600 Research Article Centriolar satellites are assembly points for implicated in human , including oral-facial- digital syndrome 1

Carla A. M. Lopes1, Suzanna L. Prosser1, Leila Romio2, Robert A. Hirst3, Chris O’Callaghan3, Adrian S. Woolf4 and Andrew M. Fry1,* 1Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK 2Nephro-Urology Unit, University College London Institute of Child Health, London WC1N 1EH, UK 3Department of Infection, Immunity and Inflammation, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester LE2 7LX, UK 4Developmental Biomedicine Research Group, University of Manchester and Royal Manchester Children’s Hospital, Manchester M13 9PT, UK *Author for correspondence ([email protected]) Accepted 22 October 2010 Journal of Cell Science 124, 600-612 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.077156

Summary Ciliopathies are caused by mutations in encoding proteins required for cilia organization or function. We show through colocalization with PCM-1, that OFD1 (the product of the mutated in oral-facial-digital syndrome 1) as well as BBS4 and CEP290 (proteins encoded by other genes) are primarily components of centriolar satellites, the particles surrounding centrosomes and basal bodies. RNA interference experiments reveal that satellite integrity is mutually dependent upon each of these proteins. Upon satellite dispersal, through mitosis or forced microtubule depolymerization, OFD1 and CEP290 remain centrosomal, whereas BBS4 and PCM-1 do not. OFD1 interacts via its fifth coiled-coil motif with the N-terminal coiled-coil domain of PCM-1, which itself interacts via its C-terminal non-coiled-coil region with BBS4. OFD1 localization to satellites requires its N-terminal region, encompassing the LisH motif, whereas expression of OFD1 C-terminal constructs causes PCM-1 and CEP290 mislocalization. Moreover, in embryonic zebrafish, OFD1 and BBS4 functionally synergize, determining morphogenesis. Our observation that satellites are assembly points for several mutually dependent ciliopathy proteins provides a further possible explanation as to why the clinical spectrum of OFD1, Bardet–Biedl and Joubert syndromes overlap. Furthermore, definition of how OFD1 and PCM-1 interact helps explain why different OFD1 mutations lead to clinically variable phenotypes.

Key words: , BBS4, Centriole, CEP290, Cilia, Laterality, PCM-1, OFD1, Zebrafish Journal of Cell Science

Introduction reported in 1954 by Papillon-League and Psaume, is the commonest, Most mammalian cells possess a single hair-like projection called occurring in 1:50,000 births. OFD1 is distinguished by its X-linked a primary , which extends into the extracellular milieu. dominant inheritance pattern (Feather et al., 1997), with prenatal These organelles act like antennae, sensing mechanical and lethality in affected males. Affected females are born with OFD chemical signals and thus coordinating cell growth, survival, dysmorphology and develop polycystic kidneys (Feather et al., 1997; polarity and differentiation (Berbari et al., 2009; Marshall and Saal et al., 2010). Mutations of OFD1, located on the X , Nonaka, 2006; Singla and Reiter, 2006). Primary cilia are generally account for most cases of OFD1 syndrome with most mutations non-motile and contain a 9+0 organization of microtubule pairs found in the first half of the gene (Ferrante et al., 2001; Macca and within the ciliary , distinct from the 9+2 organization Franco, 2009; Romio et al., 2003). Human OFD1 escapes X- found within flagella and the motile cilia of multiciliated epithelia. inactivation and affected females are probably composed of cells However, there appear to be subsets of primary cilia that, although with reduced levels of normal OFD1 (Ferrante et al., 2003). having a 9+0 arrangement, have a motile function, e.g. within the There remain, however, some OFD1 syndrome individuals for which embryonic node of mammals or the Kuppfer’s vesicle of fish. OFD1 mutations cannot be detected (Thauvin-Robinet et al., 2009). Through rotating in a clockwise manner, these nodal cilia create a In human embryos, OFD1 is expressed in many organs, including flow that is necessary for defining left–right asymmetry during those that develop abnormally in the syndrome (Romio et al., 2004; body pattern determination (Marshall and Nonaka, 2006). Romio et al., 2003). Intriguingly, OFD1 mutations have recently Much of our understanding regarding primary cilia has come been associated with other disease phenotypes, including the from studies on genetic human disorders, collectively known as nephronophthisis (NPHP)-related ciliopathy, ciliopathies, many features of which are considered to result from (Budny et al., 2006; Coene et al., 2009; and see Discussion). defects in the assembly or function of these organelles. These features The classic OFD1 syndrome is typical of a ciliopathy. Moreover, include laterality defects, digital and brain anomalies, retinal studies support the hypothesis that the developmental abnormalities degeneration, obesity and kidney cysts (Badano et al., 2006; Gerdes are, at least in part, due to abnormal cilia-dependent signalling et al., 2009; Hildebrandt and Otto, 2005). The oral-facial-digital events. In cultured cells, OFD1 is clearly required for primary cilia (OFD) syndromes are linked by dysmorphology of the mouth, face formation (Corbit et al., 2008; Graser et al., 2007; Singla et al., and digits (Macca and Franco, 2009). OFD type 1 (OFD1), first 2010). Furthermore, experimental downregulation of Ofd1 in both OFD1 regulates centriolar satellites 601

mice (Ferrante et al., 2006) and zebrafish (Ferrante et al., 2009) localization (Gerlitz et al., 2005). Furthermore, the LisH motif causes laterality defects of internal organs, including the heart, might regulate microtubule dynamics directly or indirectly via subsequent to embryonic node (Ferrante et al., 2006) and Kuppfer’s cytoplasmic (Emes and Ponting, 2001; Ferrante et al., 2003; vesicle (Ferrante et al., 2009) structural defects in cilia, the normal Gerlitz et al., 2005). Interestingly, Miller–Dieker lissencephaly and functions of which are crucial for the breaking of embryonic Treacher Collins syndrome are caused by mutations in genes symmetry (Bakkers et al., 2009). In mice lacking Ofd1, altered encoding LisH-containing proteins, and both disorders have been expression of Sonic hedgehog (Shh) pathway genes has been noted attributed to incorrect cell migration resulting from cytoskeletal (Ferrante et al., 2006). Although Ofd1 might not be involved in defects (Emes and Ponting, 2001). Hence, certain neuronal Shh signalling in zebrafish (Ferrante et al., 2009), Ofd1 functionally components of the OFD1 syndrome might involve aberrant cell synergizes in this organism with Slb/Wnt11 and Tri/Vangl2 to migration. In addition, missense mutation of the OFD1 LisH direct convergent extension movements, suggesting a role in the domain deregulates centriole elongation (Singla et al., 2010). non-canonical Wnt–planar cell polarity (PCP) signalling pathway. We set out to test the hypothesis that OFD1 contributes to the Notably, other experiments show that both Shh and PCP signalling physical organization of basal bodies and, as such, has an architectural can be initiated within cilia (Berbari et al., 2009). role in ciliogenesis. We unexpectedly found that, as well as localizing Like many proteins encoded by ciliopathy disease genes, OFD1 to centrosomes, OFD1 localizes to centriolar satellites, which are localizes to the centrosome throughout the cell cycle (Romio et al., granular structures surrounding the centrosome that are implicated 2004). The centrosome is a cytoplasmic organelle composed of two in trafficking of material required for centriole assembly (Kubo et barrel-shaped centrioles held within a proteinaceous matrix of al., 1999). Other ciliopathy disease proteins are known to localize to pericentriolar material (PCM) that together act as the primary satellites. These include the Bardet–Biedl syndrome (BBS) protein microtubule organizing centre (Nigg and Raff, 2009). During cell BBS4, one component of a large complex of BBS subunits known division, the duplicated centrosomes form the poles of the as the BBSome, and CEP290, which is a protein mutated in NPHP microtubule-based mitotic spindle. In post-mitotic cells, the and Joubert syndrome (Kim et al., 2008; Kim et al., 2004; Nachury, unduplicated centrosome moves to the apical cell surface where the 2008). We therefore addressed the dependencies of these proteins on older, or mother, centriole, docks with the plasma membrane and each other for localization to satellites by using RNA interference subtends the axonemal microtubules of the primary cilium. When and expression of truncated OFD1 constructs. Our data support a centrioles participate in ciliogenesis, they are called basal bodies model whereby centriolar satellites regulate the delivery of several and, in ciliated cells, OFD1 has been localized both to basal bodies ciliopathy disease proteins to the basal body. and the stalk of the cilium (Romio et al., 2004). Importantly, OFD1 was recently shown to localize specifically to the distal ends of Results centrioles and, in mouse ES cells lacking OFD1, centriole distal Ciliopathy disease proteins are components of centriolar ends were disturbed (Singla et al., 2010). Specifically, centrioles satellites exhibited excessive elongation and failure to properly assemble We set out to examine the regulation and function of the OFD1 distal appendages. These defects could potentially lead to problems protein at the cellular level by first revisiting its localization, primarily in attachment of the mother centriole to the apical cell surface. It in telomerase-immortalized human retinal pigment epithelial (hTERT-

Journal of Cell Science remains possible, however, that the actions of OFD1 are not confined RPE1) cells. These non-cancer-derived cells undergo normal to the generation of cilia because these appear to be present during proliferation and lack detectable cilia when subconfluent, but enter tubular cystogenesis induced by renal epithelial-specific quiescence and produce primary cilia when grown to confluence and downregulation of Ofd1 in mice (Zullo et al., 2010). OFD1 protein serum-starved. Here, we used two different polyclonal antibodies has also been detected in nuclei as part of the TIP60 chromatin raised to distinct OFD1 antigens: Ab1 from Romio and colleagues remodelling complex, and thus might play roles in regulating gene (Romio et al., 2003) was raised against a peptide representing amino expression (Giorgio et al., 2007). Supporting this idea is the acids 867–891 (so would not detect OFD1b) and used for the studies observation that downregulation of Ofd1 function leads to a striking shown in Figs 1 and 2, except where indicated. Ab2 from this study increase in levels of Ofd1 transcripts (Ferrante et al., 2009). (supplementary material Fig. S1A,B) was raised against amino acids OFD1 contains 23 exons and generates two main splice variants, 145–1012 (so should detect OFD1a and OFD1b) and used in all OFD1a and OFD1b, the latter coding for an unstudied putative other experiments. With both antibodies, we found that in proliferating protein of 367 amino acids (aa) derived from exons 1–11 (Budny hTERT-RPE1 cells the bulk of OFD1 was not primarily localized to et al., 2006). More is known about OFD1a, the protein encoded by the centrosome, but was distributed in small aggregates that mostly exons 1–23, itself with a variant lacking exon 10, with a predicted surrounded the centrosome (Fig. 1A and supplementary material Fig. molecular weight of ~110 kDa (hereafter, for simplicity, we call S1D). A similar distribution was observed in proliferating human OFD1a, ‘OFD1’). OFD1 contains an N-terminal Lis1 homology foreskin fibroblast (hFF) and renal inner medullary collecting duct (LisH) motif and an extended C-terminal domain containing what (IMCD3) cells (Fig. 1B). Moreover, in contrast to what was observed have been alternatively indicated as either five or six putative in renal proximal tubule epithelial cells (RPTEC) (Romio et al., coiled-coils (Budny et al., 2006; Coene et al., 2009; de Conciliis 2004), OFD1 was not detected along the stalk of the cilium in et al., 1998; Emes and Ponting, 2001). This C-terminal region, ciliated hTERT-RPE1 cells (Fig. 1C and supplementary material Fig. which we show as containing six coiled-coils based on SMART S1E). Localization to small pericentriolar aggregates was confirmed analysis (http://smart.embl-heidelberg.de/), is essential for localizing in hTERT-RPE1 cells overexpressing GFP- or Myc-tagged full- OFD1 to the centrosome (Romio et al., 2004). It is also essential length OFD1 protein (Fig. 1D and supplementary material Fig. S1F), for interaction with the LCA5-encoded ciliary protein, lebercilin consistent with a recent study on cells expressing enhanced cyan (Coene et al., 2009), itself mutated in Leber congenital amaurosis. fluorescent protein (eCFP)–OFD1 (Coene et al., 2009). LisH motifs are present in many proteins, where they contribute The size and distribution of OFD1 aggregates were highly to protein–protein interactions, dimerization, stability and/or reminiscent of centriolar satellites, non-membranous 70–100 nm 602 Journal of Cell Science 124 (4)

Fig. 1. OFD1 localizes to centriolar satellites. (A)hTERT-RPE1 cells were co-stained with antibodies against g-tubulin to detect the centrosome, and with two different antibodies against OFD1 (Ab1 and Ab2). (B)hFF and IMCD3 cells were co-stained with antibodies against g-tubulin and OFD1. (C)Ciliated hTERT-RPE1 cells were co-stained with antibodies against acetylated tubulin to detect the axonemal microtubules and against OFD1. (D)hTERT-RPE1 cells were transfected with constructs expressing GFP- or Myc-tagged full-length (FL) OFD1 and stained with antibodies against GFP or Myc and g-tubulin. (E)hTERT-RPE1 cells were co-stained with antibodies against OFD1 and PCM-1 to identify centriolar satellites. (F)hTERT-RPE1 cells were transfected with Myc-tagged full-length OFD1 and co-stained with antibodies against Myc to identify transfected cells and against PCM-1, BBS4, CEP290, pericentrin, Cep135 or centrin. Scale bars: 2m. Merged images are shown for all panels. Journal of Cell Science cytoplasmic granules that concentrate around the centrosome and dispersal not only of PCM-1, but also of BBS4 and CEP290 (Kim et contain the protein PCM-1 (Dammermann and Merdes, 2002; al., 2008; Kim et al., 2004; May-Simera et al., 2009). We therefore Kubo et al., 1999; Kubo and Tsukita, 2003). Using antibodies to analysed the localization pattern of OFD1 in hTERT-RPE1 cells detect endogenous OFD1 or expression of recombinant Myc– following, first, depolymerization of the microtubule network with OFD1, we found that the small aggregates that surround the nocodazole and, second, disruption of dynein function by centrosome colocalized precisely with PCM-1 (Fig. 1E,F). overexpression of the dynactin subunit, p50-dynamitin. Under both Furthermore, co-staining for two other ciliopathy disease proteins conditions, g-tubulin remained on centrosomes and the pericentriolar known to be satellite components, CEP290 and BBS4 (Kim et al., aggregates of OFD1 dispersed, which was consistent with 2008; Kim et al., 2004), also revealed colocalization with OFD1. redistribution of centriolar satellites into the (Fig. 2A,B). Note that all studies on BBS4 here were done with an antibody A fraction of OFD1 specifically on the centrosome also became raised in our laboratory (supplementary material Fig. S1A,C). more obvious to detect. Microtubule regrowth following nocodazole Interestingly, other reported satellite markers, notably centrin, washout led to re-accumulation of the pericentriolar OFD1 aggregates, pericentrin and Cep135 (Dammermann and Merdes, 2002; Ohta et which was consistent with focusing of centriolar satellites (Fig. 2A). al., 2002), did not form obvious pericentriolar aggregates in hTERT- We have previously shown that treatments that block centrosome RPE1 cells but were predominantly associated with the centrosome overduplication in S-phase-arrested Chinese hamster ovary (CHO) itself and showed no colocalization with OFD1 (Fig. 1F). Thus, we cells, including inhibition of nuclear export or Cdk activity, lead to conclude that the bulk of the ciliopathy disease proteins, OFD1, loss of detectable centriolar satellites (Prosser et al., 2009). As CEP290 and BBS4, colocalize in interphase hTERT-RPE1 cells predicted, these same treatments led to loss of the pericentriolar primarily within centriolar satellites. aggregates of OFD1 (Fig. 2C). Hence, using a variety of approaches known to disturb centriolar satellites, we have confirmed that OFD1 Redistribution of centriolar satellites disturbs OFD1 behaves as a bona fide centriolar satellite component. localization Centriolar satellites cluster around the centrosome in a manner that Redistribution of ciliopathy disease proteins during is dependent on microtubules and the microtubule-dependent motor mitotic progression protein, dynein (Dammermann and Merdes, 2002). Consistent with Through analysis of PCM-1, it has been previously reported that this, interfering with microtubule-dependent transport leads to centriolar satellites disperse in mitosis, such that in metaphase OFD1 regulates centriolar satellites 603

Fig. 2. Localization of OFD1 is disturbed following centriolar satellite dispersal. (A)hTERT-RPE1 cells were either mock-treated (Control), treated with nocodazole (+ Nocodazole) to depolymerize the microtubule network, or treated with nocodazole and then allowed to recover for 5 minutes (5 Min. Recovery). Cells were then stained with antibodies against -tubulin and either g-tubulin or OFD1. Insets show enlargements of the centrosome. (B)hTERT-RPE1 cells were transfected with Myc-tagged p50-dynamitin to disrupt dynein–dynactin function and co-stained with antibodies against Myc and against either g- tubulin or OFD1. Enlargements of g-tubulin and OFD1 signals from untransfected (1) or transfected (2) cells are shown. (C)CHO cells were treated with hydroxyurea (HU) alone for 48 hours, HU for 18 hours followed by HU and leptomycin B (LMB) for 30 hours, or HU for 18 hours followed by HU and roscovitine for 30 hours. Cells were stained with antibodies against OFD1 and g-tubulin. Merged images, including DNA stain (blue), are shown in B and C. Scale bars: 10m.

and anaphase they show little point of focus (Balczon et al., the consequence of depleting OFD1 on centriolar satellite 1994; Dammermann and Merdes, 2002). We therefore looked at organization. It has been reported that depletion of PCM-1 leads to the behaviour of OFD1 and other ciliopathy disease proteins in reduced centrin, pericentrin and ninein, but not g-tubulin, at the hTERT-RPE1 cells during mitotic progression (Fig. 3A). In the centrosome (Dammermann and Merdes, 2002), whereas depletion case of OFD1, although the satellites disappeared, the protein of BBS4 leads to dispersal of PCM-1 (Kim et al., 2004).

Journal of Cell Science was very distinctly associated with spindle poles (Fig. 3A). Interestingly, depletion of CEP290 causes PCM-1 to become CEP290 exhibited very similar behaviour and was retained on concentrated around the centrosome, whereas depletion of PCM-1 spindle poles in mitosis, although it could also be detected on causes loss of CEP290 from centriolar satellites but not the spindle microtubules in anaphase (Fig. 3A). BBS4 behaved more centrosome (Kim et al., 2008). Strikingly, we found that depletion like PCM-1 in being mainly dispersed into the cytoplasm, albeit of OFD1 led to complete loss of PCM-1 and BBS4 from the with small amounts detectable at the centrosome (Kim et al., vicinity of the centrosome (Fig. 4B). In the case of CEP290, the 2004). Pericentrin and Cep135, both proteins that are mainly satellites disappeared but weak staining of the centrosome was present on centrosomes in interphase, remained associated with apparent. We interpret this to mean that OFD1 is essential to the spindle poles in mitosis and showed no significant alteration integrity of centriolar satellites but, in their absence, CEP290 can during mitotic progression (Fig. 3A). Using recombinant Myc- be retained at the centrosome. Similarly, when PCM-1 was depleted, tagged OFD1 and co-staining for centrin, a marker of centriole the pericentriolar aggregates of OFD1, CEP290 and BBS4 distal ends (Fig. 3B), and C-Nap1, a marker of centriole proximal disappeared but OFD1 and CEP290 could be detected on ends (Fig. 3C), we confirmed that OFD1 is specifically associated centrosomes (Fig. 4C). Depletion of BBS4 led to loss of all proteins with the distal ends of the parental centrioles in mitosis. Thus, from centriolar satellites; however, CEP290 could again be weakly although certain centriolar satellite proteins are completely detected on centrosomes, but OFD1 could not (Fig. 4D). dispersed in mitosis (PCM-1, BBS4), others redistribute onto Localization of g-tubulin was not altered in response to depletion spindle poles (OFD1, CEP290). of OFD1, PCM-1 or BBS4. Interestingly, the intensity of pericentrin at the centrosome was reduced by ~50% upon depletion of PCM- Ciliopathy disease proteins are mutually dependent for 1, as previously reported (Dammermann and Merdes, 2002), and centriolar satellite localization upon depletion of BBS4 but not OFD1 (Fig. 4E). These data, To address the function of OFD1, we used siRNA oligonucleotides obtained with pooled siRNA duplexes, were verified using two to deplete the protein in hTERT-RPE1 cells (Fig. 4A). OFD1 distinct single duplexes for each protein (supplementary material depletion did not affect either the anchoring or nucleation of the Fig. S3). Hence, taken together with data published for PCM-1, microtubule network in these cells, nor did it alter the abundance BBS4 and CEP290 depletions (Dammermann and Merdes, 2002; of g-tubulin (data not shown). However, as expected, OFD1 Kim et al., 2008; Kim et al., 2004), it is clear that the localization depletion did interfere with ciliogenesis without affecting cell cycle of the centriolar satellite proteins OFD1, PCM-1, BBS4 and progression (supplementary material Fig. S2). We then addressed CEP290 are all mutually dependent with respect to the satellites. 604 Journal of Cell Science 124 (4)

Fig. 3. Redistribution of centriolar satellite proteins during mitosis. (A)hTERT-RPE1 cells were stained with antibodies against OFD1, PCM-1, CEP290, BBS4, pericentrin or Cep135. (B,C)hTERT-RPE1 cells transfected with a Myc-tagged OFD1 full-length (FL) construct were co-stained with antibodies against Myc and either centrin2 (B) or C-Nap1 (C). Cells from interphase and anaphase are shown together with enlargements of the centrosomes and spindle poles, respectively. The C-Nap1 images in anaphase are Journal of Cell Science overexposed compared to interphase due to the reduction in C-Nap1 on spindle poles. Scale bars: 5m.

However, once satellites are disturbed then the individual proteins To define the regions of OFD1 and PCM-1 required for these behave in a more complex manner with respect to their localization interactions, a series of GST-tagged OFD1 truncation constructs to centrosomes. were expressed in bacteria and mixed with extracts from hTERT- RPE1 cells transfected with GFP-tagged full-length PCM-1 or, OFD1 interacts with PCM-1 via their coiled-coil domains separately, the PCM-1 N-terminal domain encompassing its coiled- OFD1 is composed of a short N-terminal domain that contains a coils (residues 1–1468) or the C-terminal non-coiled-coil domain LisH motif and a much longer C-terminal domain containing six (residues 1468–1904) (Dammermann and Merdes, 2002) (Fig. 5B). predicted coiled-coils. Given the colocalization of OFD1 and PCM- Both full-length PCM-1 and the PCM-1 N-terminal domain 1 to centriolar satellites and their mutual dependency for this interacted strongly with the C-terminal domain of OFD1 (residues association, we investigated whether these two proteins were 145–1012), but not the N-terminal region containing the LisH capable of interaction. Following transfection of hTERT-RPE1 motif (residues 3–132) (Fig. 5C). Although the GST–OFD1 N- cells, we found that endogenous OFD1 could co-precipitate with terminal domain protein was present at lower amounts in this GFP-tagged full-length PCM-1 but not GFP alone (Fig. 5A). experiment, when analysed at equivalent amounts to the GST– Interestingly, two bands were observed for OFD1 and are likely to OFD1 C-terminal domain protein, there was still no sign of reflect the full-length OFD1 and the version lacking exon 10, interaction with the N- or C-terminal domains of PCM-1-GFP indicating that interaction with PCM-1 does not require the (supplementary material Fig. S4). Hence, these proteins interact sequence encoded by exon 10. When a co-precipitation experiment via their respective coiled-coil regions. There was no binding of was performed with GFP-tagged full-length BBS4, endogenous GFP–BBS4 with the GST-tagged N- or C-terminal OFD1 proteins OFD1 was only very weakly detected, despite good interaction of in this experiment, supporting the notion that there is unlikely to GFP–BBS4 with PCM-1 (Fig. 5A), as previously reported (Kim et be direct association between BBS4 and OFD1. Further truncation al., 2004). Hence, OFD1 exhibits a much more robust interaction of the OFD1 coiled-coil region indicated that removal of the first with PCM-1 than with BBS4. four coiled-coils had no significant effect on the interaction with OFD1 regulates centriolar satellites 605

Fig. 4. Ciliopathy disease proteins are mutually dependent for their centriolar satellite localization. (A)hTERT-RPE1 cells were either mock-depleted or depleted with pooled siRNA oligonucleotides against luciferase (GL2), OFD1, PCM-1 or BBS4 for 72 hours and depletion assessed by western blot with the antibodies indicated. (B–D) hTERT-RPE1 cells were Journal of Cell Science either mock-depleted or depleted of OFD1 (B), PCM- 1 (C) and BBS4 (D) and co-stained with antibodies against g-tubulin (green in merge) and against OFD1, PCM-1, BBS4, CEP290 or pericentrin (greyscale images and red in merge). Enlargements of centrosome are shown. Scale bar: 5m. (E)Total fluorescence intensities for the different antibodies were determined within a 2m square centred on the centrosome in cells treated with different siRNAs. The histogram presents the mean total intensities relative to that in the mock-treated sample (set at 100%); ten cells were counted in two different experiments. Error bars show s.d.

PCM-1, but the additional removal of the fifth coiled-coil led to its distinguishing between centriolar satellites and centrioles (Romio complete loss (Fig. 5C). We therefore conclude that the N-terminal et al., 2004). We therefore decided to test the localization of coiled-coil domain of PCM-1 associates with residues 601–765 of different Myc-tagged OFD1 truncation constructs following OFD1, and that although OFD1 and BBS4 might weakly associate expression in hTERT-RPE1 cells (Fig. 6A). As indicated earlier, in cells, they are unlikely to interact directly. The importance of full-length OFD1 is primarily associated with centriolar satellites these data are highlighted by the fact that several mutations reported and only weakly present at the centrosome itself (Fig. 1D). in OFD1 patients lie within the region shown to be required for Moreover, in agreement with Romio and colleagues (Romio et al., PCM-1 interaction (Fig. 5D). 2004), the C-terminal domain encompassing the coiled-coil motifs does indeed localize to the centrosome; strikingly, though, it did Expression of truncated OFD1 constructs disrupts not localize to centriolar satellites (Fig. 6B). Deleting the first two centriolar satellites coiled-coils in additional to the N-terminal domain from this Previous work had reported that the C-terminal domain of OFD1 construct (OFD1-1-365) led to the protein localizing less was necessary for localization to the centrosome, but without specifically to the centrosome, with a cytoplasmic distribution 606 Journal of Cell Science 124 (4)

Fig. 5. OFD1 and PCM-1 interact via their respective coiled-coil domains. (A)HEK293 cells were transfected with GFP alone, GFP- tagged full-length (FL) PCM-1 or GFP-tagged full-length BBS4. Cell lysates were immunoprecipitated with an anti-GFP antibody, and both the unbound supernatant (S) and immunoprecipitates (bound, B) separated by SDS-PAGE and immunoblotted with antibodies against OFD1, PCM-1 and GFP. (B)Schematic representation of GFP-tagged PCM-1 and BBS4 and GST-tagged OFD1 constructs used for interaction studies shown in C. Coiled-coil (grey), LisH (black) and TPR (red) motifs, as well as amino acid numbers, are indicated. (C)Purified GST alone or GST-tagged OFD1 constructs, as indicated, were incubated with lysates prepared from HEK293 cells transfected with PCM-1-GFP or GFP-BBS4 constructs. 10% of the input, together with the GST-bound material, were immunoblotted with an anti- GFP antibody. An anti-GST immunoblot shows the purified proteins in each fraction. (D)Schematic representation of OFD1 protein (upper diagram) is shown as in B, indicating the region (residues 601–765) essential for association with PCM-1 (red line). The lower diagram depicts the 23 exons of OFD1 gene drawn to scale so that the protein domains are aligned with their coding sequence. All point mutations identified to date are shown: frameshift (square), splice-site (triangle), nonsense (diamond) and missense (circle). Those found within the PCM-1-interacting region are shown in red and pink. The frameshift mutation in exon 16 depicted in red has been described in X-linked Simpson– Golabi–Behmel-like syndrome (Budny et al., Journal of Cell Science 2006) and the frameshift mutations in exon 21 (green) have been described in an X-linked mental retardation syndrome with Joubert-like features (Coene et al., 2009). Full descriptions of mutations have been published (Macca and Franco, 2009).

becoming obvious; however, this was diffuse with no apparent redistributed by this OFD1 construct. By contrast, in cells staining of satellites (Fig. 6B). By contrast, a protein starting at the expressing the OFD1-1-365 protein that is only weakly N-terminus and encompassing both the LisH motif and the first centrosome-associated, satellites containing PCM-1 and CEP290 four coiled-coils, hence only lacking the last two coiled-coils were dispersed, although CEP290 was still weakly detected on (OFD1-601-1012), specifically localized to centriolar satellites centrosomes (Fig. 6C). We interpret this to mean that both these suggesting that the LisH-containing N-terminal domain is crucial C-terminal OFD1 constructs can bind to PCM-1 and CEP290 and for this localization (Fig. 6B). Unfortunately, a construct containing disturb their localization to satellites, but the one lacking the first only the LisH-containing N-terminal domain could not be expressed two coiled-coils is less efficient at centrosome targeting. Finally, in hTERT-RPE1 cells, most probably as a result of unstable mRNA expression of the OFD1-601-1012 protein, which is still capable or protein. of localizing to satellites, did not obviously affect the localization We then examined the effect of expressing these truncated OFD1 of other satellite proteins (Fig. 6C). Hence, OFD1 constructs that constructs on the distribution of other satellite proteins. The OFD1 themselves mislocalize cause mislocalization of other centriolar C-terminal domain protein that localized to centrosomes but not satellite components, consistent with their interaction in cells (Fig. satellites, led to the redistribution of PCM-1 and CEP290 onto the 6D). centrosome at the expense of their localization to satellites (Fig. 6C). Even pericentrin became more intensively concentrated on Functional interaction of Ofd1 and Bbs4 in centrosomes in cells expressing this construct. This suggests that, embryogenesis even though pericentrin was not easily detected on satellites in Given the striking colocalization of OFD1, BBS4 and PCM-1 these cells, a fraction might be satellite-associated and becomes proteins in pericentriolar satellites, we elected to use a zebrafish OFD1 regulates centriolar satellites 607

Fig. 6. Ciliopathy protein localizations upon expression of truncated OFD1 constructs. (A)Schematic representation of Myc- tagged OFD1 deletion constructs. Coiled-coil (grey) and the LisH (black) motif, as well as amino acids numbers, are indicated. (B)hTERT-RPE1 cells were transfected with Myc-tagged OFD1 constructs as indicated and co- stained with antibodies against the Myc tag (greyscale and green in merge) to determine the localization of recombinant OFD1 protein and centrin2 (red in merge). Enlargements show centrosomal regions in transfected (T) and untransfected (U) cells. (C)hTERT-RPE1 cells were transfected with Myc-tagged OFD1 constructs as indicated and co-stained with antibodies against the Myc tag (green) and against PCM-1, CEP290 or pericentrin (greyscale and red in merge). Enlargements show centrosomal regions in transfected and untransfected cells. Merge images including DNA (blue) are shown in B and C. Scale bars: 10m. (D)The primary localization (+ present; – absent) of the Myc–OFD1 protein,

Journal of Cell Science PCM-1, CEP290 and pericentrin in the presence of the different OFD1 constructs is indicated. Results for each stain are based on at least 100 transfected cells for Myc-OFD1 full- length (FL), C-terminal domain (CTD) and 1-365, and 25 cells for Myc- OFD1 601-1012, which transfected with much lower efficiency. c, centrosomes; s, centriolar satellites.

model to test biological effects of downregulation of these genes embryogenesis and thus could not be used to test effects on heart using antisense morpholinos. This model also allows the assessment laterality and later phenotypes. of potential functional synergy between molecules by observing effects of administration of low doses of more than one type of OFD1 associates with amplified basal bodies in cells with morpholino. Abnormal body curvature of embryos, characteristic motile cilia of ciliopathies, was elicited by co-administration of these low dose The laterality defects seen upon depletion of Ofd1 in zebrafish morpholinos, whereas alone neither produced this effect (Fig. 7A– are suggestive of loss of function of nodal cilia. Because these D). Using heart primordium lateralization as a readout of ciliary are specialized types of primary cilia with motile function, we function, administration of 4 ng of either Ofd1 or Bbs4 morpholino decided to examine the localization of OFD1 in cultured human produced defects in 20% and 50% of embryos, respectively, multiciliated cells with motile cilia. For this purpose, primary whereas only 1% of controls had these defects (Fig. 7E,F). When human nasal epithelial cells were cultured at an air–liquid a lower dose (2 ng) of either morpholino was administered alone, interface (ALI) to induce differentiation (Hirst et al., 2010). As laterality defect frequencies were similar to controls. Strikingly, cells differentiate, they assemble around 200 basal bodies, via a co-administration of 2 ng of Ofd1 and 2 ng Bbs4 morpholinos led combination of centriolar and non-centriolar duplication to laterality defects in 28% of embryos (Fig. 7F). Unfortunately, pathways, which each then generate a motile cilium (Hagiwara Pcm-1 morpholinos caused unspecific delays in early et al., 2004). OFD1 localization was examined prior to (10 days 608 Journal of Cell Science 124 (4)

(supplementary material Fig. S5A). In differentiated cells, OFD1 was closely associated with each of the multiple basal bodies, both when the basal bodies were arranged in distinct rosette-like clusters (presumably representing a midway point in the differentiation process), and when cells were full of basal bodies (fully differentiated) (Fig. 8B,C). As in cells with primary cilia, co-staining of OFD1 with acetylated tubulin showed no evidence of OFD1 along the axonemal microtubules (Fig. 8D). It was notable that both in x–y and z-sections the OFD1 and g-tubulin signals did not precisely align, suggesting that they are part of different structures although they are both associated with basal bodies (Fig. 8B,C,E). Analysis of z-stack images revealed that OFD1 sits towards the distal ends of basal bodies between the focus of the g-tubulin signal and the axonemal microtubules (Fig. 8E). PCM-1 also localized at the sites of basal bodies in multiciliated cells but not within the ciliary (supplementary material Fig. S5B). Thus, in multiciliated cells, OFD1 and PCM-1 concentrate on basal bodies at the foot of the ciliary axoneme.

Discussion We show that the protein encoded by the OFD1 disease gene localizes not only to centrosomes and basal bodies, but also to centriolar satellites. Here, it colocalizes with other ciliopathy disease proteins, BBS4 and CEP290, and with a key centriolar satellite component, PCM-1. Our experiments further demonstrate that localization of these proteins to centriolar satellites is mutually dependent. Indeed, proteins implicated in several ciliopathies rely on each other for this particular subcellular association. Thus, centriolar satellites are crucial assembly points for proteins implicated in human ciliopathies, including OFD1. Additionally, on the basis of our zebrafish experiments, OFD1 and BBS4 functionally synergize in the whole animal (e.g. determining laterality), even if the proteins do not directly interact. Together, these observations

Journal of Cell Science provide some explanation as to why the clinical spectrum of OFD1, BBS and Joubert syndromes shows considerable overlap, for example featuring polydactyly and renal cysts. With BBS4 and CEP290, OFD1 joins a growing list of ciliopathy proteins localizing to centriolar satellites. Intriguingly, OFD1, BBS4 and CEP290 all physically interact with PCM-1. PCM-1 Fig. 7. Bbs4 and Ofd1 morpholinos synergize in disrupting body axis mutations have not yet been associated with ciliopathies, although formation and laterality in zebrafish. (A–D) Whole mount images of we suggest that it might be fruitful to seek such mutations in zebrafish embryos 28 hours after fertilization. Shown are untreated control (A) patients with apparent ciliopathies who do not have defined and embryos treated with 2 ng Bbs4 morpholino (B), 2 ng Ofd1 morpholino mutation of other genes. PCM-1 was the first component of (C) and 2 ng Bbs4 + 2 ng Ofd1 morpholino (D). Only the embryo centriolar satellites to be described and was suggested to act as the administered both Ofd1 and Bbs4 morpholinos exhibits a bent body axis. core structural element of satellites (Kubo et al., 1999; Kubo and (E)Frontal view of two zebrafish embryos (28 hours post-fertilization) Tsukita, 2003). Consistent with this, localization of OFD1, CEP290 injected with 4 ng of Ofd1 morpholino. Red vertical lines indicate the and BBS4 to centriolar satellites are all dependent on PCM-1. embryonic midline, and the arrow and black curved lines indicate the position of the heart. In the left image, the heart is positioned normally on the left side However, depletion of OFD1, BBS4 or CEP290 also leads to of the embryo, whereas in the right image the position of the heart is reversed. mislocalization of PCM-1 and other ciliopathy disease proteins (F)Histogram indicates frequency of heart laterality defects in one-day-old (see also Kim et al., 2008; Kim et al., 2004), which suggests that embryos treated with single or combined morpholinos. *P<0.001 versus removal of any satellite component destabilises this structure. control, **P<0.001 versus effects from low dose morpholinos administered Clearly, centriolar satellites are large macromolecular assemblies individually. that rely on multiple protein–protein interactions for their integrity. Our discovery that PCM-1 binds OFD1, and that the region encompassing the fifth coil-coil of OFD1 is crucial for this at ALI) and after (28 days at ALI) differentiation, and cells co- interaction, might help explain why different OFD1 mutations lead stained for g-tubulin. In contrast to results in hTERT-RPE1 cells, to clinically variable phenotypes. There exist rare males with OFD1 prior to differentiation, OFD1 was found to concentrate on mutations who do not die in early gestation nor have typical centrosomes and no distinct centriolar satellites were observed female-associated OFD1 features (Budny et al., 2006; Coene et al., (Fig. 8A). Very similar results were found for PCM-1 2009). Instead, their disorders are dominated by mental retardation, OFD1 regulates centriolar satellites 609

Journal of Cell Science Fig. 8. OFD1 localizes to basal bodies in multiciliated epithelia. (A–C) Primary human nasal epithelial cells were grown to different stages of differentiation by transfer to an air–liquid interface before fixation and co-staining with antibodies against OFD1 (red) and g-tubulin (green) (plus DNA, blue). Maximum intensity projections are shown in the upper panels. The lower panels show enlargements of the boxed areas through sequential z-sections. (D)Cells as in A–C were fixed co-stained with antibodies against OFD1 (red) and acetylated tubulin (green). (E)z-axis projections of the large panels in A–D. Cells in A are at day 10 post- transfer. Cells in B–D are at day 28 post-transfer.

sometimes associated with Joubert syndrome-like brain lesions, coil (Fig. 5D) (see also figure 7 in Coene et al., 2009). Given the with variable digital anomalies (Coene et al., 2009). Again pivotal biochemical role of PCM-1 in binding not only OFD1 but contrasting with classical OFD1 symptomatology, females carrying also BBS4 and CEP290 within satellites, we speculate that OFD1 the same mutations are healthy (Budny et al., 2006). Males affected mutations that abolish interaction with PCM-1 would result in by the atypical disease have mutations towards the end of OFD1 destabilization of PCM-1, CEP290 and BBS4 in vivo, as (frameshifts in either exon 16 or 21) and, should these mutant demonstrated by our in vitro experiments summarized in Table 1. transcipts be translated, the resulting proteins would retain the fifth This would lead to catastrophic biological effects in males, who coiled-coil domain (Fig. 5D) (see also figure 7 in Coene et al., carry only one OFD1 allele on their single , leading 2009). By striking contrast, frameshift and nonsense mutations to early embryonic lethality. By contrast, males with mutations described in females with the typical OFD1 syndrome (Macca and predicted to leave the fifth coil-coil domain intact, thus allowing Franco, 2009; Romio et al., 2003) (and presumably males in the potential interaction with PCM-1, are viable (Budny et al., 2006; same families with similar mutations and who die in early gestation) Coene et al., 2009). This, in turn, suggests that the C-terminus of would, if mutant transcripts were stable enough to be translated, OFD1, beyond the last two coiled-coil domains, has yet-to-be- overwhelmingly often generate proteins lacking the fifth coiled- defined roles, especially in brain maturation. 610 Journal of Cell Science 124 (4)

Table 1. Localization of centriolar satellite proteins in hTERT-RPE1 cells OFD1 PCM-1 Cep290 BBS4 Pericentrin Protein localization c s c s c s c s c s Interphase – + – + – + – + + – Mitosis + – – – + – – – + – +Nocodazole + – – – + – – – + – OFD1 – – – – + – – – + – PCM-1 + – – – + – – – + – BBS4 – – – – + – – – + – Cep290 n.a. n.a. + –– – n.a. n.a. + –

The predominant localization of the indicated proteins under different conditions is summarized. hTERT-RPE1 cells were observed in interphase, mitosis, after nocodazole treatment (+Nocodazole) or after depletion () using siRNAs specific for OFD1, PCM-1, BBS4 or CEP290. Presence (+) or absence (–) is indicated on the basis of results from this and previously published studies (Dammermann and Merdes, 2002; Kim et al., 2008; Kim et al., 2004). c, centrosomes; s, centriolar satellites; n.a., not analysed.

Localization of these ciliopathy disease proteins to centrosomes coiled-coil interaction. PCM-1 is also capable of oligomerization themselves is harder to pin down. In untreated interphase hTERT- via its coiled-coil region (Kubo and Tsukita, 2003); whether this RPE1 cells, OFD1 does not strongly colocalize with core competes with binding to OFD1 is not clear. By contrast, PCM-1 centrosomal markers such as centrin, Cep135 or g-tubulin. Similar interacts with BBS4 via its C-terminal non-coiled-coil region (Kim results were reported for CEP290 and BBS4 (Kim et al., 2008; et al., 2004). Hence, PCM-1 could bridge binding of OFD1 and Kim et al., 2004). These data suggest that, if there is a centrosomal BBS4, although we only detected very weak interaction between component to these proteins in cycling cells, it represents a much OFD1 and BBS4. Our interaction data is consistent with the smaller fraction of the protein than is present at centriolar satellites. consequences of overexpressing a C-terminal fragment of OFD1 PCM-1 is exclusively localized to centriolar satellites and, containing the third to sixth coiled-coil, leading to PCM-1 interestingly, upon progression through mitosis when centriolar mislocalization. This fragment also caused CEP290 mislocalization, satellites disperse, both OFD1 and CEP290 become obviously suggesting either that OFD1 directly interacts with CEP290 through associated with centrosomes whereas PCM-1 and BBS4 do not. this region or it disturbs CEP290 via PCM-1. Indeed, CEP290 co- Although we and others (Singla et al., 2010) have not observed precipitates with PCM-1, although the region of interaction is defects in mitosis in cells depleted of OFD1, the fact that the unknown (Kim et al., 2008). Intriguingly, a C-terminal fragment of protein is present on spindle poles could indicate an as-yet- OFD1 that includes the fifth and sixth coiled-coils was recently unidentified role in mitosis. In this respect, it is worth noting that found to promote interaction with SDCCAG8, the product of a OFD1 was identified as essential for mitosis in a recent genome- novel NPHP-related ciliopathy gene (Otto et al., 2010). Meanwhile, wide study by the MitoCheck consortium (Neumann et al., 2010). the section of OFD1 protein encompassing the second to fourth The ciliopathy disease proteins also display distinct patterns in coiled-coils is important for recruitment of IFT88 and association

Journal of Cell Science terms of centrosome localization following treatments that either with lebercilin, the gene for which is mutated in Leber congenital disrupt centriolar satellites or disturb their pericentriolar distribution. amaurosis (Coene et al., 2009; Singla et al., 2010). Importantly, an First, we found that CEP290 can be weakly detected at centrosomes N-terminal OFD1 fragment lacking the region required for PCM- upon depletion of OFD1, PCM-1 or BBS4, despite apparent loss of 1 interaction is localized to satellites, indicating that association satellites. However, OFD1 was detected at centrosomes upon with PCM-1 is unlikely to be the primary mechanism for this depletion of PCM-1 but not depletion of BBS4. It has been reported localization and raising the prospect that the LisH motif could that PCM-1 moves to centrosomes in the absence of CEP290 (Kim contribute to this targeting. In this regard, it is intriguing that et al., 2008), although when CEP290 localization was disturbed in satellite localization of FOR20 is also dependent on its LisH motif our hands by either OFD1 or BBS4 depletion, there was no (Sedjai et al., 2010). accumulation of PCM-1 at the centrosome. Depolymerization of the Unsurprisingly, many ciliopathy disease proteins are essential for microtubule network leads to dispersal of satellites into the cytoplasm. primary cilia formation, and not simply because of a requirement for These appear to retain OFD1 because cytoplasmic aggregates of cell cycle progression. However, a specific function for centriolar similar size to satellites could be detected. Similar results have satellites in ciliogenesis remains elusive. Certainly, satellites mediate recently been described for another LisH-containing centriolar protein transport along microtubules to the centrosome or basal satellite component, FOR20 (Sedjai et al., 2010). However, under body. For example, the incorporation of centrin, pericentrin and these conditions, OFD1 and CEP290 were also clearly detected on ninein to the centrosome depends on PCM-1 (Dammermann and centrosomes, whereas BBS4 was not (Kim et al., 2008; Kim et al., Merdes, 2002). Moreover, both BBS4 and CEP290 interact with 2004). Although these rather confusing results indicate that ciliopathy microtubule-based motor protein subunits (Chang et al., 2006; Kim disease proteins exhibit a much more complex dependency in terms et al., 2004; McEwen et al., 2007). This trafficking might provide of centrosome localization, they do suggest that in many respects the machinery for basal body biogenesis, cilia extension or OFD1 and CEP290 display remarkably similar behaviour . Indeed, both CEP290 and BBSome (summarised in Table 1). This is intriguing in light of the recent components are required for transport of Rab8 to the cilia, where this finding that mutations in both proteins can lead to Joubert syndrome GTPase enables vesicle docking and fusion events essential to (Coene et al., 2009; Kim et al., 2008). assembly of the ciliary membrane (Kim et al., 2008; Nachury, 2008). OFD1 and PCM-1 interaction required the regions encompassing Interestingly, in our experiments, mislocalization of PCM-1, either the N-terminal coiled-coil domain of PCM-1 and at least the fifth by depletion of OFD1 or expression of truncated OFD1 constructs, coiled-coil motif of OFD1, which implies the possibility of a direct did not obviously affect pericentrin levels at the centrosome. This OFD1 regulates centriolar satellites 611

suggests that there could be important differences in the way that quantification of immunofluorescence intensity, integrated pixel density was measured trafficking responds to the mislocalization or absence of PCM-1. using Openlab software (Improvision). Regarding basal body biogenesis, centriolar satellites (probably RNA interference experiments the same as structures previously termed fibrous granules) do OFD1 was depleted using two siRNA oligonucleotides (5Ј-GCUCAUAG - Ј Ј Ј accumulate in highly ciliated tissues (Hagiwara et al., 2004). CUAUUAAUUCA-3 and 5 -GAUCGAUCGUUCUGUCAAU-3 ) (Dharmacon). Two PCM-1-specific siRNA oligonucleotides were obtained from QIAGEN Moreover, we found intense staining of OFD1 and PCM-1 at the (Dammermann and Merdes, 2002). BBS4 was depleted using a pool of four basal bodies of multiciliated nasal epithelia, although discrete oligonucleotides (5Ј-GGAUAAGUGUAACCCUUUA-3Ј, 5Ј-CUGCAAGGCUG - satellites were not apparent. However, neither we, nor others UUAUCAAA-3Ј, 5Ј-CAUUAUAUCCGGAAAGAUU-3Ј and 5Ј-GCAAUGCA - CUGACUUAUGA-3Ј) (Dharmacon). Control depletions were performed with (Singla et al., 2010), observed defects in centriole duplication in luciferase (GL2) siRNAs (Dharmacon). Cells were transfected with 100 nM siRNAs cells depleted of OFD1, and PCM-1 was reported not to be essential for 72 hours using Oligofectamine (Invitrogen). for ciliogenesis in multiciliated cells (Vladar and Stearns, 2007). Preparation of cell extracts, SDS-PAGE and western blotting Nevertheless, tantalizing evidence exists for a role for OFD1 in Cells were washed once in PBS and lysed in lysis buffer (50 mM HEPES–KOH pH multiciliated cells, based on the report of a family carrying an 8.0, 100 mM KCl, 100 mM EDTA, 0.1% NP-40, 10 mM NaF, 50 mM b- OFD1 mutation. This mutation would lead to an OFD1a protein glycerophosphate, 2 mM DTT, 1ϫ Protease Inhibitor Cocktail, 10% glycerol) for 30 minutess on ice. Lysates were centrifuged for 10 minutes at 15,700 g, at 4°C, and truncated beyond the fifth coil-coil domain, but with normal protein concentrations of the cleared supernatant determined by Bradford assay. expression of the little-studied OFD1b (Budny et al., 2006). SDS-PAGE and western blotting were performed as previously described (Hames et Affected individuals had severe recurrent respiratory infections, al., 2005). Primary antibodies were against OFD1 (1:100), PCM-1 (1:1000), BBS4 and ciliary beating in respiratory epithelia was perturbed. However, (1:250), g-tubulin (0.15 g/ml), GFP (1:2000; Abcam) and GST (1:5000; Santa Cruz Biotechnology). Secondary antibodies were alkaline-phosphatase-conjugated anti- respiratory infections are not a symptom seen in classic OFD1 and rabbit or anti-mouse IgGs (1:7500; Promega) or horseradish peroxidase (HRP)- thus further work is required to define the possible roles of OFD1 labelled IgGs (Amersham). and other ciliopathy proteins in multiciliated cells. Immunoprecipitations Transfected cell extracts (4 mg) were pre-cleared for 30 minutess at 4°C with protein Materials and Methods G agarose beads (Sigma) that had been pre-washed in lysis buffer. Beads were then Plasmid constructions removed and supernatants incubated with GFP antibody (1:500; Sigma) for 1 hour OFD1 fragments were amplified by polymerase chain reaction (PCR) using full- on ice. The complexes were then captured with protein G agarose beads (again pre- length human OFD1 cDNA as the template (Romio et al., 2004). Amplified fragments washed in lysis buffer) for 1 hour at 4°C. Beads were washed four times with lysis were subcloned into the mammalian expression vector pCMV-Tag3C (Stratagene) buffer, resuspended with protein sample buffer and analysed by SDS-PAGE and and bacterial expression vectors pGEX-4T-1 (GE Healthcare) and pETM-11 (EMBL) western blotting. providing N-terminal Myc, GST or His tags, respectively. A GST-tagged C-terminal fragment of BBS4 was also generated in pGEX-4T-1 by amplifying the BBS4 GST pull-downs fragment by PCR from a GFP–BBS4 construct. All constructs were verified by DNA Purified GST-tagged protein (100 g) was incubated for 1 hour at 4°C with sequencing within the Protein and Nucleic Acid Laboratory, Leicester, UK. glutathione Sepharose 4B beads (GE Healthcare) that had been pre-washed with NETN buffer (200 mM Tris-HCl pH 8.0, 200 mM NaCl, 0.5% NP-40, 1 mM EDTA) Antibody generation containing 0.5% milk. Beads were then washed four times with NETN buffer, For production of antibodies against OFD1, rabbits were immunised with a His- resuspended in NETN buffer plus inhibitors (1 mM PMSF, 1 mM DTT, 1ϫ Protease tagged C-terminal fragment of OFD1 that spanned amino acids 145–1012. This Inhibitor Cocktail) and incubated with 1 mg of cell extract overnight at 4°C. In this fragment was expressed in Escherichia coli strain Rosetta 2 (DE3) (Novagen) and case, extracts of transfected cells were prepared by lysis with NETN buffer and

Journal of Cell Science purified under denaturing conditions according to standard protocols. For production processed as above. The next day, beads were washed four times with NETN buffer of antibodies against BBS4, rabbits were immunized with the GST-tagged BBS4 C- and resuspended in protein sample buffer for analysis by SDS-PAGE and western terminal fragment (residues 235–519) following expression in E. coli strain Rosetta blotting. 2 (DE3) (Novagen) and purification according to standard protocols. Immunizations and affinity purification of antibodies was performed by Cambridge Research Zebrafish morpholino experiments Biochemicals, Billingham, UK. Zebrafish were maintained at 28°C on a cycle of 14 hours light and 10 hours dark. Morpholino antisense oligonucleotides for Ofd1 (Spl6) and Bbs4 were injected in Cell culture and transfections one- to two-cell-stage embryos using needles pulled from glass capillary tubes, as Cells were grown in the following media: hTERT-RPE1 cells in Dulbecco’s modified previously described (Ferrante et al., 2009; Gerdes et al., 2007). In whole embryos, Eagle’s medium (DMEM)/Ham’s F12 (1:1) supplemented with 0.348% sodium the heart rudiment was visualized by direct inspection and the frequency of deviation bicarbonate solution; IMCD3 cells in DMEM/Ham’s F12 (1:1); hFF and HEK293 from normal leftward displacement of the heart cone, or cardiac ‘jogging’, was cells in DMEM; and CHO cells in Ham’s F12. All cells were supplemented with recorded as a laterality readout (Ferrante et al., 2009). Comparison between groups 10% fetal calf serum, 100 IU/ml penicillin, and 100 g/ml streptomycin. Cells were was undertaken using Fisher’s Exact Test. In addition, it was noted whether the bodies of embryos were straight or displayed abnormal curvature characteristic of grown at 37°C in a 5% CO2 atmosphere. Primary cilia formation was induced by culturing cells in serum-free medium for 48 hours. Human nasal epithelial cells were ciliary defects (Ferrante et al., 2009). obtained with ethical approval from healthy volunteers and cultured and induced to differentiate as described (Hirst et al., 2010). Transient transfections were performed Miscellaneous techniques with Lipofectamine 2000 (Invitrogen), Fugene HD (Roche) or JetPrime (Polyplus To depolymerize the microtubule cytoskeleton, cells were treated with 6 g/ml Transfection) according to the manufacturer’s instructions and analysed after 24 nocodazole for 1 hour at 37°C and then kept on ice for 30 minutes. To allow ϫ ϫ hours. microtubule regrowth, cells were subsequently washed 3 with 1 PBS and pre- warmed media added. To monitor centrosome duplication during S-phase arrest Fluorescence microscopy upon inhibition of nuclear export or Cdk2 activity, CHO cells were treated as Immunofluorescence microscopy was carried out as previously described (Fry et al., previously described (Prosser et al., 2009). For flow cytometry, cells were stained 1998). Primary antibodies were against OFD1 Ab1 (1:100) (Romio et al., 2003), with propidium iodide and analysed by flow cytometry (BD FACSCantoTM II) OFD1 Ab2 (1:100; this study), PCM-1 (1:1000) (Dammermann and Merdes, 2002), according to standard protocols. BBS4 (1:250), centrin2 (2 g/ml; N-17 Santa Cruz Biotechnology), pericentrin (2 g/ml; Abcam), acetylated tubulin (0.5 g/ml; Sigma), g-tubulin (0.15 g/ml; Sigma), We thank all members of our laboratory for useful discussion and -tubulin (0.3 g/ml; Sigma), Cep135 (1 g/ml) (Kleylein-Sohn et al., 2007), C- Stephen W. Wilson (University College London, London, UK) for Nap1 (1 g/ml) (Fry et al., 1998), CEP290 (1:200; Abcam ab84870), GFP (0.5 discussions and provision of fish facilities. We are indebted to Andreas g/ml; Sigma) and Myc (1:1000; Cell Signaling). Secondary antibodies were Alexa- Merdes (Institut des Sciences et Techniques du Médicament, Toulouse, Fluor-488- and Alexa-Fluor-594-conjugated goat anti-mouse and goat anti-rabbit IgGs (1 g/ml; Invitrogen). Images were captured using a TE300 inverted microscope France) for PCM-1 antibodies and constructs, Erich Nigg (Biozentrum, (Nikon) or Leica TCS SP5 confocal microscope equipped with a Leica DMI 6000B University of Basel, Basel, Switzerland) for Cep135 antibodies, Philip inverted microscope using a 63ϫ oil objective (numerical aperture, 1.4). For Beales (University College London, London, UK) for GFP-BBS4 and 612 Journal of Cell Science 124 (4)

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