10 (Fig.

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1), ultrastructural observations of basal bodies in dividing silenced cells revealed distinctive features. SAS6 silenced cells presented defective cartwheels devoid of a hub, a significant (ca. 20%) reduction in basal body length and complete microtubule shafts displaying an eightfold symmetry. Owing to our experimental conditions (cells observed when undergoing their first division upon silencing conditions), length reduction in old basal bodies (i.e., preexisting basal bodies) attached to the cortex can only be explained by disassembly of their proximal end. It was shown in Tetrahymena that part of the Sas6p present at basal bodies is exchangeable [Pearson et al., 2009] and in HeLa cells that the level of HsSAS-6 oscillates during the cell cycle [Kleylein-Sohn et al., 2007; Strnad et al., 2007]. Such dynamics in Paramecium could account for the shortening observed under SAS6 silencing, when the pool of protein declines. In BLD10 silenced cells, the reduction of basal body length was less marked and most anomalies concerned defective microtubule arrangement.

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These effects of BLD10 and SAS6 silencing on basal body structure, in particular the abnormal cross-sections, are strikingly similar to the anomalies found in Chlamydomonas [Hiraki et al., 2007; Nakazawa et al., 2007] in the bld10 transformants and the bld12 mutant respectively. In Paramecium however, the only abnormality observed among closed circular sections was the 8-triplet configuration in SAS6 silenced cells, while in the Chlamydomonas bld12 mutant, in addition to the predominant 8-triplet type, seven- to 11-triplet circular arrangements were also observed. These differences are likely correlated with the different in experimental conditions, early effect of gene silencing on neo-formed basal bodies versus established mutant phenotype detected in flagellar axonemes. Among the reduced number of basal bodies formed in Paramecium under Sas6p depletion (Fig. 2), the presence of the circular 8-triplet configuration supports the direct role of Sas6p in determination of the ninefold symmetry, in agreement with its function in C. elegans [Dammermann et al., 2004; Leidel et al., 2005] and with the observations on Chlamydomonas [Nakazawa et al., 2007] and Drosophila [Rodrigues-Martins et al., 2007]. As for Bld10p, the anomalies observed indicate a role in microtubule positioning and/or stability, in agreement with its localization at the tip of the cartwheel spokes. Even when not restricted to the cartwheel and present also at the distal end of centrioles, as in mammalian cells [Kleylein-Sohn et al., 2007] or of basal bodies in Drosophila [Mottier-Pavie and Megraw, 2009], Bld10/Cep135 is a peripheral protein. This localization might favor interactions not only with centriolar proteins but also with proteins around the centriolar structure, and thus lead to diverse additional functions. Functional Interdependence of Sas6p and Bld10p Despite their likely distinct primary function, a partial phenotypic overlap in basal body anomalies induced by the silencing of SAS6 and BLD10 was observed in Paramecium as in Chlamydomonas [Hiraki et al., 2007; Nakazawa et al., 2007]. In order to investigate the functional relationships between the two proteins, we examined the effect of silencing SAS6 on Bld10p and vice versa and we demonstrated a one way interaction. While the basal body localization of Bld10p is not affected by SAS6 silencing, Sas6p requires Bld10p not only to be stabilized in newly formed basal bodies but also to be maintained at mature basal bodies. Inactivation or mutation of BLD10 thus exerts a dominant negative effect on Sas6p.The stronger the inactivation of BLD10, the more phenotypic overlap with specific Sas6p deficiency would be observed, while a milder inactivation, as in Paramecium under the current gene silencing protocol, would yield more distinct phenotypes. Some overlap can be noted between the phenotypes of the bld12 and bld10 mutants in Chlamydomonas [Hiraki et al., 2007; Nakazawa et al., 2007]. The anomalies in the bld12 mutant, which include either disorganized microtubular shafts or circular shafts with abnormal numbers of triplets are also found in

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the bld10 mutant. In Paramecium, distinct phenotypes are observed: silencing of SAS6 affects the ninefold symmetry, while silencing of BLD10 perturbs the cohesion or stability of the microtubule triplet organization. Our observations, as well as the observations in Chlamydomonas [Hiraki et al., 2007], therefore support the conclusion that, in contrast to Sas6p, Bld10p is not a direct determinant of ninefold symmetry but is required for its stabilisation. Identification of Pre-Cartwheel Stages In her pioneer ultrastructural study of basal body duplication in Paramecium, Dippell [1968] described an electron dense ‘‘generative disc’’ as the first sign of assembly, followed by the sequential appearance of microtubule singlets, with a cartwheel appearing later. In contrast, studies on Chlamydomonas [Johnson and Porter, 1968; Cavalier-Smith, 1974], Tetrahymena [Allen, 1969] and epithelial cells [Lemullois et al., 1988] as well as on the de novo assembly of basal bodies in M. vestita [Hepler, 1976] or flagellates [Tamm and Tamm, 1980], all described an initial cartwheel-like structure. The question of what comes first of the ‘‘cartwheel or of the horses’’ [Dippell, 1968] that is of the hub and the disc thus remained pending. Did Paramecium use a different assembly pathway [Dutcher, 2007]? However, the molecular dissection of basal body/centriole assembly, here extended to Paramecium, shows that key proteins have a conserved localisation and function, suggesting that the physical process of assembly might also be conserved. Our observations showing that a hub (or tube-like structure) with short spokes can be seen independently of a larger electron dense ‘‘generative’’ disc, and that discs may or may not reveal a faint central hub with a ‘‘hub and spokes’’ rather suggest a conserved pathway with a ‘‘hub and spokes’’ complex as an initial scaffold —emanating from the close vicinity of the mother basal body—around which electron dense material congregates and provides a platform for cartwheel development and microtubule assembly. Re-examining Dippell’s images (where a faint hub can be seen within the generative disc) and her mentioning the occasional earlier ‘‘presence of what might be a developing hub and spoke,’’ it seems that our observations, made under facilitating conditions in cells overexpressing the cartwheel proteins Sas6p or Bld10p, are not in contradiction with those of Dippell [1968].

Conclusion Even if the disc and the peripheral nine microtubules developed before assembly of the cartwheel, what determines the ninefold symmetry has been solved in Caenorhabditis: its simpler centriole architecture facilitated the integration of cytological and functional data and promoted SAS6 as the essential gene for the establishment of the ninefold symmetry and the central tube as the first detectable structure [Pelletier et al., 2006]. Concordant studies on several organisms, including the results presented here, concur in the recognition of SAS-6 as a determinant of the nineness according to the nice word coined by Marshall [Marshall, 2007]. The

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localization of Sas6p at the hub suggests that it is homologous to the central tube in C. elegans. while the localization of Bld10p at the periphery of the cartwheel and the defects resulting from its depletion indicate that it provide a structural relay of the established symmetry for positioning and assembly of microtubule doublet or triplets. In turn, this role might be completed or relayed by SAS-4, shown to be directly involved in microtubule nucleation and stability [Dammermann et al., 2008] and in Paramecium to be essential to basal body development [Gogendeau et al, submitted]. Such a scaffolding role during centriole biogenesis has been proposed for Cep135 [Kleylein-Sohn et al., 2007]. A role of Bld10p and the cartwheel as relay and scaffold in the assembly of centriolar structures is consistent with the fact that in different biological systems, the cartwheel is lost in mature centriole or basal bodies and therefore not required for the stability of the established structure. Why it is retained in basal bodies of ciliates and lost in those of ciliated mammalian cells [review in Dawe et al., 2007] indicates that further functions of the cartwheel remain to be analyzed. Acknowledgments We thank M. Bornens, L. Sperling, and R. Basto for critical reading of the manuscript and Thomas Wassmer for providing the plasmid containing the RFP gene. This work was supported by the Statute Grant to the Nencki Institute from the Ministry of Science and Education (Poland), by the Centre National de la recherche scientifique (France) and by a grant from the Agence Nationale de la Recherche, number NT05-2_41522. D.G. was supported by the Association de la Recherche Contre le Cancer (A06/3). The excellent technical assistance of Henryk Bilski from the Laboratory of Electron Microscopy (Nencki Institute of the Polish Academy of Sciences) is kindly acknowledged.

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