Dynein-2 is required for a functional transition zone and bidirectional ciliary trafficking

The dynein-2 microtubule motor functions as the retrograde motor for intraflagellar transport and is required for the formation and maintenance of primary cilia. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 comprises a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilia function. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport, only WDR34 is essential for axoneme extension. In contrast, only WDR60 is essential for co-assembly of the other subunits. Furthermore, WDR60 cannot compensate for loss of WDR34 or vice versa. This work defines a functional asymmetry to match the subunit asymmetry within the dynein-2 motor. Analysis of causative point mutations in WDR34 and WDR60 can partially restore function to knockout cells. Our data show that Jeune syndrome is caused by defects in transition zone architecture as well as intraflagellar transport.

Introduction intraflagellar transport (IFT) (Yee and Reiter, 2015). A "transition zone" (TZ) that separates the  (Schmidts et al., 2015) have also been reported. The role of dynein-2 heavy chain has been 80 extensively studied in Chlamydomonas, C. elegans, and mice. In all cases, loss of dynein heavy chain 81 results, in short, stumpy cilia that accumulate IFT particles at the tip, consistent with a role of 82 dynein-2 in retrograde ciliary transport (Hou and Witman, 2015). Recently, more interest has been 83 focused on the role of the subunits associated with DHC2. Two studies in Chlamydomonas and in 84 human patient-derived fibroblasts revealed that LIC3 (D1bLIC in Chlamydomonas) plays an important 85 role for ciliogenesis and stability of the entire dynein-2 complex (Li et al., 2015;Taylor et al., 2015). 86 Similarly, loss of Tctex2b (TCTEX1D2) destabilizes dynein-2 and reduces IFT in Chlamydomonas 87 (Schmidts et al., 2015). 88 Previous work from our lab and others has shown that loss of function of dynein-2 intermediate 89 chains, WDR34 and WDR60, is associated with defects in cilia. Knockdown of WDR60 or WDR34 in 90 hTERT-RPE1 cells results in a reduction of ciliated cells, with an increase or decrease of the cilia 91 length, likely depending on depletion efficiency (Asante et al., 2014). Mutations in WDR34 have also 92 been shown to result in short cilia with a bulbous ciliary tip in patients fibroblast cells affected by 93 SRP (Huber et al., 2013). Consistent with the results obtained in patient cells, loss of WDR34 in mice 94 also results in short and stumpy cilia with an abnormal accumulation of ciliary proteins and defects 95 in Shh signaling (Wu et al., 2017). Similarly, mutations in WDR60 patient fibroblasts are associated with a reduction in cilia number, although the percentage of ciliated cells was variable in different 97 affected individuals (McInerney-Leo et al., 2013). These findings are all consistent with roles for 98 WDR34 and WDR60 in IFT. 99 In this study, we sought to better understand the role of dynein-2 in human cells using engineered 100 knockout (KO) cell lines for WDR34 and WDR60. We define a functional asymmetry within the 101 complex, where WDR34 is absolutely required for ciliogenesis, while WDR60 is not. In contrast, 102 WDR60 is essential to maintain the integrity of the ciliary transition zone and for retrograde 103 trafficking of IFT particles. Furthermore, by expressing HA-tagged WDR34 in WDR60 KO cells and HA-104 tagged WDR60 in WDR34 KO cells, we found that WDR34 is not required for the other subunits to 105 assemble, whereas loss of WDR60 leads to significant defects in dynein-2 holocomplex assembly. We 106 propose a model where dynein-2 requires WDR34 for axoneme extension but not for the assembly 107 of the other subunits of the complex, whereas WDR60 is crucial for dynein-2 stability, IFT, and ciliary 108 transition zone assembly and/or maintenance. Analysis of disease-causing patient mutations further 109 defines the role of dynein-2 in cilia formation and function. 110

WDR34 or WDR60 play different roles in cilia function 113
To understand the function of WDR34 and WDR60, we generated KO human telomerase-114 immortalized RPE1 (hTERT-RPE1) cells using CRISPR-Cas9. We derived two WDR34 KO clones (1 and 115 2) using guide RNAs (gRNAs) targeting exons 2 and 3, and one KO clone for WDR60, targeting exon 3. 116 Genomic sequencing of these clones identified insertion/deletion mutations on the targeted 117 sequences (Fig. S1). All cell clones were analyzed for protein expression by immunoblot using 118 polyclonal antibodies. Neither WDR34 nor WDR60 was detected in the respective KO cells compared 119 to the controls ( Fig. S2A and Fig. S2B) which provides evidence that downstream initiation sites are 120 not being used. To mitigate against the possibility of any off-target effects, we grew KO cells 121 alongside control CRISPR cells which had been transfected and treated the same way as the KO, but 122 genomic sequencing showed no mutation at the target site. These cells (WDR34 KO CTRL and 123 WDR60 KO CTRL) did not present any cilia defects when stained with Arl13b or IFT88 ( Fig. S2C and 124 S2D). Images in all figures show WT cells where indicated. Indistinguishable results were obtained 125 using these control cell lines. Defects in ciliogenesis in both WDR34 and WDR60 KO cells were 126 rescued by overexpressing WT proteins, confirming that the phenotypes we observed were not due 127 to off-target mutations (described below). 128 Loss of WDR34 severely impaired the ability of these cells to extend a microtubule axoneme (Fig. 1A,  129 B), although Arl13b localized within those few cilia that did form. In contrast, loss of WDR60 did not 130 significantly affect the ability of cells to extend an axoneme (Fig. 1B). Cilia were shorter in both 131 WDR60 and WDR34 KO cells (Fig. 1C). Next, we examined the assembly and structure of primary cilia 132 in WDR34 and WDR60 KO cells by transmission electron microscopy (EM). After 24 hr of serum 133 starvation, WT RPE1 cells extend a defined axoneme surrounded by a ciliary membrane (Fig. 1D). In 134 contrast, WDR34 KO cells failed to extend an axoneme ( Fig. 1E) but showed a large docked pre-135 ciliary vesicle, consistent with the small Arl13b-positive structures seen by immunofluorescence. 136 WDR60 KO cells showed apparently normal cilia ( Fig. 1F) with normal basal body structures and 137 axoneme extension. However, when an entire cilium was captured in WDR60 KO serial sections (Fig.  138 1G), we observed a bulged cilia tip containing accumulated electron dense particles (Fig. 1H). To our 139 surprise, we also observed the ciliary membrane bulged at a second point along the axoneme and 140 this region contained intraciliary vesicular structures (Fig. 1Hi). 141

Loss of WDR34 and WDR60 causes accumulation of proteins at the ciliary tip 142
The abnormal structure of cilia in the KO cells led us to analyze the steady-state localization of the 143 IFT machinery. After 24 hr serum starvation, IFT88 (part of IFT-B) was found almost exclusively at the base of the cilia in wild-type (WT) RPE1 cells ( Fig. 2A, quantified in Fig. 2Ai), but in WDR60 KO and 145 WDR34 KO cells IFT88 was found throughout the cilia and accumulated at the tip ( Fig. 2A, 2Aii, and  146 2Aiii). Similarly, another IFT-B component, IFT57 (Fig. 2B) was enriched at the cilia tips in WDR60 and 147 WDR34 KO cells. Quantification of the localization of these IFT-B proteins showed an accumulation 148 of both IFT88 (Fig. 2C) and IFT57 (Fig. 2D) at both the base and tip of the cilia in WDR60 KO cells. In 149 WDR34 KO cells, IFT-B proteins were seen to accumulate at the tip of cilia. The accumulation at the 150 base seen in WDR60 KO cells was not evident in WDR34 KO cells. The limited numbers of ciliated 151 WDR34 KO cells precluded further quantification. Another IFT-B protein, IFT20 (Fig. 2E), was 152 enriched at both the tip and the base of cilia in WDR60 KO cells (quantification in Fig. 2F). IFT20 is 153 the only IFT component found to localize to the Golgi until now. As expected, IFT20-GFP was found 154 at the Golgi in WT cells, although the Golgi pool of IFT20-GFP seen in WT cells was largely absent 155 from WDR60 KO cells (Fig. 2G). 156 Next, we analyzed the localization of IFT-A proteins. We found that both IFT140 ( which was also accumulated at the ciliary tip of WDR60 KO (Fig. 3E). 164 To study whether defects in dynein-2 affect the transport of membrane proteins we used GFP-165 fusions with Arl13b, somatostatin receptor type 3 (SSTR3), 5-hydroxytryptamine receptor type 6 166 (5HT6) and Rab8a. We found that in live cells Arl13b-GFP and EGFP-SSTR3 accumulate at the ciliary 167 tip ( Fig. 4A and 4B). Surprisingly, we also noticed a consistent reduction in the amount of Arl13b-GFP 168 within cilia in WDR60 KO cells ( Fig. 4C and 4Ci). The same observation was made with EGFP-SSTR3 169 ( Fig. 4D and 4Di) and EGFP-5HT6 ( Fig. 4E and 4Ei). In contrast, GFP-Rab8a localization in the cilia was 170 indistinguishable between WT and WDR60 KO cells (Fig 4F and 4Fi). To test if the reduction in Arl13b seen in our WDR60 KO was caused by a defect in the 176 transition zone, we labeled KO and WT cilia with known transition zone markers. We found that the core transition zone marker, RPGRIP1L (also known as MKS5), is no longer restricted to an area 178 adjacent to the mother centriole in WDR60 KO cells (Fig. 5A, quantified in 5Ai). Conversely, TMEM67 179 (also known as MKS3), which in WT cilia extends from the basal body through a more distal region, 180 becomes much more tightly restricted to the base of the cilium in WDR60 KO cells (Fig. 5B, 181 quantified in 5Bi). We also determined the transition zone organization in WDR34 KO cells. The few 182 cilia found in the WDR34 KO cells recapitulate the same phenotype observed in the WDR60 KO cilia 183 with an expansion of RPGRIP1L to a more distal position and a reduction of the TMEM67 domain 184 found throughout the cilium, as it was in WDR60 KO cells. Next, we tried to mimic the compound 212 heterozygosity occurring in patient cells, generating a stable cell line that expresses both WDR60 213 [T749M] and [Q631*] mutants. When the two mutants were co-expressed in the same WDR60 KO 214 cells we saw no additive effects or dominant negative effects, but cilia appeared normal with IFT88 215 only localized to the base (Fig. S3B, S3C) as was seen with the HA-WDR60[T749M] mutant. 216 In parallel, we also analyzed the phenotype of WDR34 KO cells stably expressing WT and mutant 217 forms of WDR34. We found that expression of WT mGFP-WDR34 restored ciliogenesis and axoneme 218 extension in WDR34 KO cells and that, unexpectedly, this was also true of cells expressing WDR34 219  (Fig. 8B). Next, we tested the interactions with IFT proteins, the primary cargo of the 234 ciliary motors. We found that WT WDR60 can bind to IFT140, IFT88, and IFT57; WDR60[T749M] 235 binds to all 3 IFT subunits tested but binds less well to IFT140 (Fig. 8C). In contrast, WDR60[Q631*] 236 pulled down reduced levels of IFT88 and IFT57 and did not interact with IFT140. 237

The stability of dynein-2 complex in WDR34 and WDR60 KO cells 238
It has been reported that loss of some components of dynein-2 modifies the stability of the whole 239 dynein-2 complex. In Chlamydomonas depletion or loss of LIC3 (D1bIC2) causes a reduction of DHC2 WDR60 in whole cells lysate of KO cells. Notably, we found that expression levels of WDR34 were 246 reduced in WDR60 KO cells, although not completely lost. Correspondingly, there was a reduction of 247 WDR60 expression levels in WDR34 KO whole cell lysate (Fig. 9A). Next, we sought to determine the 248 effect of WDR60 and WDR34 loss on the localization of other dynein-2 subunits. We found that LIC3 249 (DYNC2LI1) localized in cilia of WT cells, but this localization was lost in WDR34 or WDR60 cells ( Our data provide evidence that the structural asymmetry with the dynein-2 motor is matched by 283 functional asymmetry. Perhaps most strikingly, we find that WDR34 is essential for axoneme 284 extension during early steps of ciliogenesis, whereas WDR60 is not required for ciliogenesis. Both 285 subunits are necessary for maintaining proper cilia protein composition. Depletion of WDR34 using 286 RNAi is also associated with ciliary defects (Asante et al., 2013) and patient fibroblasts have shorter 287 cilia with a bulbous tip (Huber et al., 2013). Fibroblasts from WDR34 knockout mice also have 288 stumpy cilia and defects in Shh signaling (Wu et al., 2017). It is intriguing that some cells missing 289 WDR34 can still extend a rudimentary cilium but even here, ciliary protein localization is severely 290 disrupted. Since WDR60 cells can extend an axoneme, WDR34 and WDR60 clearly have distinct but 291 overlapping functions in cells. We consider that WDR34 plays an essential role in ciliogenesis to 292 ensure delivery of a key factor required for axoneme extension. It is noteworthy that in the absence 293 of WDR34, WDR60 can still assemble effectively with the other subunits of dynein-2. While these 294 interactions are likely reduced compared to the normal situation, this shows that it is specifically 295 WDR34 that is required at this early stage of ciliogenesis. Our EM data show that it acts at a stage 296 after docking of the ciliary vesicle, immediately before axoneme extension. Paradoxically, our data 297 also show that in the absence of WDR60, the dynein-2 holocomplex cannot form effectively yet 298 axoneme extension occurs normally. This raises the possibility that WDR34 is itself required for 299 axoneme extension, possibly outside of the context of the dynein-2 complex. We cannot rule out 300 that there are dynein-2-independent functions of WDR34 and WDR60 but all data provide strong 301 evidence that they co-exist in the dynein-2 holoenzyme. 302 Assembly of the dynein-2 holocomplex 303 In WDR34 and WDR60 KO cells, LIC3 is no longer detected in primary cilia, while TCTEX1 localization, 304 a dynein light chain that is also a component of dynein-1, was unperturbed. Coupled with our 305 proteomics data, this suggests that the localization of LIC3 to cilia is a good reporter of dynein-2 306 assembly. Surprisingly we found that DHC2 levels at the ciliary base are reduced in WDR34 KO cells 307 compared to WT and WDR60 KO cells. Our data show that in WDR34 KO cells, the remaining 308 subunits can coassemble into partial dynein-2 complexes. We do not, however, know if this is a 309 functional or indeed processive motor. According to the current model based on work in C. elegans suggesting that these proteins might not be effectibvvely retained within cilia, but leak out through 372 the diffusion barrier. The alternative, as discussed above, is that these proteins are less effectively 373 loaded into cilia. We did not find a difference in the intensity levels of overexpressed Rab8a in 374 WDR60 KO compared to WT cells suggesting that at least some proteins can enter normally. It seems 375 likely that the defects we see in both entry to, and exit from, cilia in these KO cells are caused by 376 defects in transition zone structure.  Fig. 3 were imaged using Leica SP8. All images were acquired as 0.5 µm z-stacks. 511 All graphs show mean and standard deviation. 512

Rescue experiments 513
For 'rescue' experiments, stable WDR60 KO cell lines overexpressing wild-type and mutants HA-514 tagged WDR60 were generated. Similarly, WDR34 KO#1 cells were stably transfected with WT and 515 mutants WDR34 tagged with a GFP. Cells were serum starved for 24 h, fixed and processed for 516 immunofluorescence analysis. 517

Electron microscopy 518
Cells were serum starved 24 h and fixed in 2.5% glutaraldehyde for 20 min. Next, the cells were 519 washed for 5 min in 0.1 M cacodylate buffer then post-fixed in 1% OsO4/0.1 M cacodylate buffer for 520 30 min. Cells were washed 3x with water and stained with 3% uranyl acetate for 20 min. After 521 another rinse with water, cells were dehydrated by sequential 10 min incubations with 70, 80, 90, 522 96, 100 and 100% ethanol before embedding in Epon™ at 70°C for 48 h. Thin (70 nm) serial sections 523 were cut and stained with 3% uranyl acetate then lead citrate, washing 3x with water after each.

Fluorescence intensity measurement 534
Quantification of fluorescence intensity was performed using original images. Measurement of 535 intensity was performed using the average projections of acquired z-stacks of the area of the ciliary 536 marker acetylated tubulin. Fluorescence intensity along the ciliary axoneme was measured using 537 ImageJ plot profile tool. Fluorescence intensity in at the ciliary base was measured drawing same 538 diameter circles at the ciliary base.

Proteomic analysis 553
For TMT Labelling and high pH reversed-phase chromatography, the samples were digested from the 554 beads with trypsin (2.5 µg trypsin, 37°C overnight), labeled with Tandem Mass Tag (TMT) six-plex 555 reagents according to the manufacturer's protocol (Thermo Fisher Scientific, Loughborough, UK) and the labeled samples pooled. The pooled sample was then desalted using a SepPak cartridge 557 according to the manufacturer's instructions (Waters, Milford, Massachusetts, USA)). Eluate from 558 the SepPak cartridge was evaporated to dryness and resuspended in buffer A (20 mM ammonium 559 hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an 560 Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was 561 loaded onto an XBridge BEH C18 Column (130 Å, 3.5 µm, 2.1 mm X 150 mm, Waters, UK) in buffer A 562 and peptides eluted with an increasing gradient of buffer B (20 mM ammonium hydroxide in 563 acetonitrile, pH 10) from 0-95% over 60 min. The resulting fractions were evaporated to dryness and 564 resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Tribrid 565 mass spectrometer (Thermo Fisher Scientific). 566

Nano-LC Mass Spectrometry 567
High pH RP fractions were further fractionated using an Ultimate 3000 nano-LC system in line with 568 an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). In brief, peptides in 1% 569 (vol/vol) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Thermo Fisher 570 Scientific). After washing with 0.5% (vol/vol) acetonitrile 0.1% (vol/vol) formic acid, peptides were 571 resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse phase analytical column (Thermo Fisher 572 Scientific) over a 150 min organic gradient, using 7 gradient segments (1-6% solvent B over 1 min., 6-573 15% B over 58 min., 15-32% B over 58 min., 32-40% B over 5 min., 40-90% B over 1 min., held at 90% 574 B for 6 min and then reduced to 1% B over 1 min.) with a flow rate of 300 nl min−1. Solvent A was 575 0.1% formic acid and Solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were 576 ionized by nano-electrospray ionization at 2.0 kV using a stainless steel emitter with an internal 577 diameter of 30 μm (Thermo Fisher Scientific) and a capillary temperature of 275°C. 578 All spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer controlled by Xcalibur 579 2.0 software (Thermo Fisher Scientific) and operated in data-dependent acquisition mode using an 580 SPS-MS3 workflow. FTMS1 spectra were collected at a resolution of 120 000, with an automatic gain 581 control (AGC) target of 400 000 and a max injection time of 100 ms. Precursors were filtered with an 582 intensity range from 5000 to 1E20, according to charge state (to include charge states 2-6) and with 583 monoisotopic precursor selection. Previously interrogated precursors were excluded using a 584 dynamic window (60 s +/-10 ppm). The MS2 precursors were isolated with a quadrupole mass filter 585 set to a width of 1.2 m/z. ITMS2 spectra were collected with an AGC target of 10 000, max injection 586 time of 70 ms and CID collision energy of 35%. 587 For FTMS3 analysis, the Orbitrap was operated at 30 000 resolution with an AGC target of 50 000 588 and a max injection time of 105 ms. Precursors were fragmented by high energy collision dissociation (HCD) at a normalized collision energy of 55% to ensure maximal TMT reporter ion yield. 590 Synchronous Precursor Selection (SPS) was enabled to include up to 5 MS2 fragment ions in the 591 FTMS3 scan. 592

Data Analysis 593
The raw data files were processed and quantified using Proteome Discoverer software v2.1 (Thermo 594 Fisher Scientific) and searched against the UniProt Human database (140000 entries) and GFP 595 sequence using the SEQUEST algorithm. Peptide precursor mass tolerance was set at 10 ppm, and 596 MS/MS tolerance was set at 0.6 Da. Search criteria included oxidation of methionine (+15.9949) as a 597 variable modification and carbamidomethylation of cysteine (+57.0214) and the addition of the TMT 598 mass tag (+229.163) to peptide N-termini and lysine as fixed modifications. Searches were 599 performed with full tryptic digestion and a maximum of 1 missed cleavage was allowed. The reverse 600 database search option was enabled and the data was filtered to satisfy false discovery rate (FDR) of 601 5%. 602      Figure 10