© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs220905. doi:10.1242/jcs.220905

RESEARCH ARTICLE RPGRIP1L helps to establish the ciliary gate for entry of Huawen Lin, Suyang Guo and Susan K. Dutcher*

ABSTRACT to the in the transition zone (Fisch and Dupuis- Mutations in transition zone change the composition of the Williams, 2011). The transition fibers may also serve as a docking ciliary proteome. We isolated new mutations in RPGRIP1L (denotated site for (IFT) proteins (Deane et al., 2001). as RPG1 in algae) that affect the localization of the transition zone At the distal end of the transition zone are structures called Y-linkers NPHP4 in the model organism Chlamydomonas reinhardtii. (reviewed in Fisch and Dupuis-Williams, 2011) and they appear to NPHP4 localization is not affected in multiple new intraflagellar anchor the doublet microtubules to the ciliary membrane. Several transport (IFT) mutants. We compared the proteome of cilia from wild- organisms, including Giardia and cycads, lack Y-linkers (Barker type and mutants that affect the transition zone (RPGRIP1L) or IFT et al., 2014). The transition zone has three protein complexes called (IFT172 and DHC1b) by mass spectrometry. The rpg1-1 mutant cilia the MKS, NPHP and CEP290 modules (see Table S1 for show the most dramatic increase in cytoplasmic proteins. These names in human, and Chlamydomonas nonciliary proteins function in translation, membrane remodeling, reinhardtii) (Williams et al., 2011; Schouteden et al., 2015). These ATP production and as chaperonins. These proteins are excluded in proteins are not essential for ciliary assembly in C. elegans since isolated cilia from fla11-1 (IFT172) and fla24-1 (DHC1b). Our data triple mutants that lack all the modules still assemble cilia support the idea that RPGRIP1L, but not IFT proteins, acts as part of (Schouteden et al., 2015). In many studies, these proteins localize the gate for cytoplasmic proteins. The rpg1-1 cilia lack only a few to the transition zone (Fliegauf et al., 2006; Garcia-Gonzalo et al., proteins, which suggests that RPGRIP1L only has a minor role of in 2011; Reiter et al., 2012; Williams et al., 2011). Mutations in these the retention of ciliary proteins. The fla11-1 mutant shows the greatest genes result in a constellation of clinical symptoms that include loss/reduction of proteins, and one-third of these proteins have a retinal, kidney, and neurodevelopmental defects as well as early transmembrane domain. Hence, IFT172 may play a role in the lethality (Czarnecki and Shah, 2012). RPGRIP1L (denoted as retention of proteins. RPG1 in algae) has been implicated by genetic studies in C. elegans as a key player in the transition zone that interacts with both the KEY WORDS: Transition zone, Cilia, , MKS and NPHP proteins (Williams et al., 2011; Schouteden et al., Chlamydomonas reinhardtii 2015), and it is required for TMEM237 localization (Huang et al., 2011). Homologs of these genes are found in the Chlamydomonas INTRODUCTION genome and in the transition zone proteome (Diener et al., 2015). Cilia are membrane-bound projections extending from the plasma BioID/proximity mapping identified 66 proteins that interact with membrane of most eukaryotic cells. Motile cilia move fluids RPGRIP1L; 11 of these are also found using FLAG-tagged protein over their surface. Primary cilia serve as antenna to receive for immunoprecipitation. These studies find that RPGRIP1L environmental signals and respond via a large number of signaling clusters with both transition zone and centrosomal appendage pathways. Cilia also send signals via ectosomes (Wood et al., 2013; proteins (Gupta et al., 2015). Mouse studies link the dosage of Long et al., 2016). Many of these signaling pathways are important RPGRIP1L with appetite control (Stratigopoulos et al., 2014). for development and homeostasis. Defects in cilia cause a group RPGRIP1L appears to be a central protein of the transition zone. of diseases called , and result in obesity, cystic The function of the transition zone as a ciliary gate has been kidney, blindness, skeletal malformations and nervous system probed in both mutants and via siRNA. In Chlamydomonas, abnormalities, as well as respiratory and laterality defects (Reiter mutations in either CEP290 (Craige et al., 2010) or NPHP4 and Leroux, 2017). (Awata et al., 2014) result in the presence of a small number of The basal body of cilia docks at the plasma membrane, inappropriate proteins in cilia and the absence of some proteins. permanently in many unicellular organisms or transiently in most In mouse embryos and hTERT-RPE1 cells, tectonic-1 (TCTN1) as metazoan tissues. The transition zone, which is recognized by the well as TCTN2 and CC2DC2A, which are part of the MKS module, change from triplet to doublet microtubules, assembles at the distal are needed to localize the membrane-associated proteins ARL13a, end of the basal body. The transition fibers in Chlamydomonas or adenylate cyclase III (AC3), Smoothened and polycystin-2 (PKD2) the distal and subdistal appendages in mammalian cells are attached to the primary (Garcia-Gonzalo et al., 2011). Knockdown of TMEM67 affects AC3 localization but not that of the other proteins (Garcia-Gonzalo et al., 2011). In C. elegans, loss of transition zone Department of Genetics, Washington University in St. Louis, St. Louis, proteins in any of the three modules causes RPI-2 (the retinitis MO 63110, USA. pigmentosa 2 homolog), and the transmembrane proteins TRAM- *Author for correspondence ([email protected]) 1a and TMEM67 to accumulate in the cilia instead of the transition zone (Williams et al., 2011; Li et al., 2016). The loss of S.K.D., 0000-0001-5689-5753 RPGRIP1L/MKS5 in C. elegans suggests that it plays a role in This is an Open Access article distributed under the terms of the Creative Commons Attribution creating a membrane diffusion barrier (Jensen et al., 2015). These License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. transition zone proteins clearly play a role in the entry of ciliary proteins as well as the exclusion of cytoplasmic proteins. In

Received 28 May 2018; Accepted 10 September 2018 addition, the ciliary gate has been proposed to behave in a similar Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs220905. doi:10.1242/jcs.220905 manner to the nuclear pore (Takao et al., 2014). Endicott and strains, called IB4, DB35, GB10 and GB24, that lack NPHP4 at the Brueckner, by using siRNA, have shown that reducing the amount basal bodies by immunofluorescence analysis of cell expressing a of the nuclear pore protein NUP98 limits the diffusion of proteins HA::NPHP4 transgene (Awata et al., 2014) (Fig. 1A). Although we greater than 70 kDa into the cilium. This protein is important for isolated the rpg1 (herein, we use RPG1 to denote Chlamydomonas import of the tubulin dimer (Endicott and Brueckner, 2018). homolog of mammalian RPGRIP1L) mutants in a screen for cells Ciliary assembly is dependent on the intraflagellar transport lacking cilia, we found that upon removal of the cell wall, the cells (IFT) system composed of at least 22 proteins (Taschner and had assembled cilia. The newly divided rpg1 mutant cells fail to be Lorentzen, 2016). In Chlamydomonas, IFT proteins have been released from the mother cell wall in a process called hatching. Cells biochemically separated into two complexes called IFT-A, which lacking cilia show a similar failure to hatch. These mutant strains plays a role in retrograde movement from the ciliary tip to the cell have no obvious mitotic or meiotic defect. They were subjected to body, and IFT-B, which plays a role in anterograde movement whole-genome sequencing and were all found to carry mutations in (Piperno and Mead, 1997; Piperno et al., 1998; Cole et al., 1998; the RPG1 gene and they were renamed as rpg1-1 through rpg1-4 Iomini et al., 2001, 2009). A conditional mutation in IFT172, which (Table 1). One of the mutants, DB35, carries mutations in both is part of the IFT-B complex (Pedersen et al., 2005), shows a RPG1 and IFT121. Through a backcross of DB35, we obtained defective retrograde transport phenotype at the permissive progeny (DB35-1) that contained only the ift121-2 mutation and temperature (Iomini et al., 2001). It has been postulated that progeny (DB35-2) that contained only the rpg1-2 mutation. Both IFT172 plays a role in IFT remodeling at the distal tip (Pedersen genotypes showed a loss of cilia. To determine whether the rpg1 et al., 2005), thereby influencing retrograde IFT. mutations are causative for the observed phenotype, we used Here, we undertook an unbiased forward genetic screen for linkage analysis (Lin and Dutcher, 2015). For each of the four rpg1 Chlamydomonas strains with ciliary assembly defects in order to mutants, we found that the lack of cilia co-segregates with the identify new mutants that may affect basal body, transition zone or mutation in RPG1 by PCR (Table 1). ciliary assembly and function. We identified 250 mutant strains. As a second line of evidence, we used reversion analysis to identify Whole-genome sequencing of a subset of these strains reveals intragenic events that restore function. Since the rpg1-3 and rpg1-4 mutations in the transition zone protein RPGRIP1L and in several alleles contain either a large insertion or a deletion, respectively, IFT genes. Comparison of the protein composition of cilia from IFT which are highly unlikely to produce intragenic events, we mutants and wild-type suggests that IFT is not required to establish mutagenized the rpg1-1 and rpg1-2 strains to isolate revertants/ the ciliary gate that keeps cytoplasmic proteins out. As observed in suppressors that restore swimming. We found one true revertant of other organisms, the gate appears to require components of the rpg1-1 and 1 pseudorevertant (E11a) of rpg1-2 that deletes 41 transition zone. nucleotides and restores the predicted reading frame with a 14 amino acid deletion, which we renamed rpg1-5 (Fig. S1). We conclude that RESULTS mutations in RPG1 are causative for the observed phenotype Isolation of mutants lacking NPHP4 in the transition zone given that we have four independent alleles with the same We performed an unbiased forward genetic screen for mutants that phenotype (Tulin and Cross, 2015), the phenotype and mutant fail to assemble cilia and a secondary screen of these strains for ones alleles co-segregate, and we were able to obtain revertants for two that fail to localize NPHP4 properly. We identified four mutant different rpg1 alleles.

Fig. 1. Localization of NPHP4 and POC2 in newly isolated mutants. (A) A strain with the NPHP4 gene tagged with the HA epitope was crossed into each mutant to determine whether NPHP4 is properly localized. Top row, wild type; wild type with no transgene; -3; ift121-2; ift172-2. Bottom row, rpg1- 1; rpg1-2; rpg1-3; rpg1-4. (B) Wild type and rpg1 mutants were stained with antibodies against POC2. Scale bars: 10 μm. Journal of Cell Science

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Table 1. Newly isolated strains and their genetic characterization Amino acid alteration/ Linkage Strain Genes Mutation/position Genomic location by PCR Revertants IB4 (rpg1-1) RPGRIP1L AAG to TAG Chr 14: 3725856 K629X Nucleotide 5056* 8 1 true revertant DB35-2 (rpg1-2) RPGRIP1L Deletion of G Chr 14: 3724543 Frame shift, M453fs 13 1 pseudorevertant Nucleotide 3743* GB10 (rpg1-3) RPGRIP1L Insertion Chr 14: 3724290 Insertion at amino acid 7 Not tested 450 Nucleotide 3490* GB24 (rpg1-4) 19 genes including Deletion of 94.5 kb genomic Deletion of entire gene 10 Not tested RPGRIP1L DNA (Chr 14: 3667847–3762396) DB35-1 (ift121-2) IFT121 GTA to ATA Chr 11: 2411434 Splice donor site change in 12 3 true revertants 5′ splice site in intron 21 Nucleotide 6221* DB31 (ift140-3) IFT140 TG to AA Chr 8: 1134675-76 K386X Nucleotides 3265–3266* 11 6 true revertants *Nulcleotide 1 is the A of the first codon; Chr, .

Diener and colleagues have previously analyzed isolated antibodies and the transgene co-segregates with the rescue of ciliary transition zones by proteomics and found that POC2 localizes to assembly. Thus, we conclude that these strains carry a null mutation this region (Diener et al., 2015). To ask whether RPGRIP1L affects in IFT172. We named this allele fla11-2. the localization of this protein, we used antibodies to POC2. We We asked whether IFT mutants affect NPHP4 localization at the found that POC2 properly localizes in the rpg1-1 strain (Fig. 1B). transition zone. We assayed the localization of NPHP4 in ift121-2, ift140-3, fla24-1 (a DHC1b mutant; Lin et al., 2013b), fla9 (-2), Localization of NPHP4 is not affected in other mutants that fla11-1 (ift172-1), fla11-2 (ift172-2), and rescued fla11-2 with lack cilia the rescuing transgene. All of these mutants show NPHP4 staining In our unbiased forward genetic screen for strains lacking cilia, we at the transition zones (Fig. 1A; Fig. S2). Hence, NHPH4 is found mutations in two IFT genes. In addition to ift121-2 in DB35 localized properly in cells lacking cilia and IFT is not needed for (Lin et al., 2018), a mutation in IFT140 (DB31) was also identified. NPHP4 localization. By using the same strategy as above, we found that the ift140-3 and ift121-2 mutations show linkage between the mutant allele and the Assaying the ciliary gate in mutants mutant phenotype (Table 1). We found three and six true revertants The transition zone serves as a gate to regulate the entry of of the ift121-2 allele and the ift140-3 allele, respectively (Table 1). cytoplasmic proteins based on the presence of a limited number of We conclude that the mutations in IFT140 and IFT121 are causative proteins assayed in animal cells (Nachury et al., 2010; Reiter et al., for the observed phenotype based on both co-segregation and 2012; Williams et al., 2011; Li et al., 2016). The region also serves reversion. In addition, we have previously shown that ift121-2 a gate preventing ciliary proteins from leaving (Li et al., 2016). is rescued upon expression of the wild-type IFT121 gene (Lin Since cilia can be easily detached from intact Chlamydomonas et al., 2018). cells and purified away from the cell bodies by centrifugation, It had been previously noted that strain CC-4348 (Goodson et al., Chlamydomonas permits a comprehensive biochemical examination 2011) lacked cilia. Here, CC-4348 was backcrossed five times, and of the role of the transition zone as a gate (Craige et al., 2010; two progeny lacking cilia were retained. The mutant phenotype Awata et al., 2014). If the transition zone serves as a gate between (lack of cilia) maps to a region of about 500 kb on chromosome 17 the and cilia, the isolated cilia from mutants would be based on ∼500 meiotic progeny. The two strains were subjected to expected to contain proteins that are not normally present in the whole-genome sequencing (Table S2) and we identified an insertion cilia. If it serves in protein transport or retention, known ciliary in exon 2 of the IFT172 gene. Primers (Fig. 2A; Table S3) proteins would be expected to be absent. Importantly, ciliary surrounding the suspected insertion amplify a 212 bp fragment from excision occurs distal to the transition zone domain, so transition wild-type but not from the CC-4348-derived strains (Fig. 2B, top zone proteins are not expected to be in the ciliary proteome (Lewin panel). This insertion in IFT172 shows linkage to the mutant and Lee, 1985). phenotype in 62 meiotic progeny from a cross of the CC-4348- We examined the ciliary protein composition of the transition derived strain with wild type (CC-125). zone mutant rpg1-1 and of temperature-sensitive mutants with In immunoblots using an antibody against IFT172 (Pedersen defects in IFT ( fla11-1 and fla24-1 after 4 h at 32°C) (Table 2). et al., 2005), we observed IFT172 in the cytoplasm of both wild type Although the rpg1-1 mutant was isolated as a strain lacking cilia, and the temperature-sensitive ift172 mutant, fla11-1, at 21°C but not cells with cilia are observed when the cell wall is removed with in the sequenced strains (Fig. 2C). A 3× FLAG-tagged IFT172 gene autolysin (Dutcher, 1995). Two independent cilia samples from (Fig. 2B, bottom panel) was introduced into wild-type cells and we each of the three strains were isolated. The number of peptides from isolated a transgenic strain that expressed the FLAG::IFT172 α-tubulin and β-tubulin is similar among all of the independent protein with the correct protein size. This transgene was introduced preparations in each group (wild type versus mutants with IFT into the CC-4348-derived strains by meiotic crosses. The ciliary defect, and wild type versus rpg1-1, Table S4), which serves an assembly defect was rescued; over 90% of the mutant cells with the internal comparison of the mass spectrometry data. transgene had cilia (n=200). By PCR, we confirmed that the tagged The most striking increase in nonciliary proteins is observed in gene co-segregated with rescue in all 18 tetrads analyzed. We the rpg1-1 mutant. A total of 40 nonciliary proteins were present in selected a parental ditype octad and a tetratype octad to ask whether the isolated rpg1-1 cilia and they range in size from 29 to 183 kDa the tagged protein co-segregates with motility (Fig. 2D,E). The (Table 2). These proteins fall into several categories (Table S5); tagged protein is recognized by both anti-FLAG and anti-IFT172 these are chaperonins (Table S5, yellow), and proteins involved in Journal of Cell Science

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Fig. 2. Characterization of the fla11-2 mutation in the IFT172 gene. (A) Diagram of the gene structure of IFT172 with the insertion in exon 2. The location of primer sets 1 and 3 used in B are indicated. (B) PCR verification of the insertion with primer set 3 (top panel) and the presence of FLAG-tag with primer set 1 in wild-type strain carrying the transgene (bottom panel). (C) Immunoblot of IFT172 in whole-cell lysates of wild-type, fla11-1 and fla11-2 strains. (D,E) Co-segregation of the insertion and rescue by the 3× FLAG::IFT172. In parental ditype and tetrad type octads, the presence of cilia is assessed (N, no cilia; Y, have cilia) and compared to the presence of the insertion (primers, 3F, 3R), the presence of the transgene (primers 1F, 1R) and immunoblots with antibodies to the FLAG tag or IFT172. The level of α-tubulin is shown as a loading control.

translation (orange), ATP production (blue), membrane remodeling wild type (Fig. 3). Since rpg1-1 cells fail to localize NPHP4, we (red) and transport (purple). The TRiC/CCT chaperonin complex, examined isolated cilia from mutants and see a 13× increase heat shock protein 70 (HSP70G) and RB60 (a protein disulfide of CCT1 in the cilia compared to wild type by immunoblotting isomerase) are present in these cilia (Table S5). The TRiC/CCT (Fig. 3). chaperonin proteins facilitate folding of ∼10% of the eukaryotic Four translation initiation factors (EIF3A, EIF3B, EIF3C and proteome (Cong et al., 2010; Kalisman and Levitt, 2010; Leitner EIF2G), a tRNA synthetase, an RNA helicase and several other et al., 2012). All of the components of the CCT/TRiC complex are proteins involved in translation are present in the rpg1-1 cilia present in rpg1-1 cilia. We performed immunoblots with antibodies (Table S5). This is consistent with results from Craige et al. (2010) to CCT1 and see an increase of 15-fold in rpg1-1 cilia compared to and Awata et al. (2014) showing that the amount of elongation factor

Table 2. Summary of proteins that show abundance changes in mutants Molecular Mutants Total Translation Energy Membranes Trans-porters FAP Other Chloro-plast No. of TM mass (kDa) Increases compared to wild type (CC-124 for rpg1-1 and CC-125 for fla11-1 and fla24-1) rpg1-1 42 10 8 4 4 3 2# 8 3 8 29-183 fla11-1 2 0 0 0 0 0 2* 0 0 0 205-269 fla24-1 2 0 0 0 0 0 2* 0 0 0 205-269 Decreases compared to wild type (CC-124 for rpg1-1 and CC-125 for fla11-1 and fla24-1) rpg1-1 2 0 0 0 0 0 2 0 0 1 157-301 fla11-1 18 0 0 0 0 0 15 3 0 6 66-357 fla24-1 2 0 0 0 0 0 2 0 0 0 50-102

#, *See Results for details. Journal of Cell Science

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(Lin et al., 2013a), we found that our CC-125 strain had acquired a point mutation that leads to a F31C change that appears to disrupt CPC1 localization to cilia. This change was confirmed by PCR followed by enzyme digestion to distinguish the difference between our CC-125 and CC-124, the other wild-type strain (Fig. S3). The cpc1 mutation is unique to our strain and is not in the CC-125 stock from the Chlamydomonas Resource Center (Fig. S3). Thus, the level of CPC1 is not increased in the mutant cilia due to an IFT defect in fla11-1 and fla24-1 but due to a mutation in our CC-125 strain. We refer to this mutation as cpc1-2. It is possible that the change in FAP42 is also related to the central pair mutation. Enolase, which interacts with CPC1, is not affected in the cpc1-2 strain, based on our mass spectrometry results, whereas it is affected in the cpc1-1 strain (Zhang and Mitchell, 2004; Mitchell et al., 2005). Our mass spectrometry data from two wild-type strains (CC-124 and CC-125) also allowed us to identify 16 novel proteins that were not previously identified in the Chlamydomonas Flagellar Proteome (Pazour et al., 2005) (Table S7). Six of these proteins are not found in the JGI Chlamydomonas version 4 genome sequence and one of them has an incomplete v4 gene model. Eight of the 16 proteins were recently added to the updated Flagellar Proteome (http:// Fig. 3. The chaperonin, CCT1, is increased in rpg1-1 and nphp4 cilia. chlamyfp.org/). They are indicated in Table S7. One protein, ALA2, μ Ciliary proteins (4 g) from wild-type, nphp4, and rpg1-1 cells were used. which is an aminophospholipid transporter, is found in the transition Proteins were separated on a 7.5% acrylamide SDS-PAGE gel. The small cross-reactive protein was used as a loading control. zone proteome (Diener et al., 2015). The TRP15 protein, a member of the transient receptor potential family, has been localized to Chlamydomonas cilia (Fujiu et al., 2011). 3 is increased in both and nphp4 cilia, and four tRNA synthetases are increased in nphp4 cilia. Four of the cytoplasmic DISCUSSION proteins found to show increased levels affect membrane Our unbiased forward genetic screen identified four strains (Table 1) remodeling and vesicle formation. Proteins of the mitochondrial that lack NPHP4 at the transition zone from a collection of 250 F0/F1 ATP synthase (ATP-2, ASB-1 and ASG-2) and several strains lacking cilia. These four mutants each carry a unique mutant protein transporters and ion channels are increased. We also observe allele of RPG1. Given the genetic heterogeneity in worms (Li et al., three chloroplast-localized proteins (Table S5, green). Thus, we do 2016; Williams et al., 2011) and the interaction network observed in not know whether these represent contamination or these proteins tissue culture cells (Sang et al., 2011; Gupta et al., 2015) at the are actively moved from the chloroplast into cilia. Surprisingly, transition zone, we are surprised that our screen did not reveal most of the flagellar-associated proteins (FAPs) that show changes additional genes. The Chlamydomonas nphp4 mutant assembles in cep290 and nphp4 (Awata et al., 2014; Aanstad et al., 2009; cilia. The rpg1 and cep290 mutants assemble cilia, but fail to release Craige et al., 2010) are not identified in our rpg1-1 dataset. cells from the mother cell wall (Craige et al., 2010). Therefore, other The main alteration in the two ift mutants is the loss or reduction transition zone mutants may assemble cilia, and not be identified in of proteins (Table 2). In fla11-1 cilia, the levels of 18 proteins are our screen. It is also possible that secondary screens for the failure to reduced, and two are reduced in fla24-1 cilia. The missing proteins localize transition zone proteins other than NPHP4 would identify range in size from 50 to 357 kDa. In fla11-1 cilia, the levels of IFT- additional transition zone defective mutants in our collection. A complex proteins IFT121, IFT122, IFT139, IFT140 and IFT144 The lack of cilia observed in rpg1 and cep290 mutants appears to are significantly reduced, whereas the sixth IFT-A subunit, IFT43, arise from the failure of cells to hatch from the mother cell wall as is not reduced. The levels of IFT-B complex proteins show no this phenotype is rescued by removal of the cell wall. We reduction in fla11-1 cilia. The IFT complex proteins are not affected hypothesize that these transition zone proteins may be involved in in fla24-1 cilia (Table S6). Six of the 13 non-IFT proteins that show the production of ectosomes that contain the lysin needed for reduced levels have transmembrane domains. This suggests hatching (Wood et al., 2013). that IFT172 may play a role in the entry or retention of proteins The use of super-resolution microscopy has revealed that associated with the ciliary membrane. multiple transition zone proteins (NPHP1, TCTN2, TMEM231 We identified changes in our wild-type strains from the and AHI1) fail to localize properly in human fibroblasts that carry proteomics data. Two FAPs, FAP102 and NSG1 (also known as RPGRIP1L mutations (Shi et al., 2017). This is consistent with our CYN7), are missing in our wild-type strain (CC-124) but are present study showing that NPHP4 is missing in the rpg1-1 mutant, given in another wild-type strain (CC-125) as well as in rpg1-1 (# in that NPHP1 and NPHP4 are part of the NPHP module. Analysis of Table 2). Analysis of the sequence for our wild-type strains does not cep290 and nphp4 mutants in Chlamydomonas (Awata et al., 2014; show any SNP or indels in these genes. We do not know the genetic Craige et al., 2010) suggested a role of these two proteins in cause. CPC1 and FAP42 (asterisks in Table 2) are missing in our preventing entry of inappropriate transmembrane domain proteins. other wild-type strain (CC-125) (Table S6). CPC1 is part of the In our analysis of rpg1-1, eight of 40 proteins have a transmembrane central pair complex (Zhang and Mitchell, 2004). Using antibodies domain. The set of cytoplasmic proteins range in size from 29 to to CPC1 (Zhang and Mitchell, 2004), we showed that CPC1 is absent 190 kDa, which suggests that RPG1 does not have a specific size from our CC-125 cilia but present in CC-124, fla11-1 and fla24-2 range for the exclusion of ciliary proteins. Comparison of protein cilia by immunoblotting (Fig. S3). By whole-genome sequencing compositions in rpg1-1 and nphp4 (Awata et al., 2014) suggest that Journal of Cell Science

5 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs220905. doi:10.1242/jcs.220905 the rpg1-1 mutant shows a stronger defect and allows the entry of available in order to test its role. The ciliary gate has been compared more nonciliary proteins. Even though the lists of enriched proteins to the nuclear pore, which allows entry of specific proteins into the found in rpg1-1 and nphp4 do not overlap, by immunoblotting we nucleus. It has been hypothesized that a subset of the nucleoporins showed that CCT1, part of the TRiC/CCT chaperonin complex, found in nuclear pores also participate in forming the ciliary gate increases in the cilia of both rpg1-1 and nphp4 gametic cells (Takao et al., 2014). Nucleoporins were not found in the (Fig. 3). We suggest that the lack of overlap may reflect differences Chlamydomonas transition zone proteome (Diener et al., 2015), in the life cycle of the cells used. Awata et al. used cells growing but these preparations are missing part of the transition zone. It will exponentially, while we used gametic cells. In the future, it will be be interesting to ask whether NUP98 is missing in the transition interesting to test how the activity of the gate varies with the stage of zone mutants, and whether the gate phenotype is a direct effect or an the life cycle. indirect effect of NUP98. In Chlamydomonas, the mitochondrial ATP synthase and the RPGRIP1L is in a unique position based on super-resolution TRiC/CCT are excluded from wild-type cilia (Table 2; Pazour et al., microscopy to act as a ciliary gate (Yang et al., 2015; Shi et al., 2005). In C. elegans, the male sensory neurons contain ATP 2017). The Chlamydomonas RPG1 protein has unique properties synthase whereas the olfactory AWA sensory neurons do not (Hu that make it an ideal candidate for forming a gate; ∼450 amino acids and Barr, 2005). Four of the TRiC/CCT subunits are found in in the last third of the protein are predicted to be an intrinsically Tetrahymena cilia (Seixas et al., 2003) and in mammalian motile disordered region (IDPs). The human RPGRIP1L also contains cilia (Seixas et al., 2003; Lopez-Fanarraga et al., 2007). The TRiC/ a disordered region. IDPs have been implicated in creating CCT subunits associate with mammalian-specific BBS proteins compartments without membranes via liquid–liquid phase (BBS6, BBS10 and BBS12), which are not present in the genome of transitions in the cytoplasm (Zhang et al., 2015), in the nucleus Chlamydomonas (Seo et al., 2010). These differences provide (Berry et al., 2015), in P-bodies in C. elegans (Brangwynne et al., striking examples that the ciliary gate can differ among cilia in the 2009), in the pericentriolar material of the (Woodruff same organism and in different organisms. These examples suggest et al., 2015), as regulators of post-translational modifications that ciliary gate is a better label than ciliary barrier as a gate can be (Das et al., 2016) and as platforms for transcriptional activation opened and closed in different tissues and cells. FOX1 is present in (Kwon et al., 2013). RPGRIP1L clearly plays a role in multiple the rpg1-1 mutant preparation (Table S5), and is in the transition organisms as part of a transition zone compartment that functions to zone proteome (Diener et al., 2015). In addition, it is found in block entry of proteins from the cytoplasm to the cilium and as a extracellular vesicles or ectosomes (Long et al., 2016). We suggest membrane diffusion barrier (Jensen et al., 2015; Williams et al., that it may be a transient protein in the ciliary compartment in wild- 2011; Huang et al., 2011). Modulation of the ciliary gate by changes type cells but becomes trapped in the rpg1-1 mutant. in the life cycle will be an interesting new area to pursue. The fla11-1 and fla24-1 mutants show similar IFT particle number and velocity defects (Iomini et al., 2001). However, more MATERIALS AND METHODS proteins are missing from the fla11-1 cilia than from the fla24-1 Strains and culture conditions cilia. Patients with defects in intraflagellar transport genes show a Strain CC-4348 was backcrossed to wild-type cells five times to remove unique set of symptoms. This syndrome is called Jeune’s the arg7-7, sta6, and cw15 mutations, as well as several mutations that asphyxiating thoracic dystrophy (JATD) or short rib polydactyly affect ciliary assembly and function that have not been identified to date. syndrome (SRPS) (Schmidts, 2014); these patients have defects in The CC-125, CC-1920 ( fla11-1), CC-3866 ( fla24-1), CC-4348 (sta6-1), CC-5116 (HA::NPHP4), and CC-1918 ( fla9) strains were obtained from cartilage and bone development. A subset of the patients with Chlamydomonas Resource Center. The CC-124 and cpc-1-2 strains were mutations in CSPP1 show reduced levels of ARL13B and ACIII maintained as in-house stocks. The tagged protein (HA::NPHP4) was (Tuz et al., 2014), which suggests a role for the proteins in the introduced into various mutant strains by meiotic crosses and detected by proper localization or retention of ciliary proteins. The JATD PCR. All mutants and epitope tags were verified by PCR (Table S3). patients often show nonskeletal symptoms that develop later in childhood; these include kidney, retinal degeneration and liver Molecular mapping and whole-genome sequencing fibrosis symptoms, as well as symptoms seen in Bardet–Biedl Progeny of CC-4338 lacking cilia with an intact cell wall were crossed to the syndrome patients (Bujakowska et al., 2014; Halbritter et al., highly polymorphic strain CC-1952 (S1C5), and 500 progeny was used in 2013). Some of these phenotypes are associated with defects in the mapping. Crude DNA preparation, PCR and digestion of individual progeny for meiotic mapping were performed as previously described (Lin and transition zone (Waters and Beales, 2011). Interestingly, this Dutcher, 2015). No recombinants were found with the primers at 0.57 Mb constellation of later-appearing symptoms is observed in all of the and 1.09 Mb. Two independent progeny lacking cilia (4348-1 and 4348-2) JATD patients with the exception of those with mutations in were subjected to whole-genome sequencing (Lin and Dutcher, 2015). We the heavy chain (Schmidts et al., 2013). We suggest that the used Softsearch (Hart et al., 2013) to find breakpoints, which would indicate difference in the transmembrane ciliary composition between >200 bp insertions/deletions, within the mapped region of chromosome 17. fla11-1 and fla24-1 may be key in understanding the appearance of In the 4348-1 strain, 379 breakpoints were identified; in the 4348-2 strain, the adolescent phenotypes. 272 breakpoints were identified. Common breakpoints found in both strains Several studies in mammalian cells have found that restricted cilia were compared to 292 breakpoints found in a wild-type strain (CC-124) in entry occurs for proteins in the 40–70 kDa range through the same interval. experiments using an in vitro, permeabilized cell model (Breslow Chlamydomonas et al., 2013) or by using labeled dextran (Kee et al., 2012). Lin and Plasmid DNA constructs and transformation colleagues, by using a chemically inducible trap, found that proteins The BAC DNA 4P11 (Chlamydomonas Resource Center), was digested with XmaI and HindIII. A 10.9 kb fragment, which contains the full-length up to 7.9 nm could enter the cilia (Lin et al., 2013c). The IFT172 gene, was gel-purified and ligated into a pBlueScript vector cytoskeletal protein septin is involved in a diffusion barrier in yeast (Stratagene) digested with the same enzymes to form the pBS-IFT172 (Takizawa et al., 2000; Barral et al., 2000). Septins play a role in plasmid. The 3× FLAG tag was inserted immediately after the start ATG regulating membrane protein movement in cilia (Hu et al., 2010). codon via nested PCR. In the first round of PCR, three different PCR

Unfortunately, mutants in the homolog in Chlamydomonas are not products were generated. The first PCR product, a 474 bp fragment, was Journal of Cell Science

6 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs220905. doi:10.1242/jcs.220905 amplified by primers HindIII-F and IFT172-start-R (see Table S3 for primer (CC-124 and CC-125) samples were converted into JGI Chlamydomonas sequences) with pBS-IFT172 as a template. The second PCR product, a v4 protein IDs from the file provided by Phytozome and compared to the 95 bp fragment that contains the 3× FLAG tag, was amplified by primers Flagellar Proteome JGI v4 IDs. Sequences of proteins not found in the IFT172-start-FLAG-F and IFT172-start-FLAG-R with pBS-3×FLAG (Lin Flagellar Proteome were subjected to BLAST analysis against the v4 and Goodenough, 2007) as a template. The third PCR product, a 329 bp genome sequences to query whether the proteins were predicted in the v4 fragment, was amplified by primers start-FLAG and BsmI-R with pBS- genome. Some proteins do not have a match in the v4 genome. Some IFT172 as a template. In the second round of PCR, the 474 bp fragment and proteins have v4 protein IDs but are not found in the flagellar proteome. the 95 bp fragment were combined as templates. A 550 bp fragment was These proteins are considered novel ciliary-associated proteins. amplified with primers IFT172-HindIII-F and IFT172-start-FLAG-R. In the third round of PCR, the 550 bp fragment from the second round and the Isolation of mutants lacking cilia 329 bp fragment from the first round were combined as templates. Primers Wild-type cells (∼5×108) were mutagenized with ultraviolet light (750 μJ) IFT172-HindIII-F and IFT172-BsmI-R were used to generate an 863 bp and resuspended in 48 25×150 mm culture tubes at 25°C. Cells at the bottom fragment, which was digested with HindIII and BsmI, and cloned into pBS- of the tubes were transferred into 20 ml of fresh medium after 2–3 days. This IFT172 digested with the same enzymes. The resultant plasmid pBS-FLAG- was repeated six times. Single colonies were obtained by plating ∼100 cells IFT172 was subjected to Sanger sequencing to verify the sequence. onto solid medium from cultures with cells found primarily at the bottom of Chlamydomonas transformation was performed as previously described the culture tubes. Single colonies from ∼250 independent mutant cultures (Lin et al., 2013a). Wild-type cells (CC-125), were transformed with 1 µg were examined for the presence or absence of the HA-tagged NPHP4 of pBS-FLAG-IFT172 and 1 µg of pSI103, which confers resistance to protein at the basal bodies by immunofluorescence. paromomycin (Sizova et al., 2001); 80 paromomycin-resistant colonies were obtained. Seven FLAG-positive colonies were identified using PCR. Acknowledgements These were subjected to immunoblotting with an anti-FLAG antibody. One We are extremely grateful to Dr Ursula Goodenough for suggesting that we examine positive strain, which contains expressed FLAG–IFT172 proteins, was the CC-4348 strain. We thank Nick Nauman for help during the initial mapping of the identified and subsequently crossed to the fla11-2 and fla11-1 mutants. fla11-2 mutant in CC-4348 and Gervette Penny for many useful comments on the manuscript. Antibodies were provided by Dr Dennis Diener for POC2, by Dr Doug Immunoblotting and immunofluorescence Cole for IFT172 and by Dr David Mitchell for CPC1. We thank the Genome Technology Access Center in the Department of Genetics at Washington University SDS-PAGE and immunoblotting were performed as previously described School of Medicine for help with genomic analysis. The Center is partially supported (Lin et al., 2013a). Primary antibodies used in this study were: anti-FLAG by NCI Cancer Center Support Grant #P30 CA91842 to the Siteman Cancer Center antibody (Lin and Goodenough, 2007), M2 monoclonal, Sigma-Aldrich and by ICTS/CTSA Grant # UL1TR000448 from the National Center for Research (F2555, SIG1-25, 1:2000 dilution), anti-IFT172.1 antibody (Pedersen Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH et al., 2005, 1:200 dilution), anti-CPC1 antibody (Zhang and Mitchell, Roadmap for Medical Research. 2004, 1:100 dilution), anti-CCT1 antibody (Sigma-Aldrich, HPA027337, 1:200 dilution), and anti-α-tubulin antibody (Sigma-Aldrich, T6199, lot Competing interests number 091M4813, 1:2000 dilution). Secondary antibodies include The authors declare no competing or financial interests. horseradish peroxidase (HRP)-conjugated goat anti-mouse-IgG antibody (BioRad, 1721011, 1:5000 dilution) and HRP-conjugated goat anti-rabbit-IgG Author contributions antibody (Sigma-Aldrich, A6154, 1:5000 dilution). For immunofluorescence, Conceptualization: S.K.D.; Methodology: S.K.D., H.L.; Software: H.L.; Validation: cells were treated with autolysin for 30 min to remove cell walls. Cells were H.L., S.K.D.; Formal analysis: H.L., S.K.D.; Investigation: H.L., S.G., S.K.D.; Resources: S.K.D.; Data curation: H.L.; Writing - original draft: S.K.D.; Writing - allowed to adhere to poly-L-lysine-coated glass slides for 2 min at room review & editing: H.L., S.K.D.; Supervision: H.L., S.K.D.; Project administration: temperature in the dark, lysed with 1% Nonidet P-40 for 2 min, fixed with 4% S.K.D.; Funding acquisition: S.K.D. paraformaldehyde for 15 min at room temperature and then subjected to − fixation in cold methanol for 5 min at 20°C. Primary antibodies used include Funding anti-HA High Affinity antibody (3F10, Roche, 11867423001, lot number This work is supported by a grant from the National Institute of General Medical 13500600, 1:200 dilution) and anti-acetylated-α-tubulin (Sigma-Aldrich, Science to S.K.D. (NIH R01-GM32843). This publication is solely the responsibility T7451, lot number 036M4856V, 1:250 dilution). Secondary antibodies used of the authors and does not necessarily represent the official view of NCRR or NIH. include Alexa Fluor 488-conjugated goat anti-rat-IgG antibody (Invitrogen Deposited in PMC for immediate release. API83P, lot number 2915317, 1:500 dilution) and Alexa Fluor 594-conjugated chicken anti-mouse-IgG antibody (Invitrogen, A21201, lot number 737674, Data availability 1:500 dilution). Whole-genome sequencing reads are deposited at BioProject IDs PRJNA407207 (for DB35), PRJNA473171 (for 4342-1 and 4348-2) and PRJNA473163 (for the remaining strains) at the National Center for Bioinformatics Information (NCBI). Cilia isolation, fractionation, silver staining, mass spectrometry and data analysis Supplementary information The rpg1 cells were treated with autolysin to release cells from the mother Supplementary information available online at cell wall before cilia isolation (Craige et al., 2010; Kubo et al., 2015). Cilia http://jcs.biologists.org/lookup/doi/10.1242/jcs.220905.supplemental were detached from cell bodies by pH shock and isolated as previously described (Lin et al., 2013b). Isolated cilia were resuspended in HMDEK References buffer (Awata et al., 2014). Proteins (5 μg) from each sample were subjected Aanstad, P., Santos, N., Corbit, K. C., Scherz, P. J., Trinh, L. A., to in-solution tryptic digestion and mass spectrometry (Donald Danforth Salvenmoser, W., Huisken, J., Reiter, J. F. and Stainier, D. Y. R. (2009). Plant Science Center, St Louis, MO). The extracellular domain of Smoothened regulates ciliary localization and is We used the following criteria to construct the lists of proteins that required for high-level Hh signaling. Curr. Biol. 19, 1034-1039. changed in the mutant compared to wild-type preparations: (1) for a protein Awata, J., Takada, S., Standley, C., Lechtreck, K. F., Bellve, K. D., Pazour, G. J., to be considered as increased in the mutant, it must have greater or equal to Fogarty, K. E. and Witman, G. B. (2014). NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone. J. Cell Sci. 10 peptides on average from two biological replicates while it has no 127, 4714-4727. peptides in the wild-type preparation, and (2) for a protein to be considered Barker, A. R., Renzaglia, K. S., Fry, K. and Dawe, H. R. (2014). Bioinformatic as decreased in the mutant, it must show greater than a five-fold reduction analysis of ciliary transition zone proteins reveals insights into the evolution of compared to the number of peptides found in wild-type cilia and there must networks. BMC Genomics 15, 531. be greater than or equal 10 peptides in the wild-type preparation. Barral, Y., Mermall, V., Mooseker, M. S. and Snyder, M. (2000). The Phytozome Chlamydomonas v5.5 accession numbers of proteins that Compartmentalization of the cell cortex by septins is required for maintenance have an average number of five or more peptides in all four wild-type of cell polarity in yeast. Mol. Cell 5, 841-851. Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs220905. doi:10.1242/jcs.220905

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