JASN Express. Published on August 2, 2006 as doi: 10.1681/ASN.2005121351

Nephrocystin Specifically Localizes to the Transition Zone of Renal and Respiratory Cilia and Photoreceptor Connecting Cilia

Manfred Fliegauf,* Judit Horvath,* Christian von Schnakenburg,* Heike Olbrich,* Dominik Mu¨ller,† Julia Thumfart,† Bernhard Schermer,‡ Gregory J. Pazour,§ ʈ Hartmut P.H. Neumann,‡ Hanswalter Zentgraf, Thomas Benzing,‡ and Heymut Omran* *Department of Pediatrics and Adolescent Medicine and ‡Renal Division, University Hospital Freiburg, Freiburg, ʈ †Department of Pediatric Nephrology, Charite, Berlin, and Deutsches Krebsforschungszentrum, Heidelberg, Germany; and §Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts

Nephronophthisis (NPHP) is a hereditary cystic kidney disorder that causes renal failure in children and young adults and can be associated with various extrarenal disorders, including retinitis pigmentosa. Six NPHP , whose functions are disrupted by autosomal recessive mutations in patients with NPHP, have been identified. The majority of patients with NPHP carry homozygous deletions of NPHP1 encoding nephrocystin. Previous data indicate that nephrocystin forms a complex at cell junctions and focal adhesions. Here, it is shown that nephrocystin specifically localizes at the ciliary base to the transition zone of renal and respiratory cilia and to photoreceptor connecting cilia. During in vitro ciliogenesis of primary human respiratory epithelial cells, nephrocystin can be detected first with a diffuse cytoplasmic localization as soon as cell polarization starts, and translocates to the transition zone when cilia are formed. In columnar respiratory cells, nephrocystin is attached tightly to the axonemal structure of the transition zone at a region that contains the calcium-sensitive cilia autotomy site. In patients with homozygous NPHP1 deletions, nephrocystin is absent from the entire respiratory cell, including the transition zone, which might be of interest for future diagnostic approaches. Cilia formation is not altered in primary nephrocystin-deficient respiratory cells, which is consistent with previous findings obtained for the Caenorhabditis elegans ortholog. In addition, it is shown that the localization pattern of and nephrocystin differs, suggesting distinct functional roles. In conclusion, nephrocystin deficiency or dysfunction at the transition zone of renal monocilia and the photoreceptor connecting might explain renal failure and retinal degeneration that are observed in patients with NPHP1. J Am Soc Nephrol ●●: ●●●-●●●, ●●●●. doi: 10.1681/ASN.2005121351

he nephronophthisis (NPHP) complex comprises a ge- NPHP cases (1,11) and is one of the most frequent genetic netically heterogenous group of renal cystic disorders causes of ESRD in children and young adults. In the majority T with an autosomal recessive inheritance pattern. NPHP (94%) of patients with NPHP1, large homozygous deletions of can be associated with extrarenal disease manifestations, in- approximately 290 kb involving the NPHP1 locus (on chromo- cluding ocular motor apraxia, retinitis pigmentosa, Leber con- some 2q12-q13) can be detected, whereas only some patients genital amaurosis, cerebellar vermis aplasia, liver fibrosis and carry point mutations in combination with a heterozygous cone-shaped epiphyses, and rarely situs inversus (1,2). Five deletion (3,4,11). NPHP genes have been identified to date; they are responsible NPHP1 encodes nephrocystin, a 733–amino acid with for infantile (type 2, NPHP2), juvenile (type 1, NPHP1; type 4, an N-terminal coiled-coil domain, an adjacent Src homology 3 NPHP4; and type 5, NPHP5), and adolescent (type 3, NPHP3) domain flanked by two highly acidic E-rich domains, and a forms and differ in the onset of ESRD (2–8). In addition, conserved nephrocystin homology domain that encompasses CEP290/NPHP6 mutations in patients with a novel disease vari- the C-terminal two thirds of the protein (3,4). A number of ant (, NPHP6) were identified recently (9,10) protein interaction partners, including p130CAS, proline-rich NPHP type 1 (NPHP1; OMIM #256100) accounts for 62% of tyrosine kinase 2, and tensin, that are supposed to function in focal adhesion complexes or at sites of cell–cell contact in polarized MDCK cells have been identified (12,13). In addition, Received December 20, 2005. Accepted June 26, 2006. the proteins that are involved in the NPHP2, NPHP3, and Published online ahead of print. Publication date available at www.jasn.org. NPHP4 have been shown to associate with nephrocystin, sug- Address correspondence to: Dr. Heymut Omran, Department of Pediatrics and gesting assembly into a large, multiprotein complex (2,6,7,14). Adolescent Medicine, Mathildenstrasse 1, 79106 Freiburg, Germany. Phone: ϩ49- 761-270-4301; Fax: ϩ49-761-270-4344; E-mail: heymut.omran@uniklinik- Various cystic kidney disorders are associated with dysfunc- freiburg.de tion of renal monocilia (15), which are localized on the epithe-

Copyright © ●●●● by the American Society of Nephrology ISSN: 1046-6673/●●●●-0001 2 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●-●●●, ●●●● lial surface of nephron segments, where they extend into the Technologies/Invitrogen (Karlsruhe, Germany). Cells were grown on lumen of the kidney tubules and possibly act as fluid flow or coverslips for 8 d past confluence to allow for epithelial cell polarization chemosensors (16). Proteins that are involved in renal mono- and cilia formation, washed with PBS, and subjected to immunofluo- cilia function include the ciliary proteins polycystin-1 and -2 rescence staining as described below. and (mutated in autosomal dominant and autoso- mal recessive polycystic kidney disease, respectively) as well as Immunoblotting the BBS1 through BBS8 proteins (mutated in Bardet-Biedl syn- Protein extracts from Epstein-Barr virus–2-transformed B-lympho- cytes, HEK293T, MDCK, and respiratory epithelial cells were prepared drome [BBS]), which localize to the basal bodies of cilia (16–22). by standard procedures using NP-40 or RIPA lysis buffers. Axonemal The localization of the newly identified BBS9 through BBS11 high-salt protein extracts were obtained from a fresh pig trachea as proteins has not yet been investigated. Furthermore, nephro- described previously (25–27). Samples were separated on NuPAGE 4 to cystin (NPHP1), inversin (NPHP2), and nephrocystin-4 12% bis-tris gels (Invitrogen) and blotted onto polyvinylidene difluo- (NPHP4) localize to primary cilia predominantly at the ciliary ride membranes (Amersham). Blots were processed for ECL plus (Am- base in renal epithelial cells (2,14). ersham/GE Healthcare, Freiburg, Germany) detection using rabbit an- Most of our knowledge about cilia structure and function ti-nephrocystin (1:2500) and anti-rabbit–horseradish peroxidase originates from studies of the biflagellate unicellular alga (1:2500) antibodies (Santa Cruz, Heidelberg, Germany). Chlamydomonas rheinhardtii. The axonemal architecture of the motile Chlamydomonas flagella, composed of nine peripheral Immunofluorescence Analysis doublet microtubules that surround two central single micro- Respiratory epithelial cells were obtained by transnasal brush biopsy tubules, is highly reminiscent of motile respiratory cilia lining (Cytobrush Plus, Medscand, Malmo¨, Sweden) and suspended in RPMI the upper and lower airways. Each flagellum and cilium ex- 1640 medium without supplements. Cells were spread onto glass Ϫ tends from a specialized centriole, the . The centrio- slides, air-dried, and stored at 80°C until use. A pig eye was obtained from a local butchery, and the retina was removed carefully using a lar triplet microtubular structure converts within the transition scalpel. Cryosections (10 ␮m) were prepared according to standard zone into the axonemal doublet microtubular structure of the methods. Samples were treated with 4% paraformaldehyde, 0.2% Tri- cilium. We recently showed that nephrocystin is present at the ton-X 100, and 5% skim milk (all in PBS) before incubation with ciliary base of human respiratory cilia, where it co-localizes primary (at least 2 h) and secondary (30 min) antibodies at room with the retinitis pigmentosa GTPase regulator (RPGR) and the temperature. Slides were washed with PBS after each step. Appropriate phosphoacidic cluster sorting protein-1 (PACS-1) (23). controls were performed omitting the primary antibodies. Antibodies In this study, we analyze the dynamic expression of nephro- were mouse anti–acetylated-␣-tubulin and mouse anti–␥-tubulin cystin during ciliogenesis and evaluate its functional role. We (Sigma, Taufkirchen, Germany), mouse anti–␤-tubulin (Abcam, Cam- show that nephrocystin is absent in undifferentiated respira- bridge, UK), mouse anti–PACS-1, mouse anti-tensin, and mouse anti- cas tory cells, becomes cytoplasmically expressed during early p130 (Transduction Laboratories, BD Biosciences, Heidelberg, Ger- phases of in vitro ciliogenesis, and is localized predominantly many). Polyclonal rabbit antibodies against DNAH5, nephrocystin, IFT88, and IFT20 as well as mouse anti-nephrocystin antibodies have within the apical cytoplasmic area when the cell starts to po- been described previously (13,23,27–29). Secondary antibodies (Alexa larize. With the onset of budding, nephrocystin be- Fluor 488, Alexa Fluor 546) were from Molecular Probes (Invitrogen). comes tightly localized at the ciliary base and exclusively lo- DNA was stained with Hoechst 33342 (Sigma). Confocal images were calizes to the ciliary transition zone of mature respiratory taken on a Zeiss laser scanning microscope (Axiovert 200 LSM510 epithelial cells. We further show that nephrocystin co-localizes META) using a 63 ϫ 1.2 numerical aperture water immersion or a 100 ϫ with p130CAS and tensin in fully differentiated respiratory cells 1.3 numerical aperture oil immersion objective. A four-channel, eight- and thus possibly participates in a multimeric protein complex bit multitracking scan mode was used with a 1024 ϫ 1024 frame size at the transition zone. In nephrocystin-deficient respiratory and four-fold average line scan settings. Images were processed with cells of patients with homozygous NPHP1 deletions, we ana- the Zeiss LSM510 software. lyzed the effect on ciliogenesis and motile cilia function. In addition, we demonstrate localization of nephrocystin within High-Speed Video Analysis for Ciliary Beat Assessment the photoreceptor connecting cilium, which is consistent with Ciliary beat frequency was assessed with the SAVA system (30). retinitis pigmentosa that is observed in a subset of patients with Transnasal brush biopsies were viewed immediately with an Olympus ϫ NPHP1 (24). IMT-2 microscope ( 40 phase contrast objective) equipped with a Redlake ES-310Turbo monochrome high-speed video camera (Redlake, San Diego, CA) set at 125 frames per second. The ciliary beating pattern Materials and Methods was evaluated on slow-motion playbacks. Signed and informed consent was obtained from probands using protocols that were approved by the Institutional Ethics Review Board Electron Microscopy at the University of Freiburg. A mouse eye was cut into slices using a scalpel. Ultrathin retina sections were prepared according to standard methods and subjected to Cell Culture transmission electron microscopy using a Zeiss EM 900. MDCK and HEK293T cells were cultured in DMEM/10% FCS, mu- rine inner medullary collecting duct (mIMCD3) and LLC-PK1 (porcine Ciliogenesis kidney) were cultured as recommended by American Type Culture Respiratory epithelial cells from nasal conchae or polyps were ob- Collection (Manassas, VA). All cell culture reagents were from Life tained from patients who underwent ear, nose, and throat surgery or J Am Soc Nephrol ●●: ●●●-●●●, ●●●● Nephrocystin Localizes to the Ciliary Transition Zone 3

after nasal brushing biopsy. Primary cell culture was performed essen- tially as described previously (31). Briefly, cells were isolated from tissue samples with pronase (Sigma) and grown to confluent monolay- ers on collagen-coated tissue flasks in F12/DMEM/2% Ultroser G (Pall Life Sciences, Cergy-Saint-Christophe, France). Cell layers were treated with collagenase, cut into pieces, and cultured in HamЈs F12/DMEM/ 10% NU-serum (BD Biosciences) on a rotary shaker. After 10 d, most of the cells were organized in spheroids covered with motile cilia.

ϩ Ca2 -Dependent Deciliation Respiratory epithelial cells from brush biopsies were collected by centrifugation (300 ϫ g, 5 min) and resuspended in deciliation buffer that contained 15.7 mM Tris-Cl (pH 7.5), 79.1 mM NaCl, 1.56 mM

EDTA, 0.1% Triton-X 100, 15.8 mM CaCl2, and protease inhibitors (32). Deciliation (30 to 60 min at room temperature with occasional shaking) was monitored under a microscope. Aliquots were removed, cells were pelleted, and cilia were collected from the supernatant by centrifuga- tion (16,000 ϫ g, 5 min). Samples were spread onto glass slides, air- dried, and used for immunofluorescence staining as described above.

Results Nephrocystin Associates with the Axonemal Structure at the Transition Zone We recently demonstrated that nephrocystin localizes to re- nal monocilia in polarized MDCK cells (2). Here, more detailed analyses by high-resolution immunofluorescence imaging identifies that nephrocystin predominantly localizes to the cil- iary base of renal monocilia in MDCK cells (Figure 1A). Similar results were obtained using mIMCD-3 and LLC-PK1 cells (data not shown). Co-staining with ␥-tubulin, a marker of the micro- tubule organizing centers (MTOC), which are located adjacent to the basal bodies, demonstrates that nephrocystin localizes in renal monocilia of mIMCD-3 cells distal to the MTOC within the transition zone (Figure 1B).

Co-staining of ␥-tubulin, a component of the microtubule or- ganizing centers (MTOC), and nephrocystin in mIMCD3 cells confirms that nephrocystin localizes to the transition zone but not to the basal bodies. (C) Co-staining of human respiratory epithelial cells with antibodies against acetylated ␣-tubulin (green) and rabbit anti-nephrocystin antibodies (red). Specific nephrocystin staining is observed only at the ciliary bases. (D) Co-staining with antibodies against the axonemal outer arm heavy chain DNAH5 (red) and mouse anti-nephrocystin antibodies (green). DNAH5 localizes to the entire length of the ciliary and to the MTOC at the ciliary basal bodies Figure 1. Nephrocystin localizes to the transition zone of renal (27). Nephrocystin localizes distally to the MTOC at the prox- monocilia and human respiratory epithelial cilia. High-resolu- imal end of the cilia. The cilia section between the basal bodies tion confocal immunofluorescence imaging of polarized conflu- and the axoneme is the transition zone. (E and F) Partial co- ent MDCK cells, murine inner medullary collecting duct localization of nephrocystin (red) and its interaction partners (mIMCD3) cells, and human ciliated respiratory epithelial cells p130cas and tensin (green) at the transition zone. (G and H) The that were obtained by transnasal brush biopsy. Cells were intraflagellar transport proteins (IFT) IFT20 and IFT88 (red) stained using the indicated antibodies; nuclei were stained with localize to the MTOC and, with a speckled pattern, to the ciliary Hoechst 33342. Merged and overlay images are shown on the axonemes. Nephrocystin (green) shows a partially overlapping right. Bars ϭ 10 ␮m. (A) Co-staining of MDCK cells with localization with the IFT proteins at the ciliary base but does antibodies against the cilia marker acetylated ␣-tubulin (green) not co-localize with the IFT proteins at the MTOC and the and rabbit anti-nephrocystin antibodies (red). Specific nephro- ciliary axonemes. (I) At the ciliary base, nephrocystin also lo- cystin staining is observed at the base of each monocilium. (B) calizes in close proximity to ␤-tubulin. 4 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●-●●●, ●●●●

Consistent with these findings, nephrocystin also localizes to the transition zone of respiratory cilia (Figure 1C). To analyze further the role of nephrocystin in respiratory cilia, we tested whether it co-localizes with well-characterized ciliary proteins. Co-staining of the axonemal outer dynein arm motor protein DNAH5, which localizes to the MTOC and to the axonemal doublet structure of cilia (27), confirms that nephrocystin spe- cifically localizes to the transition zone, distally from the MTOC and proximally from the axonemes (Figure 1D). On the basis of interaction with tensin and p130cas,ithas been speculated that nephrocystin functions at focal adhesions (12,13). In contrast, we find that in human ciliated respiratory epithelial cells, tensin and p130cas localize at the apical cyto- plasmic area, predominantly at the MTOC region, where their signals overlap at the proximal end of the transition zone with nephrocystin (Figure 1, E and F). Because the transition zone is thought to act as the docking site for the intraflagellar transport Figure 2. Nephrocystin localizes to photoreceptor-connecting (IFT) particles, we next tested whether nephrocystin co-local- cilia at the junction of the inner segment (IS) and outer segment (OS). (A) Electron microscopic image of a mouse photoreceptor izes with the IFT proteins IFT88 and IFT20. Similarly, as in demonstrating the position of the connecting cilium (cc) and the Chlamydomonas (33), both IFT proteins are localized predomi- basal body (bb) between the OS and the IS. Bar ϭ 1 ␮m. (B) nantly at the ciliary base and with a speckled pattern along the Immunofluorescence staining of a cryosectioned pig retina ciliary axonemes in respiratory epithelial cells (Figure 1, G and demonstrates that nephrocystin (red) co-localized with acety- H). Overlapping staining of nephrocystin with IFT proteins is lated ␣-tubulin (green), a marker of the photoreceptor-connect- observed only at the proximal part of the transition zone but ing cilia. (C) Co-staining of a cryosectioned pig retina with not within the ciliary axoneme. In addition, antibodies directed antibodies against nephrocystin (red) and ␥-tubulin (green) against ␤-tubulin stained the entire axonemes. At the proximal shows that nephrocystin localizes in close proximity to ␥-tubu- axonemal end, ␤-tubulin localizes in close vicinity of nephro- lin which marks the positions of the basal body/centrioles. The cystin (Figure 1I). OS and the IS, the outer nuclear layer (ONL), the outer plexi- Because retinitis pigmentosa can be found in patients with form layer (OPL), and the inner nuclear layer (INL) are indi- cated. Nuclei were stained with Hoechst 33342 (blue). Bars ϭ 10 NPHP1, we tested whether nephrocystin localizes to the con- ␮m in B and C. necting cilium that bridges the outer and inner photoreceptor segments (Figure 2A). Co-staining of retinal cryosections with antibodies against nephrocystin and either acetylated ␣-tubulin axonemal structure. This also is supported by Western blot or ␥-tubulin demonstrates that nephrocystin exclusively local- analysis, which identified nephrocystin in high-salt protein izes to the connecting cilium in close proximity to the basal extracts from purified demembranated respiratory ciliary bodies (Figure 2, B and C). axonemes. To analyze whether nephrocystin is associated tightly with the axonemal structure at the ciliary base, we took advantage of Nephrocystin Expression During Ciliogenesis the microtubule severing mechanism by which eukaryotic cells We next addressed the question of whether the localization ϩ excise cilia and flagella upon treatment with Ca2 -containing of nephrocystin changes during the process of respiratory cell buffers. The deflagellation mechanism in Chlamydomonas in- differentiation and in vitro ciliogenesis of primary human re- ϩ volves the Ca2 binding protein centrin, which is part of the spiratory cells. Dissociated cells from nasal polyps were grown contractible stellate fibers within the transition zone (34). Treat- to confluence on collagen layers and subsequently were differ- ment of human respiratory epithelial cells with deciliation entiated into ciliated spheroids (cell conglomerates) in suspen- buffer resulted in complete or partial cilia autotomy in most sion cultures (31). cells (Figure 3). Isolated autotomized cilia showed specific At an early stage of in vitro ciliogenesis, cell morphology of punctual nephrocystin staining at the proximal end (Figure spheroids is characterized by a symmetric cell body and a 3A). All partially deciliated cells (Figure 3, B and C) as well as central nucleus in most spheroids (data not shown). These cells all completely deciliated cell remnants (Figure 3D) showed do not express nephrocystin and do not carry cilia as evidenced specific nephrocystin staining at the apical cellular surface. by the absence of acetylated ␣-tubulin staining. A few cells ϩ Thus, Ca2 -dependent deciliation in respiratory epithelial cells within each spheroid then start to express nephrocystin with a occurs within the region where nephrocystin is localized. This diffuse cytoplasmic localization (Figure 4, A and B), which specific staining pattern is observed even after prolonged treat- precedes cilia formation (4 to 8 d). In the next period of respi- ment of the cells with deciliation buffer that contains detergent, ratory cell differentiation (6 to 8 d), most cells in each spheroid which removes the membranes from cell remnants and cilia show diffuse cytoplasmic nephrocystin expression (Figure 4B) (25). We therefore conclude that nephrocystin is not a compo- that becomes apically enhanced in those cells that exhibit cell nent of the ciliary membrane but associated tightly with the polarization, recognizable by a more prolonged cell body and J Am Soc Nephrol ●●: ●●●-●●●, ●●●● Nephrocystin Localizes to the Ciliary Transition Zone 5

Figure 3. The nephrocystin-positive transition zone contains the ciliary autotomy site. Human ciliated respiratory cells were ϩ subjected to Ca2 -dependent deciliation by microtubule severing within the transition zone. (A) Purified autotomized cilia show punctuate nephrocystin staining (red) at the proximal end. Axonemes were stained with antibodies against acetylated ␣-tubulin. Completely (B) and partially (C) deciliated cell remnants stain positive for nephrocystin (red) at the sites where cilia autotomy occurs. (D) Co-staining of a deciliated cell with antibodies against DNAH5 (red) and nephrocystin (green). DNAH5 staining at the MTOC and nephrocystin staining distally from the MTOC in deciliated cell remnants demonstrate that microtubule severing occurs within the nephrocystin-positive transition zone. Bars ϭ 10 ␮m in A and B. downward placement of the nucleus. The concentration of E). Staining of nephrocystin at the basolateral side of respira- nephrocystin at the apical cell region coincides with expression tory cells never was observed. of acetylated ␣-tubulin that specifically localizes to well- defined spots beneath the apical plasma membrane in these Nephrocystin Deficiency Does Not Alter Cilia Formation cells, indicating the beginning of axoneme budding and cilia Because nephrocystin translocalizes from the to formation (Figure 4, B and C). During later stages of ciliogen- the transition zone during respiratory epithelial cell differenti- esis (8 to 12 d), the punctual localization of nephrocystin at the ation, we next tested whether absence of nephrocystin affects base of each growing and mature axoneme and proximally to ciliogenesis or cilia function. We analyzed respiratory epithelial the acetylated ␣-tubulin remains unchanged (Figure 4, D and cells from patients with NPHP1 (patients ON-21, ON-23, ON- 6 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●-●●●, ●●●●

43, ON-45II1, ON45II2, and ON-50), who had received a diag- nosis before for homozygous NPHP1 deletions according to routine molecular genetic testing of juvenile NPHP (11). We first confirmed the deletion of genomic NPHP1 sequences by PCR (data not shown) and the absence of nephrocystin by Western blot in four of these patients using Epstein-Barr virus– transformed B-lymphocytes (Figure 5H). We then confirmed by immunofluorescence imaging that respiratory epithelial cells from all six patients are deficient for nephrocystin (Figure 5, A and B). Because cell and cilia mor- phology were indistinguishable from controls, nephrocystin deficiency does not change the overall integrity of respiratory cilia. To test whether nephrocystin localization also is altered in patients with other forms of NPHP, we analyzed respiratory epithelial cells from two patients who had adolescent NPHP and carried compound heterozygous NPHP3 mutations (pa- tients F23II1 and F23II2) and one patient who had NPHP type 5 and carried compound heterozygous NPHP5 mutations (pa- tient A19), which were reported previously (7,8). In these pa- tients, cell morphology and nephrocystin localization were nor- mal (data not shown), demonstrating that NPHP3 and NPHP5 mutations in the analyzed patients do not alter nephrocystin localization. In addition, we found normal nephrocystin local- ization in two patients with BBS, five patients with autosomal recessive polycystic kidney disease, and three patients with NPHP (for which NPHP1 deletions have been excluded) and unknown mutations (data not shown). These results demon- strate that absence of nephrocystin from the transition zone is a specific finding in patients who have NPHP with NPHP1 de- letions rather than a common alteration in cystic kidney dis- eases. Next, we asked whether nephrocystin is essential for correct targeting of ciliary/basal body proteins. However, staining of nephrocystin-deficient respiratory cells with antibodies against PACS-1 (Figure 5, C and D); ␥-tubulin, a component of the pericentriolar material around the basal bodies; and IFT88 (Fig- Figure 4. Nephrocystin translocalizes from the cytoplasm to the ure 5, E and F) revealed normal localization of all three proteins ciliary transition zone during respiratory epithelial cell polar- at the ciliary base. These results demonstrate that nephrocystin ization. Confocal immunofluorescence imaging of human re- spiratory epithelial cells that were subjected to spheroidal is not required for targeting of these proteins to the ciliary base growth and in vitro ciliogenesis. Co-staining with the antibodies and that the structural integrity of the ciliary base remains against cilia-specific acetylated ␣-tubulin (green) and nephro- unaffected in nephrocystin-deficient cells. Furthermore, be- cystin (red). Nuclei were stained with Hoechst 33342. Bars ϭ 10 cause the localization IFT88 along the ciliary axonemes also ␮m. (A) In early stages of spheroidal growth, only a few cells remains unaffected in nephrocystin-deficient cells, intraflagel- express nephrocystin with a diffuse cytoplasmic localization. lar transport per se obviously is not impaired by the absence of Cells show a round appearance with no indication of polariza- nephrocystin from the transition zone. tion. No specific acetylated ␣-tubulin staining is detectable. (B) We also tested whether the loss of nephrocystin impairs the At the beginning of cell polarization, indicated by cone-shaped function of respiratory cilia because three of the patients with cells, diffuse cytoplasmic nephrocystin expression becomes lo- NPHP1 deletions (ON-21, ON-23, and ON-50) reported mild cally enriched at the apical cytoplasmic region. In these cells, ␣ respiratory symptoms with chronic sinusitis and rhinitis sug- acetylated -tubulin is detectable at the apical plasma mem- brane, indicating the beginning of axoneme budding. (C and D) gestive of a cilia dysmotility defect. High-speed video-micro- In later stages of cell polarization, nephrocystin becomes con- scopic analyses of respiratory epithelial cells from these pa- centrated at the apical cell surface and is tightly associated with tients showed normal ciliary beat frequencies (5 to 9 Hz at room the bases of the growing respiratory cilia. (E) In heavily ciliated temperature) and normal beat amplitudes. However, evalua- spheroids, nephrocystin shows a speckled localization pattern tion of slow-motion playbacks revealed that ciliary motility is at the ciliated apical cell side. Nephrocystin localizes exclu- slightly irregular (Supplementary Videos 1 and 2). sively to the base of cilia, and cytoplasmic localization is no To examine whether the observed dysmotility is caused by longer detectable. J Am Soc Nephrol ●●: ●●●-●●●, ●●●● Nephrocystin Localizes to the Ciliary Transition Zone 7

secondary effects, we grew respiratory epithelial cells that were obtained by transnasal brush biopsies from patient ON-50 to heavily ciliated spheroids in vitro, which bypasses secondary ciliary dyskinesia (35). Nephrocystin-deficient spheroids (Fig- ure 5G) had normal morphology and normal ciliogenesis when compared with control cells (Figure 4) as well as normal ciliary beat frequencies and amplitudes. Although we noted a slightly irregular beating pattern, the degree of the observed abnormal- ity was not as severe as usually observed in primary ciliary dyskinesia (Supplementary Videos 3 and 4).

Discussion In recent years, a link between cystic kidney disease and renal monocilia dysfunction became evident by the discovery that genes that are mutated in cystic kidney disorders all en- code cilia-related proteins that localize either to the ciliary axoneme or to the ciliary base. Although a number of disease- related genes have been identified within the NPHP complex, the molecular mechanisms underlying the disease process largely have remained elusive. Importantly, NPHP often occurs in association with extrarenal manifestations such as ocular motor apraxia (type Cogan), retinitis pigmentosa (Senior-Løken syndrome), liver fibrosis, cone-shaped epiphyses (Mainzer- Saldino syndrome), and cerebellar vermis aplasia (Joubert’s syndrome type B). These associations indicate that NPHP pro- teins play a functional role in a variety of distinct cell types, which also is consistent with their broad expression (3,6,7,36). Here we show that nephrocystin specifically localizes to a well-defined region at the ciliary base, the transition zone, of renal monocilia and respiratory cilia (Figure 1). This is consis- tent with previous observations in which nephrocystin local- ization was strongest at the ciliary base in renal monocilia (2) and respiratory cilia (23). In contrast to previous studies using high-stringency staining conditions and avoiding background signals, we cannot demonstrate nephrocystin localization clearly within the ciliary axoneme. We confirm our results using distinct polyclonal and monoclonal anti-nephrocystin an- tibodies and demonstrate antibody specificity in nephrocystin-

Figure 5. Nephrocystin-deficient respiratory epithelial cells form normal cilia and basal bodies. Confocal immunofluores- cence imaging of ciliated respiratory epithelial cells from ponent of the MTOC, and the IFT88 in nephrocystin-deficient healthy probands (control) and patients with nephronophthisis cells indicates that the integrity of the ciliary bases and the (NPHP) with NPHP1 deletions. Nuclei were stained with intraflagellar transport processes per se are not disrupted by the Hoechst 33342 (blue). Bars ϭ 10 ␮m. (A and B) Localization of absence of nephrocystin from the ciliary transition zones. (G) In nephrocystin (red) at the transition zone of respiratory cilia in vitro ciliogenesis of respiratory epithelial cells organized in control cells. Cilia were stained with anti-acetylated ␣-tubulin spheroids from a healthy donor (left) and from a patient with antibodies (green). No specific nephrocystin staining (red) is NPHP with NPHP1 deletions (right). Nephrocystin deficiency observed in respiratory epithelial cells from patients with does not inhibit epithelial cell polarization and cilia formation. NPHP with NPHP1 deletions. In B, microscope settings of the (H) By Western blot, nephrocystin is detected in crude lysates red channel were adjusted to visualize background staining. from tracheal epithelial cells and in high-salt extracts from No red signals are observed when identical settings as in A are isolated demembranated respiratory ciliary axonemes, indicat- used. Nephrocystin deficiency does not impair respiratory ep- ing that nephrocystin is associated tightly with axonemal struc- ithelial cell polarization and cilia formation. (C and D) Co- tures. MDCK cells were used as a control (top). Nephrocystin localization of phosphoacidic cluster sorting protein-1 (PACS-1; can be readily detected in protein extracts from Epstein-Barr green) and nephrocystin (red) at the transition zone of respira- virus–transformed B-lymphocytes from healthy donors (con- tory cilia in control cells. Normal localization of PACS-1 in trol) but is absent in these cells that were derived from patients nephrocystin-deficient cells indicate normal integrity of the with NPHP1 (ON-21, ON-23, ON-43, and ON-50). HEK293T ciliary base. (E and F) Normal localization of ␥-tubulin, a com- cells were included as a control (bottom). 8 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●-●●●, ●●●● deficient cells by Western blot and high-resolution confocal BBS proteins and inversin (NPHP2), which are involved in the imaging (Figures 1 and 5). However, we point out that small pathogenesis of BBS and infantile NPHP, respectively (40,41). nephrocystin pools within the ciliary axoneme might be not In nephrocystin-deficient respiratory cells from patients with detectable by immunofluorescence staining. homozygous NPHP1 deletions (Figure 5), expression and local- In Caenorhabditis elegans, the homologs of nephrocystin and ization of PACS-1, a binding partner of nephrocystin, was nephrocystin-4 also localize to the cilia transition zone of sen- normal, indicating that nephrocystin is not essential for the sory neurons but were not detected within the ciliary axoneme localization of PACS-1 to the ciliary base. In addition, because (37), which indicates evolutionary conservation of subcellular the localization of ␥-tubulin and IFT88 at the ciliary base and localization from C. elegans to human. In addition, we demon- along the ciliary axoneme, respectively, is normal in nephro- strate for the first time that nephrocystin localizes to the pho- cystin-deficient cells, the structural integrity of the cilia and the toreceptor-connecting cilium (Figure 2), which might explain intraflagellar transport per se obviously is unaffected by the why a subset of patients with NPHP1 exhibit retinal degener- absence of nephrocystin from the transition zone. Furthermore, ation (24). It is interesting that the photoreceptor-connecting in vitro ciliogenesis of nephrocystin-deficient respiratory cells cilium is the analogous structure of the ciliary transition zone, did not reveal any obvious abnormalities, indicating that where the interaction partners retinitis pigmentosa GTPase reg- nephrocystin is essential neither for establishment of cell polar- ulator (RPGR) and NPHP5 also are localized (8,38). In addition, ity nor for cilia formation in respiratory epithelial cells. Similar NPHP4 and NPHP6 have been localized to the ciliary base results have been obtained in C. elegans, where nphp-1 and (9,14,39). This indicates that nephrocystin possibly participates nphp-4 mutants did not exhibit abnormalities of cilia morphol- in the function of these proteins at the ciliary base. ogy in sensory neuronal cells (37). Defects of the intraflagellar transport cause shortened flagella To increase our understanding of nephrocystin function, we in Chlamydomonas and shortened renal monocilia and cystic analyzed primary human respiratory cells, which are readily kidney disease in tg737orpk mice (15,42). The observation of accessible, carry multiple instead of single cilia on their surface, normal cilia morphology in nephrocystin-deficient cells argues and allow in vitro ciliogenesis. Furthermore, nephrocystin- against a function of nephrocystin as an intraflagellar transport deficient cells from patients with juvenile NPHP can be ob- protein that is essential for transport of ciliary proteins across tained, obviating analyses of genetically manipulated cell sys- the compartment border and along the axonemes (43). In ad- tems. dition, the subcellular localization of the intraflagellar transport Because nephrocystin physically interacts with a number of proteins IFT88 and IFT20, typically within the MTOC and with proteins, including p130cas, tensin, PACS-1, ␤-tubulin, and a punctuate pattern along the ciliary axonemes, differs from the nephrocystin-2, -3, and -4, previous data suggested that these localization of nephrocystin, which is confined to the transition proteins form a complex at cell junctions, at focal adhesions, or zone (Figure 1). It is interesting that IFT88 and IFT20 localize in within the ciliary axoneme (2,12,13,19). Our data indicate, how- close proximity to nephrocystin with a narrow overlapping ever, that these proteins probably assemble into a functional localization at the proximal end of the transition zone. Thus, protein complex at the ciliary base, which is supported by the nephrocystin exactly localizes to the ciliary substructure, where cas ␤ subcellular localization of p130 , tensin, -tubulin (Figure 1, E, protein transfer to and from the ciliary compartment occurs. F, and I) and PACS-1 (Figure 5, C and D) with overlapping Nephrocystin might be a component of a supramolecular struc- staining patterns or localization in close proximity to nephro- ture called the ciliary (flagellar) pore complex, which is marked cystin in respiratory epithelial cells. by transition fibers in Chlamydomonas (43). We show that nephrocystin localization extends proximally Three studied patients with homozygous NPHP1 deletions and distally to the ciliary autotomy site (Figure 3), within the reported symptoms that also occur in primary ciliary dyskine- 2ϩ transition zone where Ca -dependent microtubule severing sia (44,45), suggesting a possible cilia motility defect. Although occurs (34), where it is tightly attached to the axonemal struc- the beating patterns of respiratory cilia were slightly irregular ture. This robust, detergent-resistant attachment possibly is in these patients, we do not consider the observed findings to mediated by interaction with the axonemal structural compo- be diagnostic for primary ciliary dyskinesia, in absence of other nent ␤-tubulin (2) and indicates that nephrocystin is neither a typical symptoms such as bronchiectasis. component of the ciliary membrane nor a transiently bound Our finding that nephrocystin deficiency can be readily iden- molecule at the ciliary base. During in vitro ciliogenesis (Figure tified by immunofluorescence microscopy in nasal respiratory 4), we found that nephrocystin is not detectable in undifferen- cells that are obtained by noninvasive brush biopsy might tiated respiratory cells but is expressed with a diffuse cytoplas- contribute to the development of novel diagnostic tools in mic localization as soon as establishment of cellular polarity cystic kidney disorders. This suggests that DNA diagnosis by becomes evident. Simultaneously with axoneme budding and molecular genetic approaches may be directed depending on a the appearance of respiratory cilia, nephrocystin completely characteristic aberrant staining pattern. translocalizes to the ciliary bases. Although reminiscent of a possible role for establishment or maintenance of cell polarity, Acknowledgments we did not find evidence for obviously disrupted respiratory J.H. received research grants from the German Academic Exchange epithelial cell polarization in nephrocystin-deficient cells (Fig- Service. This work was supported in part by grants from the “Deutsche ure 5). In contrast, such a role was demonstrated previously for Forschungsgemeinschaft” (SFB592 and DFG Om6/2 to H.O.; DFG J Am Soc Nephrol ●●: ●●●-●●●, ●●●● Nephrocystin Localizes to the Ciliary Transition Zone 9

Be2212 to T.B.) and from the Zentrum Klinische Forschung of the Williams DS, Heckenlively JR, Swaroop A: An in-frame University Freiburg. deletion in a novel centrosomal/ciliary protein CEP290/ We are grateful to the patients for participation in this study and for NPHP6 perturbs its interaction with RPGR and results in the support of the Arbeitsgemeinschaft fuer Paediatrische Nephrologie. early-onset retinal degeneration in the rd16 mouse. Hum We thank Roland Nitschke and Sabine Haxelmans, Life Imaging Cen- Mol Genet 15: 1847–1857, 2006 ter, Institute for Biology I, University Freiburg, for excellent support 10. Sayer JA, Otto EA, O’Toole JF, Nurnberg G, Kennedy MA, with confocal imaging and Julia Kalnitski, Karin Sutter, Carmen Kopp, Becker C, Hennies HC, Helou J, Attanasio M, Fausett BV, and Myriam Krome for technical assistance. Utsch B, Khanna H, Liu Y, Drummond I, Kawakami I, Kusakabe T, Tsuda M, Ma L, Lee H, Larson RG, Allen SJ, Wilkinson CJ, Nigg EA, Shou C, Lillo C, Williams DS, References Hoppe B, Kemper MJ, Neuhaus T, Parisi MA, Glass IA, 1. Hildebrandt F, Omran H: New insights: Nephronophthi- Petry M, Kispert A, Gloy J, Ganner A, Walz G, Zhu X, sis-medullary cystic kidney disease. Pediatr Nephrol 16: Goldman D, Nurnberg P, Swaroop A, Leroux MR, Hilde- 168–176, 2001 brandt F: A novel centrosomal protein nephrocystin-6 is 2. Otto EA, Schermer B, Obara T, O’Toole JF, Hiller KS, mutated in Joubert syndrome and activates transcription Mueller AM, Ruf RG, Hoefele J, Beekmann F, Landau D, factor ATF4/CREB2. Nat Genet 38: 674–681, 2006 Foreman JW, Goodship JA, Strachan T, Kispert A, Wolf 11. Hildebrandt F, Rensing C, Betz R, Sommer U, Birnbaum S, MT, Gagnadoux MF, Nivet H, Antignac C, Walz G, Drum- Imm A, Omran H, Leipoldt M, Otto E; Arbeitsgemein- mond IA, Benzing T, Hildebrandt F: Mutations in INVS schaft fur Paediatrische Nephrologie (APN) Study Group: encoding inversin cause nephronophthisis type 2, linking Establishing an algorithm for molecular genetic diagnos- renal cystic disease to the function of primary cilia and tics in 127 families with juvenile nephronophthisis. Kidney left-right axis determination. Nat Genet 34: 413–420, 2003 Int 59: 434–445, 2001 3. Hildebrandt F, Otto E, Rensing C, Nothwang HG, Vollmer 12. Donaldson JC, Dempsey PJ, Reddy S, Bouton AH, Coffey M, Adolphs J, Hanusch H, Brandis M: A novel en- RJ, Hanks SK: Crk-associated substrate p130(Cas) interacts coding an SH3 domain protein is mutated in nephronoph- with nephrocystin and both proteins localize to cell-cell thisis type 1. Nat Genet 17: 149–153, 1997 contacts of polarized epithelial cells. Exp Cell Res 256: 168– 4. Saunier S, Calado J, Heilig R, Silbermann F, Benessy F, 178, 2000 Morin G, Konrad M, Broyer M, Gubler MC, Weissenbach J, 13. Benzing T, Gerke P, Hopker K, Hildebrandt F, Kim E, Walz Antignac C: A novel gene that encodes a protein with a G: Nephrocystin interacts with Pyk2, p130(Cas), and tensin putative src homology 3 domain is a candidate gene for and triggers phosphorylation of Pyk2. Proc Natl Acad Sci familial juvenile nephronophthisis. Hum Mol Genet 6: 2317– USA98: 9784–9789, 2001 2323, 1997 14. Mollet G, Silbermann F, Delous M, Salomon R, Antignac C, 5. Otto E, Hoefele J, Ruf R, Mueller AM, Hiller KS, Wolf MT, Saunier S: Characterization of the nephrocystin/nephro- Schuermann MJ, Becker A, Birkenhager R, Sudbrak R, cystin-4 complex and subcellular localization of nephro- Hennies HC, Nurnberg P, Hildebrandt F: A gene mutated cystin-4 to primary cilia and centrosomes. Hum Mol Genet in nephronophthisis and retinitis pigmentosa encodes a 14: 645–656, 2005 novel protein, nephroretinin, conserved in evolution. Am J 15 Pazour GJ, Dickert BL, Vucica Y, Seeley ES, Rosenbaum JL, Hum Genet 71: 1161–1167, 2002 Witman GB, Cole DG: Chlamydomonas IFT88 and its 6. Mollet G, Salomon R, Gribouval O, Silbermann F, Bacq D, mouse homologue, polycystic kidney disease gene tg737, Landthaler G, Milford D, Nayir A, Rizzoni G, Antignac C, are required for assembly of cilia and flagella. J Cell Biol Saunier S: The gene mutated in juvenile nephronophthisis 151: 709–718, 2000 type 4 encodes a novel protein that interacts with nephro- 16. Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, cystin. Nat Genet 32: 300–305, 2002 Elia AE, Lu W, Brown EM, Quinn SJ, Ingber DE, Zhou J: 7. Olbrich H, Fliegauf M, Hoefele J, Kispert A, Otto E, Volz A, Polycystins 1 and 2 mediate mechanosensation in the pri- Wolf MT, Sasmaz G, Trauer U, Reinhardt R, Sudbrak R, mary cilium of kidney cells. Nat Genet 33: 129–137, 2003 Antignac C, Gretz N, Walz G, Schermer B, Benzing T, 17. Pazour GJ, San Agustin JT, Follit JA, Rosenbaum JL, Wit- Hildebrandt F, Omran H: Mutations in a novel gene, man GB: Polycystin-2 localizes to kidney cilia and the NPHP3, cause adolescent nephronophthisis, tapeto-retinal ciliary level is elevated in orpk mice with polycystic kidney degeneration and hepatic fibrosis. Nat Genet 34: 455–459, disease. Curr Biol 12: R378–R380, 2002 2003 18. Yoder BK, Tousson A, Millican L, Wu JH, Bugg CE Jr, 8. Otto EA, Loeys B, Khanna H, Hellemans J, Sudbrak R, Fan Schafer JA, Balkovetz DF: Polaris, a protein disrupted in S, Muerb U, O’Toole JF, Helou J, Attanasio M, Utsch B, orpk mutant mice, is required for assembly of renal cilium. Sayer JA, Lillo C, Jimeno D, Coucke P, De Paepe A, Rein- Am J Physiol Renal Physiol 282: F541–F552, 2002 hardt R, Klages S, Tsuda M, Kawakami I, Kusakabe T, 19. Watnick T, Germino G: From cilia to cyst. Nat Genet 34: Omran H, Imm A, Tippens M, Raymond PA, Hill J, Beales 355–356, 2003 P, He S, Kispert A, Margolis B, Williams DS, Swaroop A, 20. Wang S, Luo Y, Wilson PD, Witman GB, Zhou J: The Hildebrandt F: Nephrocystin-5, a ciliary IQ domain pro- autosomal recessive polycystic kidney disease protein is tein, is mutated in Senior-Loken syndrome and interacts localized to primary cilia, with concentration in the basal with RPGR and calmodulin. Nat Genet 37: 282–288, 2005 body area. J Am Soc Nephrol 15: 592–602, 2004 9. Chang B, Khanna H, Hawes N, Jimeno D, He S, Lillo C, 21. Ansley SJ, Badano JL, Blacque OE, Hill J, Hoskins BE, Parapuram SK, Cheng H, Scott A, Hurd RE, Sayer JA, Otto Leitch CC, Kim JC, Ross AJ, Eichers ER, Teslovich TM, EA, Attanasio M, O’Toole JF, Jin G, Shou C, Hildebrandt F, Mah AK, Johnsen RC, Cavender JC, Lewis RA, Leroux MR, 10 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●-●●●, ●●●●

Beales PL, Katsanis N: Basal body dysfunction is a likely tifies basal body transitional fibers as the docking site for cause of pleiotropic Bardet-Biedl syndrome. Nature 425: IFT particles. Curr Biol 11: 1586–1590, 2001 628–633, 2003 34. Sanders MA, Salisbury JL: Centrin plays an essential role in 22. Badano JL, Teslovich TM, Katsanis N: The centrosome in microtubule severing during flagellar excision in Chlamy- human genetic disease. Nat Rev Genet 6: 194–205, 2005 domonas reinhardtii. J Cell Biol 124: 795–805, 1994 24. Schermer B, Hopker K, Omran H, Ghenoiu C, Fliegauf M, 35. Jorissen M, Willems T, Van der Schueren B, Verbeken E: Fekete A, Horvath J, Kottgen M, Hackl M, Zschiedrich S, Secondary ciliary dyskinesia is absent after ciliogenesis in Huber TB, Kramer-Zucker A, Zentgraf H, Blaukat A, Walz culture. Acta Otorhinolaryngol Belg 54: 333–342, 2000 G, Benzing T: Phosphorylation by casein kinase 2 induces 36. Otto E, Kispert A, Schatzle, Lescher B, Rensing C, Hilde- PACS-1 binding of nephrocystin and targeting to cilia. brandt F: Nephrocystin: and sequence EMBO J 24: 4415–4424, 2005 conservation between human, mouse, and Caenorhabditis 24. Caridi G, Murer L, Bellantuono R, Sorino P, Caringella DA, elegans. J Am Soc Nephrol 11: 270–282, 2000 Gusmano R, Ghiggeri GM: Renal-retinal syndromes: Asso- 37. Winkelbauer ME, Schafer JC, Haycraft CJ, Swoboda P, ciation of retinal anomalies and recessive nephronophthi- Yoder BK: The C. elegans homologs of nephrocystin-1 and sis in patients with homozygous deletion of the NPH1 nephrocystin-4 are cilia transition zone proteins involved locus. Am J Kidney Dis 32: 1059–1062, 1998 in chemosensory perception. J Cell Sci 118: 5575–5587, 2005 25. Hastie AT: Isolation of respiratory cilia. Methods Cell Biol 38. Hong DH, Pawlyk B, Sokolov M, Strissel KJ, Yang J, Tul- 47: 93–98, 1995 loch B, Wright AF, Arshavsky VY, Li T: RPGR isoforms in 26. Reed W, Carson JL, Moats-Staats BM, Lucier T, Hu P, photoreceptor connecting cilia and the transitional zone of Brighton L, Gambling TM, Huang CH, Leigh MW, Collier motile cilia. Invest Ophthalmol Vis Sci 44: 2413–2421, 2003 AM: Characterization of an axonemal dynein heavy chain 39. Roepman R, Letteboer SJ, Arts HH, van Beersum SE, Lu X, expressed early in airway epithelial ciliogenesis. Am J Re- Krieger E, Ferreira PA, Cremers FP: Interaction of nephro- spir Cell Mol Biol 23: 734–741, 2000 cystin-4 and RPGRIP1 is disrupted by nephronophthisis or 27. Fliegauf M, Olbrich H, Horvath J, Wildhaber JH, Zariwala Leber congenital amaurosis-associated mutations. Proc MA, Kennedy M, Knowles MR, Omran H: Mislocalization Natl Acad Sci U S A 102: 18520–18525, 2005 of DNAH5 and DNAH9 in respiratory cells from patients 40. Ross AJ, May-Simera H, Eichers ER, Kai M, Hill J, Jagger with primary ciliary dyskinesia. Am J Respir Crit Care Med DJ, Leitch CC, Chapple JP, Munro PM, Fisher S, Tan PL, 171: 1343–1349, 2005 Phillips HM, Leroux MR, Henderson DJ, Murdoch JN, 28. Pazour GJ, Baker SA, Deane JA, Cole DG, Dickert BL, Copp AJ, Eliot MM, Lupski JR, Kemp DT, Dollfus H, Tada Rosenbaum JL, Witman GB, Besharse JC: The intraflagellar transport protein, IFT88, is essential for vertebrate photo- M, Katsanis N, Forge A, Beales PL: Disruption of Bardet- receptor assembly and maintenance. J Cell Biol 157: 103– Biedl syndrome ciliary proteins perturbs planar cell polar- 113, 2002 ity in vertebrates. Nat Genet 37: 1135–1140, 2005 29. Baker SA, Freeman K, Luby-Phelps K, Pazour GJ, Besharse 41. Simons M, Gloy J, Ganner A, Bullerkotte A, Bashkurov M, JC: IFT20 links kinesin II with a mammalian intraflagellar Kronig C, Schermer B, Benzing T, Cabello OA, Jenny A, transport complex that is conserved in motile flagella and Mlodzik M, Polok B, Driever W, Obara T, Walz G: Inversin, sensory cilia. J Biol Chem 278: 34211–34218, 2003 the gene product mutated in nephronophthisis type II, 30. Sisson JH, Stoner JA, Ammons BA, Wyatt TA: All-digital functions as a molecular switch between Wnt signaling image capture and whole-field analysis of ciliary beat fre- pathways. Nat Genet 37: 537–543, 2005 quency. J Microsc 211: 103–111, 2003 42. Yoder BK, Hou X, Guay-Woodford LM: The polycystic 31. Laoukili J, Perret E, Willems T, Minty A, Parthoens E, kidney disease proteins, polycystin-1, polycystin-2, polaris, Houcine O, Coste A, Jorissen M, Marano F, Caput D, and cystin, are co-localized in renal cilia. J Am Soc Nephrol Tournier F: IL-13 alters mucociliary differentiation and 13: 2508–2516, 2002 ciliary beating of human respiratory epithelial cells. J Clin 43. Rosenbaum JL, Witman GB: Intraflagellar transport. Nat Invest 108: 1817–1824, 2001 Rev Mol Cell Biol 3: 813–825, 2002 32. Ostrowski LE, Blackburn K, Radde KM, Moyer MB, 44. Ibanez-Tallon I, Heintz N, Omran H: To beat or not to beat: Schlatzer DM, Moseley A, Boucher RC: A proteomic anal- Roles of cilia in development and disease. Hum Mol Genet ysis of human cilia: Identification of novel components. 12: R27–R35, 2003 Mol Cell Proteomics 1: 451–465, 2002 45. Van’s Gravesande KS, Omran H: Primary ciliary dyskine- 33. Deane JA, Cole DG, Seeley ES, Diener DR, Rosenbaum JL: sia: Clinical presentation, diagnosis and genetics. Ann Med Localization of intraflagellar transport protein IFT52 iden- 37: 439–449, 2005

Supplemental information for this article is available online at http://www.jasn.org.