CEP290 is essential for the initiation of ciliary transition zone assembly

Zhimao Wu; Nan Pang; Yingying Zhang; Huicheng Chen; Ying Peng; Jingyan Fu; Qing Wei

doi:10.1371/journal.pbio.3001034

. 2020 Dec 28;18(12):e3001034. doi: 10.1371/journal.pbio.3001034

CEP290 is essential for the initiation of ciliary transition zone assembly

Zhimao Wu ^1,^2,³, Nan Pang ⁴, Yingying Zhang ^1,^2,³, Huicheng Chen ^1,^2,³, Ying Peng ⁵, Jingyan Fu ⁴, Qing Wei ^3,^*

Editor: Renata Basto⁶

PMCID: PMC7793253 PMID: 33370260

Abstract

Cilia play critical roles during embryonic development and adult homeostasis. Dysfunction of cilia leads to various human genetic diseases, including many caused by defects in transition zones (TZs), the “gates” of cilia. The evolutionarily conserved TZ component centrosomal protein 290 (CEP290) is the most frequently mutated human ciliopathy gene, but its roles in ciliogenesis are not completely understood. Here, we report that CEP290 plays an essential role in the initiation of TZ assembly in Drosophila. Mechanistically, the N-terminus of CEP290 directly recruits DAZ interacting zinc finger protein 1 (DZIP1), which then recruits Chibby (CBY) and Rab8 to promote early ciliary membrane formation. Complete deletion of CEP290 blocks ciliogenesis at the initiation stage of TZ assembly, which can be mimicked by DZIP1 deletion mutants. Remarkably, expression of the N-terminus of CEP290 alone restores the TZ localization of DZIP1 and subsequently ameliorates the defects in TZ assembly initiation in cep290 mutants. Our results link CEP290 to DZIP1-CBY/Rab8 module and uncover a previously uncharacterized important function of CEP290 in the coordination of early ciliary membrane formation and TZ assembly.

Dysfunction of cilia leads to various human genetic diseases, including many caused by defects in transition zones (TZs), the “gates” of cilia. A study in Drosophila reveals that the cilia TZ core protein CEP290 coordinates early ciliary membrane formation and TZ assembly; the N-terminus of CEP290 recruits DZIP1, which in turn recruits Rab8 and CBY to promote early ciliary membrane formation.

Introduction

Cilia are microtubule-based organelles that extend from the surface of most eukaryotic cells. These protrusions are important for diverse cellular functions, including cell signaling, sensory perception, cell motility, and extracellular fluid movement [1–4]. In humans, almost every cell has at least 1 cilium. Due to the ubiquitous distribution and important functions of cilia, ciliary dysfunction results in a wide variety of human genetic disorders, collectively termed ciliopathies [5,6].

Cilia arise from the distal ends of basal bodies (BBs) that are derived from mother centrioles. Depending on the cell type, cilia form through 1 of 2 pathways: the intracellular pathway and the extracellular pathway [7,8]. Recent studies on mammalian cells have revealed that, in both pathways, ciliogenesis begins with the docking of Myo-Va–associated small vesicles to mother centriole distal appendages (DAs) [9]. These small vesicles, termed preciliary vesicles (PCVs), then fuse into a large ciliary vesicle (CV) in a process mediated by the membrane-shaping proteins EH domain-containing protein 1 (EHD1) and EHD3 [10]. Then, the Rab11-Rabin8-Rab8 signaling cascade mediates the formation of the early ciliary shaft membrane [9–12]. Perhaps at the same time, the centriole cap protein CP110 is removed, followed by the subsequent assembly of the transition zone (TZ) which is the fundamental ciliary base structure for gating the ciliary compartment [13,14], and then the elongation of the axoneme which is mediated by the evolutionarily conserved intraflagellar transport (IFT) machinery [15].

The TZ is characterized by Y-link–like structures under electron microscopy, which connect proximal axonemal microtubules to the ciliary base membrane and may organize the ciliary necklace composed of annular intramembrane particles on the TZ membrane surface. It has been proposed that the TZ functions as a diffusion barrier to gate the ciliary compartment [16–18]. Dozens of proteins have been identified as TZ components from various model organisms [6,14,19]. Elegant genetic studies on C. elegans have grouped known TZ proteins into 3 functional modules, namely, Meckel–Gruber syndrome (MKS), Nephronophthisis (NPHP), and CEP290 [20–22]. Although great progress has been made in understanding the molecular composition and hierarchical assembly of TZs, little is known about how TZ assembly is initiated after basal body docking, or about how early ciliary shaft membrane formation is coordinated with TZ assembly.

CEP290, an important TZ component, is evolutionarily conserved from unicellular Chlamydomonas to humans [20,21,23–25] and is one of the most intriguing cilia genes. Mutations in CEP290 cause several ciliopathies ranging from nonsyndromic retinal degeneration, i.e., Leber Congenital Amaurosis (LCA), to syndromic disorders including, Senior–Loken syndrome (SLS), Joubert syndrome (JBTS), MKS, and Bardet–Biedl syndrome (BBS) [26]. Over 100 CEP290 mutations have been identified in patients, and these mutations cause a wide variety of phenotypes, ranging from isolated blindness to embryonic lethality [26]. The broad spectrum of diseases and phenotypes associated with CEP290 mutations highlight the multiple and critical roles of CEP290 in cilia. However, the molecular mechanisms of CEP290 in TZ assembly are still not fully understood.

Here, we discover that CEP290 is essential for TZ assembly initiation in Drosophila. Complete deletion of CEP290 blocks ciliogenesis at the stage of TZ assembly initiation in both ciliated sensory neurons and spermatocytes, 2 main ciliated cell types of Drosophila. Further studies reveal that the N-terminus of CEP290 directly interacts with DAZ interacting zinc finger protein 1 (DZIP1) and recruits DZIP1 to the TZ. Interestingly, deletion of DZIP1 results in a phenotype similar to cep290 deletion mutants. Furthermore, we demonstrate that DZIP1 functions upstream of CBY and Rab8 to mediate ciliary membrane assembly. We propose that ciliary membrane assembly is a prerequisite for TZ assembly and that CEP290 coordinates the formation of early ciliary membranes with the assembly of the TZ.

Results

Both N-terminal and C-terminal truncation mutants of CEP290 localize to the TZ in Drosophila

To determine the domain that mediates the TZ localization of Drosophila CEP290, we constructed several green fluorescent protein (GFP)-tagged CEP290 truncation constructs and assessed their subcellular localization in various types of cilia in Drosophila: auditory cilia, olfactory cilia, and spermatocyte cilia. It has been proposed that CEP290 bridges axonemal microtubules and ciliary membranes, where its C-terminus contains a microtubule binding domain and N-terminus possesses a highly conserved membrane-binding amphipathic α-helix motif (Fig 1A) [23,27,28]. Consistent with these findings, the C-terminal fragment of CEP290 containing the microtubule binding domain (CEP290-C, from aa 1385 to the end) localized to the TZ in all examined cilia in Drosophila (Fig 1A–1E), and the N-terminal fragment of CEP290 (CEP290-N, comprising aa 1–650) alone was also located at the TZ, but the middle fragment (CEP290-M, aa 651–1384) was not (Fig 1A–1E). The fact that CEP290-N alone targets to the TZ suggests that the N-terminus of CEP290 has a TZ target signal independent of the C-terminal microtubule binding domain. Notably, wild-type (WT) flies with overexpression of the N-terminus of CEP290 showed defective motility (Fig 1F), a phenotype commonly associated with cilia dysfunction.

Fig 1 — (A) Schematic representations of CEP290 truncations used in (B–E). (B) Localization patterns of various GFP-tagged CEP290 truncations in auditory cilia. 21A6 marks the ciliary base (red). Both CEP290-N and CEP290-C localized to the ciliary base, but CEP290-M did not. Bar, 1 μm. (C) The localization of various GFP-tagged CEP290 truncations in olfactory cilia. 21A6 marks the ciliary base (red). Both CEP290-N and CEP290-C localized to the ciliary base, but CEP290-M did not. Bar, 1 μm. (D) The localization of various GFP-tagged CEP290 truncations in spermatocytes. γ-Tubulin was used to label the BBs (red). Both CEP290-N and CEP290-C localized to the ciliary base, but CEP290-M did not. Bar, 1 μm. (E) 3D-SIM images of the localization of CEP290, CEP290-N, and CEP290-C in auditory cilia (upper panel), and the corresponding quantification of signal length (lower panel). FBF1 was used to indicate the ciliary base. n = 20. Bar, 1 μm. (F) Analysis of the moving behavior of flies overexpressing of CEP290 truncations. Climbing assay was used to assess the fly motility. Motility of flies with overexpression of CEP290-N and CEP290-N+M were significantly compromised. Uncropped images for panels B and C can be found in S1 Raw Image. Numerical data for panel E and F can be found in the file S1 Data. 3D-SIM, three-dimensional structured illumination microscopy; BB, basal body; CEP290, centrosomal protein 290; FBF1, Fas binding factor 1; GFP, green fluorescent protein; ns, not significant; WT, wild-type.

The length of the TZ in different types of cilia is variable in Drosophila. TZs of auditory cilia are significantly longer than those of olfactory cilia and spermatocyte cilia [27]. TZ in auditory cilia contains 2 longitudinal subdomains: The proximal one comprises all TZ components, whereas the distal one comprises CEP290 only [27]. We did see that the CEP290 signal in auditory cilia was longer than that in other cilia (Fig 1B). However, we observed that the distribution of CEP290-N signal was significantly more restricted than the full-length CEP290 signal (Fig 1B), suggesting that CEP290-N is restricted toward the proximal part of the TZ. On the other hand, we observed that the signal of CEP290-C was significantly longer than that of CEP290 (Fig 1B), which was also seen in olfactory cilia (Fig 1C). Different distributions of CEP290, CEP290-N, and CEP290-C in TZs of auditory cilia were further confirmed by three-dimensional structured illumination microscopy (3D-SIM) (Fig 1E). These results suggest that the N-terminus of CEP290 might have an inhibitory effect on the localization of its C-terminus.

CEP290 is essential for the initiation of TZ assembly in spermatocytes

The fact that a short N-terminal truncation mutant of CEP290 was targeted to the TZ suggests that the previously reported cep290 mutant (cep290^mecH, lacking the C-terminal sequence from aa 1471 to the end) may be not a null mutant [24]. To better understand the function of CEP290, especially its N-terminus, we employed CRISPR-mediated genome engineering and generated a putative null mutant, cep290¹ (c.469-1174Del), in which site-specific deletion leads to a frameshift, leaving only 156 aa of the N-terminus (Fig 2A and S1A Fig). In order to compare the phenotype differences between the cep290¹ mutants and C-terminus deletion mutants, we additionally generated cep290^ΔC (c.4156delG), in which a deletion results in a frameshift and C-terminus (aa 1386-end) loss (Fig 2A and S1B and S1C Fig). As expected, immunofluorescence staining of CEP290 in spermatocytes with an antibody against its N-terminal showed that the N-terminus of CEP290 was completely lost in cep290¹ mutants, but it was still present in cep290^ΔC mutants (S1D Fig). Notably, compared with WT, the signal intensity of CEP290 in cep290^ΔC mutant was significantly reduced (S1D Fig). This is consistent with the transcript level of cep290 in cep290^ΔC mutant, which was also significantly lower than that of WT (S1B Fig), probably due to nonsense-mediated mRNA decay.

Similar to previously reported alleles [24,29], both cep290¹ and cep290^ΔC were severely uncoordinated during walking and flying, and they had severe defects in both touch sensitivity and hearing sensation (S1E Fig), typical phenotypes of cilia deficiency mutants. Importantly, all these defects could be rescued by expression of UAS-cep290 under the pan-neural driver elav-GAL4 (S1E Fig).

First, we focused on cilia in spermatocytes. Drosophila spermatocyte ciliogenesis has several unique features (Fig 2B): (1) Cilia grow from both mother and daughter centrioles [30]; (2) cilia have only a TZ region, with no axoneme above the TZ [31]; (3) cilia are assembled on the cell surface in the early G2 phase but are internalized into the cytosol along with BBs in later stages, as BBs need to be used to assemble the spindle for meiosis [32,33]; and (4) during spermatid elongation after meiosis, cilia (also known as ciliary caps at this stage) extend and move away from BBs [24].

To analyze cilia/TZs formation in spermatocytes, we examined the localization of TZ core proteins (MKS1 and MKS6) and TZ-related proteins (CBY and DILA/CEP131). CBY and Dilatory (DILA) cooperate to build the TZ in Drosophila [34], but their localization in the TZ is very different; CBY completely colocalizes with MKS1 and MKS6 near the TZ membrane in spermatocytes, whereas DILA is inside the lumen of the TZ axoneme. Consistent with previous reports [24], we observed that all these proteins were present at the tips of BBs in cep290^ΔC mutant, although the signal intensity of MKS1, MKS6, and CBY was significantly reduced (Fig 2C). However, surprisingly, we observed that MKS1, MKS6, and CBY were completely lost from the tips of BBs in cep290¹ mutants (Fig 2C), while the distribution of DILA was normal. These results indicate that CEP290 is essential for TZ assembly.

Strikingly, we observed that, compared with that in WT flies and cep290^ΔC mutants, the signal of Uncoordinated (UNC, the homolog of mammalian OFD1) was aberrantly elongated in cep290¹ mutants (Fig 2C). Colabeling of UNC with the transition fiber (TF) protein Fas binding factor 1 (FBF1) indicated that the elongation occurred above the TF (Fig 2C), suggesting that the ciliary axoneme, rather than the centriole, was elongated. To confirm this, we labeled primary cilia axonemes with CG6652-GFP, which specifically labels axoneme in cilia of spermatocytes and ciliary caps of round spermatids [34]. Indeed, as shown in Fig 2D, axonemes extended abnormally in cep290¹ mutants, but not in WT flies or cep290^ΔC mutants.

Abnormal spermatocyte ciliary axoneme elongation has previously been observed in cby;dila double mutants and has been attributed to defects in basal body docking and the absence of a TZ membrane cap [34]. Accordingly, we observed that the association between BBs (as shown by UNC-GFP) and the plasma membrane (labeled with CellMask, Invitrogen, Carlsbad, California, United States of America) was defective in later spermatocytes and spermatids in cep290¹ mutants, but not in WT flies or cep290^ΔC mutants (Fig 2E). However, in the early spermatocyte stage, we observed that BBs were still attached/close to the membrane (Fig 2E). To further confirm this result, we employed transmission electron microscopy (TEM). Consistent with our immunofluorescence observations, BBs were able to migrate to the plasma membrane in cep290¹ mutants, but no cilia-like structures and TZs formed (Fig 2F). Taken together, we proposed that in cep290¹ mutants, BBs initially migrate to the plasma membrane, but the TZ assembly initiation is completely blocked, such that primary cilia/TZs never form. During the process of BB internalization in later stages, due to the absence of the ciliary cap, the stable association between the BB and the cell membrane was unable to be maintained, and the BB’s microtubules were abnormally elongated within the cytoplasm (Fig 2G).

In addition to the initiation of TZ assembly, spermatogenesis was also very different between cep290¹ mutants and cep290^ΔC mutants. As previously reported [24], cep290^ΔC mutants had mild defects in spermatid development and male fertility (S1F and S1G Fig); however, sperm cysts were severely defective in elongation in cep290¹ mutants, although the overall sizes of their testes were comparable (S1F Fig). Subsequently, no mature sperms were produced, and males were completely infertile in cep290¹ mutants (S1G Fig). TEM examination of testes showed that flagellar axonemes were almost completely lost in cep290¹ mutants (S1H Fig). Similar severe defects in the spermatid axoneme were also observed in TZ absent mutants [34], but the underlying mechanism remains poorly understood.

CEP290 is critical for the initiation of TZ assembly in sensory neurons

Next, we asked whether CEP290 is also involved in TZ assembly initiation in sensory neurons. Previously, EM data have shown that the auditory cilium in cep290^mecH mutants lacks the TZ and ciliary axoneme [27]. Consistent with this, complete loss of cilia was observed in cep290¹ and cep290^ΔC mutants by both immunofluorescence and electron microscope (EM) assays (Fig 3A and 3B). Our EM data showed that BBs were very close to the plasma membrane, but no apparent TZs formed in either cep290¹ or cep290^ΔC mutants (S1I Fig). Although, under EM, we did not find any obvious differences in cilia base structures between cep290¹ and cep290^ΔC mutants; we found that, similar to that of spermatocyte cilia, the localization of TZ components in auditory cilia were drastically different in different cep290 mutants. All TZ proteins examined were present at the BB in cep290^ΔC mutants, although their signal intensities were severely reduced compared to WT. However, remarkably, these signals were completely lost in cep290¹ mutants (Fig 3C). Similar results were also observed in olfactory cilia (Fig 3C). These results suggest that in sensory neurons, the CEP290 N-terminus may be essential for initial TZ assembly, whereas TZ elongation or maturation may involve a tissue-specific requirement for the C-terminus of CEP290.

Fig 3 — (A) Cilium morphology in auditory cilia as labeled by *nompC-Gal4; UAS-GFP* in WT flies and *cep290*¹ mutants. Cilia were completely lost in *cep290*¹ mutants. (B) EM analysis of cross sections of auditory cilia. In WT flies, each scolopidium possessed 2 ciliary axonemes (arrows), but no ciliary axonemes were observed in the scolopidia of *cep290* mutants. Bar, 200 nm. (C) Localization of various TZ proteins in auditory cilia and olfactory cilia in WT flies, *cep290*^ΔC mutants, and *cep290*¹ mutants, and corresponding relative fluorescence intensity quantitation. In *cep290*^ΔC mutant, TZ proteins, MKS1-GFP, MKS6-GFP, and CBY-GFP were still present at the ciliary base, although the signals were severely reduced. However, these signals were completely lost in *cep290*¹ mutants. 21A6 (red) was used to label the ciliary base. Uncropped images for panel C can be found in S1 Raw Image. Numerical data for panel C can be found in the file S1 Data. The bars and error bars represent the means and SDs, respectively. n = 50 basal bodies over 5 flies. Scale bars, 1 μm. CEP290, centrosomal protein 290; EM, electron microscope; SD, standard deviation; TZ, transition zone; WT, wild-type.

Taken together, our results indicate that CEP290 is essential for the initiation of TZ assembly in both sensory neurons and spermatocytes. Of note, without a robust antibody to survey expression to validate levels of CEP290 protein expression within our alleles, we cannot fully unpick how much of our phenotypes are due solely to reduced levels versus requirement for specific domains. We suspect it is likely a combination of both.

DZIP1 is a TZ protein interacting with the N-terminus of CEP290

To elucidate the role of CEP290 in TZ assembly initiation, we employed a yeast two-hybrid assay to search for TZ-related proteins that interact with the N-terminus of CEP290 and identified DZIP1. Drosophila DZIP1 is the ortholog of mammalian DZIP1 and DZIP1L (S2A–S2D Fig), both have been implicated in ciliogenesis [35–38]. In our yeast two-hybrid assay, DZIP1 interacted with the N-terminus of CEP290, but did not interact with its C-terminus (Fig 4A). The interaction was further confirmed by the glutathione S-transferase (GST) pull-down assay (Fig 4B and S3A Fig). Both the N-terminus and C-terminus of DZIP1 interacted with the CEP290 N-terminus. DZIP1-N (aa 1–293) interacted with both CEP290 N (aa 401–650) and CEP290 N (aa 651–887); DZIP1-C (aa 294–737) interacted with CEP290 N (aa 651–887) (S3B Fig). To further confirm the interaction, we expressed GFP-tagged CEP290 and HA-tagged DZIP1 in cultured Drosophila S2 cells and performed coimmunoprecipitation (Co-IP) using GFP-trap beads. Western blots of the GFP-pulldowns confirmed the binding between CEP290 and DZIP1 (Fig 4C).

Fig 4 — (A) Yeast two-hybrid assay results showing that DZIP1 interacts with CEP290-N (aa 1–887), but not with CEP290-ΔN (Δ aa 1–887). (B) GST pull-down assay confirmed the interaction between CEP290-N and DZIP1 in vitro. Both DZIP1-N (aa 1–293) and DZIP1-C (aa 294–737) interacted with CEP290-N. Upper panel, blotting with anti-His antibodies. Lower panel, Ponceau S staining of GST, GST-DZIP1-N, or GST-DZIP1-C. (C) CEP290-N and DZIP1 were coimmunoprecipitated in S2 cells. S2 cells were transiently transfected with CEP290-N-GFP and DZIP1-HA; 48 h later, cells were subjected to Co-IP using GFP-trap beads. (D, E) 3D-SIM images of the localization of DZIP1 on TZs in different types of cilia. (D) In all cilia, DZIP1 localized distal to the TF protein FBF1 and surrounded the C-terminal GFP-tagged CEP290. (E) In spermatocytes, DZIP1 overlapped with MKS1 and CBY. In sensory neurons, DZIP1 localized above FBF1, below MKS1, and partially overlapped with MKS1. Bars, 500 nm. (F) The TZ proteins MKS1-GFP, MKS6-GFP, and CBY-GFP were completely lost from the ciliary base in sensory neurons in *dzip1*^ΔC mutants. CEP290-GFP and UNC-GFP were still present at the ciliary base. Notably, the signal of CEP290 was abnormally expanded in auditory cilia. 21A6 (red) marks the ciliary base. Bar, 1 μm. (G) MKS1, MKS6, and CBY were almost completely lost in spermatocytes of *dzip1*^ΔC mutants. CEP290 and UNC were still present, but the signals were longer in mutants than in WT flies. γ-Tubulin was used to label the BBs (red). Bar, 1 μm. (H) Representative images of aberrantly elongated and branched spermatocyte ciliary axonemes (white arrow) labeled by CG6652-GFP in *dzip1*^ΔC mutants. γ-Tubulin was used to label the BBs (red). Bar, 2 μm. (I) Live imaging of the connection between BBs and the PM in WT flies and *dzip1*^ΔC mutants. The membrane was labeled with CellMask, and the BBs were indicated by UNC-GFP. In *dzip1*^ΔC mutants, the BBs migrated to the PM in the early spermatocyte stage but failed to maintain the cilium-PM connection in subsequent processes. Bar, 10 μm. Numerical data for panels D and E can be found in the file S1 Data. Uncropped images for panel F can be found in S1 Raw Image. Uncropped immunoblots for panels B and C can be found in S2 Raw Image. 3D-SIM, three-dimensional structured illumination microscopy; BB, basal body; CBY, Chibby; CEP290, centrosomal protein 290; Co-IP, coimmunoprecipitation; DZIP1, DAZ interacting zinc finger protein 1; FBF1, Fas binding factor 1; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, the hemagglutinin (HA) tag; MKS1, Meckel syndrome type 1; MKS6, Meckel syndrome type 6; PM, plasma membrane; TZ, transition zone; UNC, Uncoordinated; WT, wild-type.

While the role of Drosophila DZIP1 in ciliogenesis was unknown when we started this project, a recent study showed that DZIP1 was localized to the TZ and was required for ciliogenesis in Drosophila [29]. Consistent with this report, we observed that Drosophila DZIP1 localized to the basal body in all cilia examined (S4A and S4B Fig). In spermatocytes, DZIP1 was found to localize to the tips of BBs. In spermatids, like TZ proteins, DZIP1 migrated along with the ciliary cap to the flagellar tip. To more accurately determine the subcellular localization of DZIP1, we employed 3D-SIM and observed that in all types of cilia, DZIP1 localized above the TF protein FBF1, surrounded the C-terminal GFP-tagged CEP290 which labels the center of the TZ [27] (Fig 4D). In spermatocytes, DZIP1 overlapped completely with the TZ protein MKS1 and CBY along TZ membranes (Fig 4E). In spermatids, DZIP1 was enriched at the ring centriole like CBY, but did not extend distally along the whole ciliary cap like MKS1 and MKS6 (S4C Fig). In sensory neurons, there was an obvious gap between the TF protein FBF1 and the TZ component MKS1 (S4D Fig). We observed that DZIP1 was above FBF1, but clearly below MKS1 and partially overlapped with MKS1, indicating that DZIP1 occupied the gap region between FBF1 and MKS1, and further extended distally, partially colocalizing with MKS1 (Fig 4E). Therefore, there are tissue-specific differences in DZIP1-related ciliary base structures. Overall, Drosophila DZIP1 is a TZ protein interacting with the N-terminus of CEP290.

dzip1 deletion mutants mimic the phenotype of cep290 mutants

To investigate the functional relationship between CEP290 and DZIP1, we generated a deletion mutant of dzip1^ΔC (c.1069-1529Del), in which the C-terminus (aa 357-end) of DZIP1 is lost (S5A–S5C Fig). Consistent with the recent report [29], dzip1 mutant exhibited typical cilia mutant phenotypes. The flies were completely uncoordinated, unable even to stand, and were severely deficient in touch and hearing (S5D Fig). Introducing a WT DZIP1 cDNA transgene into the mutants fully rescued these deficiencies (S5D Fig), indicating that DZIP1 was the gene responsible for these defects.

Interestingly, we found that our dzip1 mutants mimicked the phenotype of cep290¹ mutants. In sensory cilia of dzip1 mutants, TZ components MKS1, MKS6, and CBY were completely absent from the TZ, but the recruitment of CEP290 and UNC was not affected (Fig 4F). And cilia were almost completely lost in auditory cilia of dzip1^ΔC mutant as demonstrated by both immunofluorescence and EM assays (S5E and S5F Fig). In spermatocytes of dzip1 mutants, MKS1, MKS6, and CBY were almost completely lost but CEP290 persisted (Fig 4G). Furthermore, similar to cep290¹ mutants, ciliary axonemes extended aberrantly in dzip1 mutants, as shown by UNC-GFP and CG6652-GFP (Fig 4G and 4H). Additionally, deletion of DZIP1 resulted in the separation of the BBs and the plasma membrane in late spermatocytes (Fig 4I). All these observations are consistent with the recent report [29], indicating that like CEP290, DZIP1 is essential for TZ assembly initiation.

CEP290 recruits DZIP1 to regulate TZ assembly

cep290¹ and dzip1 mutants have similar phenotypes, and DZIP1 is dispensable for the recruitment of CEP290 (Fig 4F and 4G). Interestingly, the DZIP1 signal was completely lost in both spermatocyte cilia and sensory neuron cilia in cep290¹ mutants, but it is still existed in the unc, dila, and cby mutants (Fig 5A and 5B). It has been reported that CEP290 is lost in cby;dila double mutants [34]; accordingly, no DZIP1 was observed in this double mutants (Fig 5A and 5B). Notably, in cep290^ΔC mutants, DZIP1 was present in all types of cilia, suggesting that the remaining N-terminus of CEP290 has a role in recruiting DZIP1 to the TZ. However, compared with WT, the signal intensity of DZIP1 decreased significantly in sensory cilia in cep290^ΔC mutants, and it extended abnormally into the whole cilia/TZs in spermatids (Fig 5B and 5C), suggesting that the CEP290 C-terminus may have tissue-specific roles in regulating the localization of DZIP1.

Fig 5 — (A) Localization of DZIP1 in spermatocytes in the indicated genetic background. DZIP1-GFP was completely lost in *cep290*¹ mutants and *cby;dila* double mutants, but existed in other mutants, including *cep290*^ΔC mutants. γ-Tubulin (red) was used to label the BBs. Bar, 1 μm. (B) Localization of DZIP1 in sensory neurons in the indicated genetic background. In *cep290*¹ mutants, DZIP1-GFP was completely lost. In *cep290*^ΔC mutants, DZIP1 was present, but the signal was severely reduced in sensory neurons. 21A6 staining indicates the ciliary base in sensory neurons. Uncropped images can be found in S1 Raw Image. Bars, 1 μm. (C) The C-terminus of CEP290 is required to limit the DZIP1 signal at the ring centriole in spermatids. Bar, 1 μm. (D) Overexpression of the CEP290 N-terminus (aa 1–650) ameliorated the localization defects of DZIP1 in *cep290*¹ mutants. Bars, 1 μm. (E) Overexpression of the CEP290 N-terminus rescued the localization of MKS1 in spermatocytes and spermatids in *cep290*¹ mutants. Bars, 1 μm. (F) Overexpression of CEP290 N-terminus restored the connection between the BBs and the membrane in *cep290*¹ mutants. CellMask was used to label the membrane, and UNC-GFP indicates the BBs. Bar, 10 μm. BB, basal body; CEP290, centrosomal protein 290; DZIP1, DAZ interacting zinc finger protein 1; MKS1, Meckel syndrome type 1; TZ, transition zone.

Because the N-terminus of CEP290 directly interacts with DZIP1 (Fig 4A–4C), we hypothesized that CEP290-N may directly recruit DZIP1 to regulate TZ assembly. If so, expressing the N-terminus of CEP290 should ameliorate the defects in TZ assembly initiation in cep290¹ mutants. To test this hypothesis, we expressed CEP290 truncations in cep290¹ mutants individually. Interestingly, we found that overexpression of CEP290-N restored the signal of DZIP1 on the TZ in cep290¹ mutants, but the middle and C-terminal fragments did not (Fig 5D). Remarkably, we observed the return of MKS1 signal in both spermatocytes and spermatids, indicating that overexpression of the N-terminus correspondingly attenuated the defects in TZ formation in cep290¹ mutants (Fig 5E). Accordingly, the BB docking defects in late spermatocytes were also ameliorated after overexpression of CEP290-N (Fig 5F). Notably, the flies with overexpression of CEP290-N were still uncoordinated; this is because that CEP290 has other roles independent of its N-terminal and DZIP1; CEP290 is not only required for the initiation of ciliogenesis and TZ assembly, but also required for TZ maturation and axoneme formation in sensory neurons.

DZIP1 regulates TZ assembly upstream of CBY and Rab8

Lapart and colleagues reported that Drosophila DZIP1 directly interacts with CBY and function upstream of CBY-FAM92 module to regulate the TZ assembly [29]. However, dzip1 mutant has much more severely defective phenotypes than cby or fam92 single mutants. Ciliogenesis is completely blocked in our dzip1 mutants, but it is partially affected in cby or fam92 single mutants [29,39]. These results suggest that defects caused by deletion of DZIP1 cannot be attributed to CBY-FAM92 pathway only, other binding factors of DZIP1 should also be involved.

It has been reported that mammalian DZIP1 interacts with ciliary membrane formation regulator Rab8 [40]. Rab8 is a core regulator of membrane vesicle trafficking whose role in ciliary membrane formation has been extensively studied [9,41]. To determine if DZIP1 interacts with Rab8 in Drosophila, we performed the GST pull-down assay. Interestingly, we found that the N-terminus of DZIP1 interacted with Rab8, while the C-terminus of DZIP1 did not (Fig 6A). Consistent with what has been reported in mammals [40], DZIP1 preferentially interacted with the Rab8^GDP-mimicking mutant Rab8^T22N. To further confirm the interaction, we transiently transfected Drosophila S2 cells with DZIP1-GFP and Rab8-HA. GFP-pulldowns from cell extracts confirmed that DZIP1 did interact with Rab8 (Fig 6C). Therefore, Rab8 is a binding factor of DZIP1 in Drosophila.

Fig 6 — (A) The N-terminus of DZIP1 interacted with Rab8 in a GST pull-down assay, but the C-terminus did not. And DZIP1 preferentially bound to Rab8^T22N. (B) DZIP1 interacted directly with CBY in a GST pull-down assay. (C) DZIP1-GFP and Rab8-HA, DZIP1-GFP, and CBY-HA were coimmunoprecipitated in cultured *Drosophila* S2 cells. (D) Schematics of *cby* and *fam92*. *cby* (c.121delC) are predicted to encode a truncated protein lacking the C-terminal region from aa 41 to the end. *fam92* (c. 4-789Del) deletes a large portion (262 out of 395 amino acids) of FAM92 protein. (E) Localization of MKS1-GFP in spermatocytes in the indicated genetic background (upper panel) and the corresponding quantitative relative fluorescence intensities (lower panel). Compared with any single mutants, MKS1-GFP was significantly reduced in *rab8*¹; *cby* double mutants and in *rab8*¹; *fam92* double mutants. The bars and error bars represent the means and SDs, respectively. n = 50 centrioles over 5 flies. Scale bars, 1 μm. (F) Images of CG6652-GFP signals in late spermatocytes in WT flies, *rab8*¹ mutants, *cby* mutants, *fam92* mutants, *rab8*¹; *cby* double mutants, and *rab8*¹; *fam92* double mutants. Compared to WT, *rab8*¹, *cby*, and *fam92*, the percentage of centriole pairs with abnormally elongated CG6652 signal increased significantly in *cby; rab8* double mutant and in *rab8*¹; *fam92* double mutants. Overexpression of FAM92 rescued the elongated CG6652 phenotype of *fam92*, *rab8*¹ double mutants. n = 2,000 over 10 flies for *rab8* mutants, n = 500 over 5 flies for others. Scale bars, 2 μm. Uncropped immunoblots can be found in S2 Raw Image. Numerical data can be found in the file S1 Data. CBY, Chibby; DZIP1, DAZ interacting zinc finger protein 1; FAM92, Family with sequence similarity 92; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, the hemagglutinin (HA) tag; MKS1, Meckel syndrome type 1; SD, standard deviation; TZ, transition zone; WT, wild-type.

The function of Rab8 in Drosophila ciliogenesis has not been investigated yet. We observed that Rab8 localized to the ciliary base in both auditory neurons and olfactory neurons. And consistent with what is observed in mammals, the Rab8^GTP-mimicking mutant RAB8^Q67L was frequently observed inside cilia (S6A and S6B Fig). Interestingly, DZIP1 and CEP290 were necessary for the localization of Rab8 in both auditory and olfactory cilia (S6C–S6E Fig). However, we were disappointed to find that auditory cilium morphology looked normal in rab8¹ mutants (S6F Fig), supported by the behavior readouts that no defects were observed in larval hearing and touch sensitivity (S6G Fig). However, we did observe that a small proportion of spermatocyte centriole pairs have elongated CG6652-GFP signal in every rab8 mutant fly we checked (Fig 6F). Elongated CG6652 signal in spermatocytes is usually attribute to ciliary cap/TZ defects. So, Rab8 is indeed involved in ciliogenesis in Drosophila, even though the effect is relatively weak, probably due to the redundancy with other factors, as has been reported for Rab8 and Rab10 in mammalian ciliogenesis [42].

Multiple reports indicated that the CBY-FAM92 module facilitate ciliary membrane formation and BB docking [43,44]. Since both CBY-FAM92 module and Rab8 are involved in ciliary membrane formation, we hypothesized that these 2 DZIP1 binding partners may jointly promote early ciliary membrane formation and ciliogenesis in Drosophila. To test our hypothesis, we first confirmed the interaction between CBY and DZIP1 in Drosophila (Fig 6B and 6C). Then, we created a cby deletion mutants (Fig 6D), and then generated the cby; rab8 double mutants. Interestingly, we found that the signal of the TZ component MKS1 was dramatically decreased, whereas the percentage of centrioles with abnormally elongated CG6652 signal was significantly increased in spermatocytes of cby; rab8 double mutant, compared with either cby or rab8 single mutants (Fig 6E and 6F).

Furthermore, as CBY and FAM92 function in a same pathway to regulate ciliogenesis. The TZ localization of CBY and FAM92 is interdependent, and fam92 mutants mimic the phenotype of cby mutants. We wondered whether deletion of rab8 could also aggravate the phenotype of fam92 mutants. Then, we created fam92 deletion mutants and generated the fam92; rab8 double mutants. Indeed, as expected, MKS1 signal decreased dramatically, and the percentage of centrioles with abnormally elongated CG6652 signal increased significantly in spermatocytes of fam92; rab8 double mutant (Fig 6E and 6F).

All these results strongly indicated that CBY and Rab8 do function downstream of DZIP1 and jointly contribute to TZ formation.

Discussion

Ciliogenesis begins with the BB docking, followed by the subsequent formation of the early ciliary shaft membrane and assembly of the TZ, which together form the ciliary bud. However, how early ciliary membrane formation coordinates with TZ assembly is still not clear. In this work, we link the TZ core component CEP290 to the DZIP1-CBY/Rab8 ciliary membrane formation module and uncover a novel function of CEP290 in coordinating early ciliary membrane formation and TZ assembly. We propose that the N-terminus of CEP290 recruits DZIP1, which then recruits Rab8 and CBY to promote early ciliary membrane formation (Fig 7), whereas the C-terminus of CEP290 associates with axonemes and regulates TZ elongation and/or maturation. Our observation that TZ assembly is completely blocked in dzip1 mutants suggests that early ciliary membrane formation is a prerequisite for TZ assembly.

Fig 7 — Proposed model for the initiation of TZ assembly based on data from *Drosophila* spermatocytes. Thin fibers with unknown components were observed to mediate the early interaction between the BB and the PM in *Drosophila* spermatocytes [50]. CEP290 localizes at the distal end of centriole, with its C-terminus toward the centriole microtubules. After the BB migrates to the membrane, the N-terminus of CEP290 binds to the membrane and recruits DZIP1, which then recruits CBY and Rab8. CBY together with FAM92 to remodel the membrane and facilitate Rab8-mediated ciliary membrane formation, and then the TZ assembly begins. In *cep290*^ΔC mutant, the remaining N-terminus of CEP290 is functional; therefore, the transition zone assembly is initiated. In *cep290*¹ and *dzip1* mutant, ciliogenesis is blocked at the stage of early ciliary membrane formation. BB, basal body; CBY, Chibby; CEP290, centrosomal protein 290; DZIP1, DAZ interacting zinc finger protein 1; FAM92, family with sequence similarity 92; PM, plasma membrane; TZ, transition zone.

In agreement with previous reports [24,27], TZs fail to form in sensory cilia in cep290^ΔC mutants, but they still form in spermatocyte cilia, suggesting that CEP290 has a tissue-specific functional difference in TZ assembly of sensory cilia and spermatocyte cilia, 2 types of ciliated cells in Drosophila. This difference may be correlated with the significant reduction of DZIP1 signal in sensory cilia but not in spermatocyte cilia in cep290^ΔC mutants (Fig 5A and 5B). It has been reported that ciliary base structures are different in sensory neurons and spermatocytes [27]. Our SIM analysis of the TZ protein localization showed that there is an obvious gap, where DZIP1 is located, between TF and TZ core components in sensory neurons, but no gap exists between TF and TZ core components in spermatocytes (Fig 4D and 4E). All these structural differences may contribute to the tissue-specific functional difference of CEP290 in Drosophila. In addition, the TZ in Drosophila sensory neurons are longer than most cells and are suggested to be homologous to the specialized TZ, “connecting cilium” [45]. It is also possible that CEP290 plays a specific role in the formation of this kind of TZ. Interestingly, mammalian CEP290 may also have a specific role in the formation of “connecting cilium” of photoreceptors. In cep290 null mice, photoreceptor cells lack cilia, while ependymal cells retain cilia [46]. In CEP290 patients, cilia defects are often evident in photoreceptors [26,47]. Therefore, there may be an evolutionary conservative mechanism of CEP290 in the formation of “connecting cilium” which is worthy of further investigation.

From our TEM analysis, we observed that BBs are attached/very close to the plasma membrane in cep290¹ mutant, similar to what Jana and colleagues has proposed [27]. However, technically, it is very difficult to determine whether BBs are docked or just apposed to the plasma membrane. In mammalian cells, the emerging model of the BB is as follows: Myo-Va–associated preciliary vesicles dock to distal appendages of the mother centriole, fuse into a large ciliary vesicle, and then the large ciliary vesicle associated with the BB migrates to and fuses with the plasma membrane [9]. However, to date, no evidence indicates that this mechanism is conserved in invertebrates. In Drosophila, the centriole pericentrin-like protein (PLP) regulates the migration of centriole/BB [48,49]. But how the centriole attaches to the plasma membrane remains unclear. Recently, thin fibers with unknown components were observed to mediate the early interaction between the BB and the plasma membrane in Drosophila spermatocytes [50]. In sensory neurons, there are Drosophila-specific structures called dense fibers that connect the proximal centriole (daughter centriole) to the membrane [51,52]. It is likely that those thin fibers in spermatocytes and dense fibers in sensory neurons are involved in the initial BB membrane docking. More work will be needed to understand the mechanisms of BB docking and the role of CEP290 in BB docking in Drosophila.

By using the simple Drosophila model, we demonstrated that Rab8 and CBY-FAM92 module function downstream of DZIP1 and jointly regulate TZ assembly initiation. The fact that rab8; cby double mutants and rab8; fam92 double mutants show synthetic defects suggests that they have synergetic roles in ciliogenesis. It is tempting to speculate that CBY-FAM92 module promotes membrane remodeling and then facilitate the Rab8 and its yet unknown compensatory mechanism regulating the ciliary membrane formation. The data available so far suggest that the function of DZIP1-Rab8 module and DZIP1-CBY-FAM92 module in ciliogenesis is largely conserved from Drosophila to mammals [53]. The interaction between DZIP1 and Rab8, between DZIP1/DZIP1L and CBY, and between CBY and FAM92 are conserved [36,37,40,43], and all of them have been implicated in ciliogenesis. Similar to what we observed in Drosophila, Dzip1 or Dzip1l knockout (KO) mice have much more severe defects than Cby1 KO mice or Fam92a KO mice, supporting the notion that CBY-FAM92 is not the solely downstream pathway of DZIP1 or DZIP1L [35,36,44,54]. However, there are 2 DZIP1 orthologs (DZIP1 and DZIP1L), 2 FAM92 orthologs (FAM92A and FAM92B), and 3 CBY orthologs in vertebrates, the existence of so many paralogs makes it very complicated to study their genetic relationship.

For the first time, using the Drosophila model, we demonstrated that TZ core protein CEP290 directly interacts with DZIP1 and controls TZ assembly initiation by regulating DZIP1-mediated early ciliary membrane formation. It has been reported that mammalian DZIP1/DZIP1L interacts with Rab8 and CBY [36,37,40], and mammalian DZIP1L is required for ciliary bud formation and TZ assembly. Mother centrioles with docked vesicles but no ciliary buds have been observed in Dzip1l mutant mice [36]. Thus, the function of DZIP1/DZIP1L in early ciliary membrane formation is likely conserved in mammals [53]. Although there is no reports connecting CEP290 to DZIP1 in mammals yet, it has been suggested that mammalian CEP290 is involved in ciliary vesicle formation [55], and defects in early ciliary membrane formation have been observed in fibroblasts of CEP290-deficient patients [47]. In addition, our preliminary data show that human CEP290 interacts with DZIP1L (S7 Fig). Therefore, it is highly possible that the function of CEP290-DZIP1 module in TZ assembly initiation is conserved in mammals. It will be interesting to investigate and confirm our overall model for TZ assembly initiation in mammalian cells.

Methods

Fly stocks

The fly strains used in the study are as follows: w¹¹¹⁸ (FBal0018186), elav-GAL4 (BDSC 458), unc² (BDSC 7424), rab8¹ (BDSC 26173), Df(3L)ED228 (BDSC 8086), DJ-GFP (BDSC 5417), UASp-YFP-Rab8^T22N (BDSC 9780), UASp-YFP-Rab8^Q67L (BDSC 9781), and UASp-YFP-Rab8 (BDSC 9782); CG6652-GFP, UNC-GFP, MKS1-GFP and MKS6-GFP were provided by Dr. Bénédicte Durand; nompC-Gal4; UAS-GFP was provided by Dr. Wei Zhang. The flies were raised on standard media at 25°C.

Generation of transgenic flies

Transgenic lines were generated by the Core Facility of Drosophila Resources and Technology, Shanghai Institute of Biochemistry and Cell Biology (SIBCB), Chinese Academy of Sciences (CAS). To construct the FBF1-GFP, CBY-GFP, and DZIP1-GFP plasmids, the entire coding sequences (CDSs) with their promoter regions were separately cloned and inserted into the HindIII and BamHI sites of PJFRC2 vectors using a one-step cloning method. To construct the UAS-DZIP1-GFP and UAS-CEP290-GFP plasmids, we cloned the entire CDSs into the BglII and BamHI sites of PJFRC2 vectors. The DILA-GFP, truncated CEP290, and truncated CEP290-GFP plasmids were prepared similarly. We first subcloned the ubiquitin promoter region from pWUM6 into the HindIII and BglII sites of PJFRC2 vector or PJFRC2-APEX vector, and then individually cloned each gene cDNA sequence into the NotI and BamHI sites of the vector. Transgenes of CEP290, DILA, and UNC were inserted at the attP site of the 25C6 locus on chromosome 2, and transgenes of FBF1 and DZIP1 were inserted at the attP site of 75B1 locus on chromosome 3. All CDS fragments were amplified from cDNA reverse transcribed from total RNA. The primers used are listed in S1 Table.

Generation of deletion mutants in Drosophila

The CRISPR/Cas9 system was used to generate the deletion mutants. Briefly, CRISPR targeting sites were designed using Target Finder (http://tools.flycrispr.molbio.wisc.edu/targetFinder). Customized single-guide RNA (gRNA) oligos were synthesized and then cloned into the BbsI site of the pEASY-PU6-BbsI-chiRNA vector. All the constructed plasmids were then injected into vasa-Cas9 embryos by the Core Facility of Drosophila Resources and Technology, SIBCB, CAS following a standard protocol. The mutant fly lines were screened via PCR. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the transcription level. The rp49 gene was used as an internal reference. The primers used are listed in S1 Table.

Immunofluorescence

For staining of sensory cilia or testes, pupae, or juveniles were collected 36–48 h after puparium formation (APF). The collected flies were dissected and washed with 1× PBS. The dissected tissues were then placed on slides and squashed with coverslips. The slides were submerged in liquid nitrogen for 1 min, after which coverslips were removed. After incubation in cold methanol (−20°C) for 15 min followed by cold acetone (−20°C) for 10 min, the slides were washed 2 times for 10 min each in 1× PBS at room temperature, and then blocked for at least 1 h in blocking buffer (0.3% Triton X-100, 3% bovine serum albumen in PBS) at room temperature and stained with primary antibodies overnight in a moisture chamber at −4°C. After washing with 1× PBS, the slides were incubated with secondary antibodies for 3 h at room temperature.

Cell membrane staining

Testes from juvenile flies were dissected in PBS and placed on slides. The testes were stained in CellMask Deep Red (Invitrogen, Carlsbad, CA, USA) solution (40 μg/mL) for 5 min at room temperature.

Antibodies

Rabbit antibodies against fly DZIP1 (aa 451–737) and CEP290 (aa 292–541) were produced by YOUKE Biotech (Shanghai, China). Mouse anti-GFP (1:200, 11814460001, Roche, Basel, Switzerland), rabbit anti-GFP (1:500, ab290, Abcam), mouse anti-γ-tubulin (1:500, T5326, Sigma-Aldrich), and mouse anti-21A6 (1:200, AB528449, DSHB) were also used. The secondary antibodies (1:1000 dilution) were goat anti-mouse Alexa Fluor 488/594 or goat anti-rabbit Alexa Fluor 488/594.

Microscopy and image analysis

Most slides were visualized using a fluorescence microscope (Nikon Ti, Tokyo, Japan) or a confocal microscope (Olympus FV1000a, Tokyo, Japan) with a 100 × (1.4 NA) oil-immersion objective. SIM images were taken using a Delta Vision OMX SR (GE Healthcare, Buckinghamshire, United Kingdom) with a 60 × (1.42 NA) oil-immersion objective. All data analysis was performed using ImageJ (National Institutes of Health, Bethesda, USA). For quantification of signal intensity of TZ proteins, the fluorescence intensity is calculated by subtracting the sum of background signals of the same size from the sum of the signals in the region of interest, n = 50 centrioles or BBs over 5 flies.

Transmission electron microscopy

Testis and antenna samples were prepared according to published protocols. Briefly, tissues were fixed in 2.5% glutaraldehyde buffer for at least 24 h and post fixed in OsO₄. The samples were dehydrated using a diluted ethanol series and embedded in epoxy resin. Ultrathin sections (approximately 70 nm) were cut using an Ultracut S ultramicrotome (Zeiss, Jena, Germany). The samples were stained with uranyl acetate and lead citrate. Electron microscopy observations were made with Hitachi H-7650 transmission electron microscope (Tokyo, Japan) at 80 kV.

Male fertility test

Single newly enclosed males were crossed with a virgin w¹¹¹⁸ females for 3 days. The fertility ratio was quantified, and the progeny were counted.

Negative geotaxis assay

For the adult climbing assay, 10 flies aged 3 to 5 d were placed in a graduated cylinder. At the beginning of each test, the flies were gently tapped to the bottom of the cylinder. The flies that climbed above the 8-cm height mark in 10 s were then counted, and counting was repeated 10 times. The data for each genotype are the averages of 5 biological replicates.

Larval touch assay

The larval touch assay was performed as previously described [52]. A single third instar larva was gently touched on its thoracic segments with a slim eyelash during bouts of linear locomotion on an agar plate. A score was assigned according to the response of the larva: A score of 0 indicated that the larva did not respond and continued to crawl, a score of 1 indicated that the larva hesitated or immediately stopped, a score of 2 indicated that the larva anteriorly retracted with or without anterior turns, a score of 3 indicated that the larva retracted with 1 full wave of the body, and a score of 4 indicated that the larva retracted with 2 or more full waves of the body. The score for each larva was determined 5 times, and the results were summed. In each group, 10 larvae were tested, and there were 5 groups for each genotype.

Larval hearing assay

The larval hearing assay was processed as previously described [56]. In each assay, 5 third instar larvae were placed on an agar plate. Larvae were observed for contraction responses before and during a tone of 1k Hz at 30 s intervals, repeated 5 times. Five groups were tested for each genotype. The data are presented as box plots.

Yeast two-hybrid assay

DZIP1 was introduced into the pGBKT7 vector (Clontech, Mountain View, CA, USA), and CEP290-N (aa 1–887) and CEP290-ΔN (aa 888–1978) were individually inserted into the pGADT7 vector (Clontech). The constructs were subsequently transferred into strain AH109 (Takara Bio, Tokyo, Japan) by the LiCl-polyethylene glycol method. Yeasts were grown on SD-Leu-Trp plates at 30°C. After 3 to 4 d of incubation, positive colonies were tested on SD-Ade-Leu-Trp-His plates and incubated for 5 d at 30°C.

Immunoprecipitation

To construct the DZIP1-GFP, CEP290-N-GFP (aa 1–878), DZIP1-HA, CBY-HA, and Rab8-HA plasmids, we cloned the CDSs into the Drosophila Gateway vector PAWG or PAWH, respectively. GFP was cloned into pAWM as a control. One microgram of total DNA was transfected into S2 cells or HEK293 cells in a 60-mm plate with Effectene (Qiagen, Duesseldorf, Germany). After 48 h, the cells were lysed in ice-cold Co-IP lysis buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, 1× protease inhibitor) at 4°C for 45 min and centrifuged at 20,000 × g for 10 min at 4°C. The supernatants were added to anti-GFP nanobody agarose beads (Chromotek, Planegg-Martinsried, Germany), and the beads were tumbled end over end for 1 h at 4°C. After western blotting, the polyvinylidene fluoride (PVDF) membrane was incubated with anti-GFP antibody (Roche) and anti-HA antibody (Invitrogen) or anti-Flag antibody (Sigma). Full scans of the western blots are presented in S1 Raw Image.

GST pull-down assay

To generate the GST-DZIP1-N (aa 1–293), GST-DZIP1-C (aa 294–737), His-CEP290-N (aa 1–887), His-CEP290-ΔN (aa 888–1978), His-CBY, His-Rab8, His-Rab8^T22N, and His-Rab8^Q67L plasmids, we cloned the CDSs into the pET28a or pGEX-4 T-1 vector. The constructs were transformed into BL21 (DE3) E. coli. The proteins were expressed and purified using His resin or Glutathione Sepharose. The purified His-CEP290-N, His-CEP290-ΔN, His-CBY, His-Rab8, His-Rab8^T22N, and His-Rab8^Q67L proteins were incubated with either GST, GST-DZIP1-N, or GST-DZIP1-C in binding buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% Triton X-100, 1 mM dithiothreitol, 10% glycerol and protease inhibitors) overnight at 4°C. After western blotting, the PVDF membrane was incubated with anti-His antibodies (Invitrogen) and stained with Ponceau S. Full scans of the western blots are presented in S1 Raw Image.

Statistics

The results are presented in all figures as the means and SDs. Statistical analyses were performed with two-tailed unpaired Student t tests. (GraphPad Prism 5, Version X; La Jolla, CA, USA; ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Supporting information

S1 Fig. Identification and phenotype analysis of cep290 mutants.

(A) Genotyping of cep290¹ mutants by PCR of CEP290 fragment spanning the deletion region using whole fly genomic DNA. The amplification products were 1,754 bp long for w¹¹¹⁸ and 929 bp long for cep290¹ mutants. (B) RT-PCR analysis of the splice isoforms and transcription level of cep290 gene in cep290^ΔC mutants. Amplification of the C-terminal half of cep290 CDS (2662–5937) did not show other splice forms caused by the deletion mutation. Semiquantitative RT-PCR analysis of the transcription level of cep290 CDS using primer F2 and R1 showed that the transcription level was reduced in cep290^ΔC mutant, probably due to nonsense-mediated mRNA decay. Housekeeping gene rp49 was used as the internal control. (C) cDNA sequencing verification of cep290^ΔC mutant. (D) Upper panel: Immunofluorescence staining with an anti-CEP290 N (aa292-541) antibody confirmed that the CEP290 N-terminal signal was reduced in cep290^ΔC mutants but completely lost in cep290¹ mutants. The corresponding relative fluorescence intensity was quantitatively displayed in lower panel. The bars and error bars represent the means and SDs, respectively. n = 50 centrioles over 5 flies. Scale bars, 1 μm. (E) Cilia-related behavior analysis of cep290 mutants. Both cep290^ΔC and cep290¹ mutant flies have severe defects in hearing (left panel) and touch sensitivity (right panel). Expression of CEP290 rescued these defects. For the hearing assay, 5 larvae as a group, and at least 5 groups of flies were tested. For the touch sensitivity assay, n = 50. (F) Testes of WT flies and cep290 mutants. The mitochondrial protein DJ was used to label sperm cysts. Compared to those in WT flies and cep290^ΔC mutants, sperm cysts in cep290¹ mutants were severely defective in elongation. The arrow indicates that the cysts failed to elongate. Bar, 200 μm. (G) Male fertility assay in WT flies and cep290 mutants. We first rescued the severely uncoordinated phenotype of cep290 mutants by expressing CEP290 driven by elav-GAL4. The percentage of fertile males was then quantified, and the number of offspring was calculated. cep290¹ males were completely infertile, while more than 65% of cep290^ΔC males were fertile. n = 50. (H) EM images of testis cross sections from WT flies and cep290¹ mutants. There were 64 spermatids in each cyst in WT flies, whereas axonemes were almost completely lost in cep290¹ mutants. The arrows show defective axonemes. (I) EM images of longitudinal sections of auditory cilia in WT flies and cep290 mutants. Bars, 200 nm. Numerical data for panels D, E, and G can be found in the file S1 Data. CDS, coding sequence; CEP290, centrosomal protein 290; DBB, distal basal body; DJ, Donjuan; EM, electron microscope; PBB, proximal basal body; rp49, ribosomal protein 49; RT-PCR, reverse transcription-polymerase chain reaction; TZ, transition zone; WT, wild-type.

(TIF)

Click here for additional data file.^{(31.8MB, tif)}

S2 Fig. DZIP1 is the ortholog of mammalian DZIP1 and DZIP1L in Drosophila.

(A) DZIP1 is evolutionarily conserved in Holozoans. There is only 1 DZIP1 gene in invertebrates, while there are 2 genes in vertebrates, DZIP1 and DZIP1L, possibly because of gene duplication. (B) Phylogenetic tree of DZIP1. Drosophila DZIP1 is orthologous to both DZIP1 and DZIP1L. (C) Schematic representation of the protein structure of Drosophila DZIP1 and human DZIP1 and DZIP1L. All share highly conserved domain that includes Dzip_like-N and ZnF_C2H2. (D) Table showing the sequence similarity and identity between Drosophila DZIP1 and human DZIP1 and DZIP1L.

(TIF)

Click here for additional data file.^{(26.1MB, tif)}

S3 Fig. CEP290–DZIP1 interaction analysis.

(A) DZIP1 did not interact with CEP290-ΔN (Δ aa 1–887) in the GST pull-down assay. (B) The GST pull-down assay was used to narrow down the interaction region between CEP290 and DZIP1. DZIP1-N (aa 1–293) interacts with both CEP290 N (aa 401–650) and CEP290 N (aa 651–887), and DZIP1-C (aa 294–737) interacts with CEP290 N (aa 651–887). Uncropped immunoblots can be found in S2 Raw Image. CEP290, centrosomal protein 290; DZIP1, DAZ interacting zinc finger protein 1; GST, glutathione S-transferase.

(TIF)

Click here for additional data file.^{(31.4MB, tif)}

S4 Fig. The subcellular localization of DZIP1 in Drosophila.

(A) Subcellular localization of DZIP1 during spermatogenesis in Drosophila. Upper panel: DZIP1-GFP; lower panel: anti-DZIP1. DZIP1-GFP and anti-DZIP1 staining showed similar localization patterns. No DZIP1 was present on centrioles in spermatogonia. DZIP1 began to appear on the tips of centrioles in early spermatocytes. In round spermatids, DZIP1 migrated together with the ring centriole to the tip of flagella. The centrioles/BBs were labeled with γ-Tubulin (red). Bar, 2 μm. (B) DZIP1 localized to the ciliary base in both auditory and olfactory cilia. Ciliary bases were labeled with 21A6 (red). Anti-DZIP1 staining showed a localization pattern similar to that of DZIP1-GFP. Bar, 1 μm. (C) 3D-SIM images of DZIP1 localization in spermatids, DZIP1 colocalized with CBY at the ring centriole, but did not extend like MKS1. Bar, 500 nm. (D) 3D-SIM images of spatial relationship between TF marker FBF1 and TZ core protein MKS1 in various types of cilia. Notably, there is a gap (arrows) between FBF1 and MKS in sensory cilia but not in spermatocyte cilia. Bar, 500 nm. 3D-SIM, three-dimensional structured illumination microscopy; BB, basal body; CBY, Chibby; DZIP1, DAZ interacting zinc finger protein 1; FBF1, Fas binding factor 1; GFP, green fluorescent protein; MKS, Meckel–Gruber syndrome; MKS1, Meckel syndrome type 1; TF, transition fiber; TZ, transition zone.

(TIF)

Click here for additional data file.^{(27.4MB, tif)}

S5 Fig. Identification and phenotype analysis of dzip1 mutants.

(A) Generation of the dzip1 deletion mutant. Schematic of the dzip1 gene. The gRNA target sites are represented by scissors. dzip1^ΔC has a deletion of cDNA from nt 1,069 to 1,529, resulting in a frameshift and a premature stop codon and leading to the loss of the C-terminus. (B) Genotyping of the dzip1 mutant. The sizes of the PCR products are as follows: 1,070 bp in w¹¹¹⁸ flies and 609 bp in dzip1^ΔC flies. (C) Anti-DZIP1 staining confirmed that DZIP1, at least the C-terminus of DZIP1, was completely lost in dzip1^ΔC mutants. Bars, 1 μm. (D) dzip1^ΔC mutant flies showed defects in hearing and touch sensitivity, but expression of DZIP1-GFP driven by its own promoter rescued these defects. The retraction index is shown as the median and interquartile range. Numerical data can be found in the file S1 Data. The bars and error bars represent the means and SDs, respectively. n = 50. (E) nompC-Gal4; UAS-GFP was used to mark auditory cilia. Cilia completely disappeared in dzip1^ΔC mutants. (F) EM images of cross sections of cilia showing that no axonemes existed in dzip1^ΔC mutants. The black arrows indicate ciliary axonemes in WT flies. Bars, 200 nm. DZIP1, DAZ interacting zinc finger protein 1; EM, electron microscope; GFP, green fluorescent protein; gRNA, guide RNA.

(TIF)

Click here for additional data file.^{(31.2MB, tif)}

S6 Fig. Subcellular localization of Rab8 in sensory cilia and phenotype of rab8 mutants.

(A) Rab8-GFP and Rab8^Q67L-GFP colocalized with DZIP1 at the ciliary base in auditory cilia and olfactory cilia. Notably, Rab8^Q67L-GFP was frequently observed in cilia. Bar, 1 μm. (B) 3D-SIM images of the colocalization of Rab8^Q67L-GFP and DZIP1 at the base of auditory cilia. Rab8 ^Q67L-GFP surrounded DZIP1 at TZ and expanded into cilia. Bar, 1 μm. (C) DZIP1 was critical for Rab8-GFP localization at the ciliary base of olfactory cilia. Live imaging showed that Rab8-GFP exhibited a clear 2-dot pattern at the ciliary base in WT flies, but the signal was dispersed in dzip1 mutants. Interestingly, these dispersed signals completely disappeared in our immunofluorescence assay, most likely due to fixation. Bars, 5 μm. (D) DZIP1 was required for Rab8 ^Q67L-GFP localization at the ciliary base of auditory cilia. Bar, 1 μm. (E) Rab8-GFP was completely lost from the ciliary base of both auditory cilia and olfactory cilia in cep290¹ mutants, indicating that CEP290 is essential for Rab8 localization. Bar, 1 μm. (F) Cilium morphology was normal in rab8¹ mutants. (G) rab8 mutants exhibited normal hearing responses and normal touch sensitivity. Numerical data can be found in the file S1 Data. 3D-SIM, three-dimensional structured illumination microscopy; DZIP1, DAZ interacting zinc finger protein 1; GFP, green fluorescent protein; TZ, transition zone; WT, wild-type.

(TIF)

Click here for additional data file.^{(27.4MB, tif)}

S7 Fig. Human CEP290 N-terminal was coimmunoprecipitated with DZIP1L.

The interaction between human CEP290 and DZIP1L was analyzed using an immunoprecipitation assay. Human CEP290-N-GFP and Flag-DZIP1L were transiently transfected into HEK293 cells; 48 h later, cells were lysed and subjected to Co-IP using GFP-trap beads. Uncropped immunoblots can be found in S2 Raw Image. CEP290, centrosomal protein 290; Co-IP, coimmunoprecipitation; GFP, green fluorescent protein.

(TIF)

Click here for additional data file.^{(25.5MB, tif)}

S1 Table. Primers were used in this paper.

(TIF)

Click here for additional data file.^{(25.5MB, tif)}

S1 Data. Excel file of numerical data.

(XLSX)

Click here for additional data file.^{(83.7KB, xlsx)}

S1 Raw Image. Uncropped images of immunofluorescence staining of sensory cilia.

(TIF)

Click here for additional data file.^{(28.5MB, tif)}

S2 Raw Image. Full scans of western blots.

(TIF)

Click here for additional data file.^{(26.6MB, tif)}

Acknowledgments

We thank Dr. Bénédicte Durand from Université Claude Bernard Lyon 1 and Dr. Wei Zhang from Tsinghua University for strains. We thank core facilities of Drosophila Resource and Technology (SIBCB, CAS), confocal imaging core facilities (SIPPE, CAS), and EM core facilities (SIPPE, CAS) for their technical support.

Abbreviations

3D-SIM: three-dimensional structured illumination microscopy
APF: after puparium formation
BB: basal body
BBS: Bardet–Biedl syndrome
CDS: coding sequence
CEP290: centrosomal protein 290
Co-IP: coimmunoprecipitation
CV: ciliary vesicle
CBY: Chibby
DA: distal appendage
DILA: Dilatory
DZIP1: DAZ interacting zinc finger protein 1
EHD1: EH domain-containing protein 1
EM: electron microscope
FBF1: Fas binding factor 1
GFP: green fluorescent protein
gRNA: guide RNA
GST: glutathione S-transferase
IFT: intraflagellar transport
JBTS: Joubert syndrome
LCA: Leber Congenital Amaurosis
MKS: Meckel–Gruber syndrome
NPHP: Nephronophthisis
PCV: preciliary vesicle
PLP: pericentrin-like protein
PVDF: polyvinylidene fluoride
RT-PCR: reverse transcription-polymerase chain reaction
SLS: Senior–Loken syndrome
TEM: transmission electron microscopy
TF: transition fiber
TZ: transition zone
UNC: Uncoordinated
WT: wild-type

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by National Natural Science Foundation of China (http://www.nsfc.gov.cn/), Grant 31871357, 32070692 and 31671549 to Q.W. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Mitchison HM, Valente EM. Motile and non-motile cilia in human pathology: from function to phenotypes. J Pathol. 2017;241:294–309. 10.1002/path.4843 [DOI] [PubMed] [Google Scholar]
2.Anvarian Z, Mykytyn K, Mukhopadhyay S, Pedersen LB, Christensen ST. Cellular signalling by primary cilia in development, organ function and disease. Nat Rev Nephrol. 2019;15:199–219. 10.1038/s41581-019-0116-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Nachury MV, Mick DU. Establishing and regulating the composition of cilia for signal transduction. Nat Rev Mol Cell Biol. 2019;20:389–405. 10.1038/s41580-019-0116-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Shinohara K, Hamada H. Cilia in Left-Right Symmetry Breaking. Cold Spring Harb Perspect Biol. 2017;9. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. N Engl J Med. 2011;364:1533–43. 10.1056/NEJMra1010172 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Reiter JF, Leroux MR. Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol. 2017;18:533–47. 10.1038/nrm.2017.60 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Sorokin S. Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Biol. 1962;15:363–77. 10.1083/jcb.15.2.363 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Sorokin SP. Reconstructions of Centriole Formation and Ciliogenesis in Mammalian Lungs. J Cell Sci. 1968;3:207 [DOI] [PubMed] [Google Scholar]
9.Wu CT, Chen HY, Tang TK. Myosin-Va is required for preciliary vesicle transportation to the mother centriole during ciliogenesis. Nat Cell Biol. 2018;20:175–85. 10.1038/s41556-017-0018-7 [DOI] [PubMed] [Google Scholar]
10.Lu Q, Insinna C, Ott C, Stauffer J, Pintado PA, Rahajeng J, et al. Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nat Cell Biol. 2015;17:228–40. 10.1038/ncb3109 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Knödler A, Feng S, Zhang J, Zhang X, Das A, et al. Coordination of Rab8 and Rab11 in primary ciliogenesis. Proc Natl Acad Sci U S A. 2010;107:6346–51. 10.1073/pnas.1002401107 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Westlake CJ, Baye LM, Nachury MV, Wright KJ, Ervin KE, et al. Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome. Proc Natl Acad Sci U S A. 2011;108:2759–64. 10.1073/pnas.1018823108 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Gonçalves J, Pelletier L. The Ciliary Transition Zone: Finding the Pieces and Assembling the Gate. Mol Cells. 2017;40:243–53. 10.14348/molcells.2017.0054 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Garcia-Gonzalo FR, Reiter JF. Open Sesame: How Transition Fibers and the Transition Zone Control Ciliary Composition. Cold Spring Harb Perspect Biol. 2017;9:a028134 10.1101/cshperspect.a028134 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Ishikawa H, Marshall WF. Ciliogenesis: building the cell's antenna. Nat Rev Mol Cell Biol. 2011;12:222–34. 10.1038/nrm3085 [DOI] [PubMed] [Google Scholar]
16.Reiter JF, Blacque OE, Leroux MR. The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO Rep. 2012;13:608–18. 10.1038/embor.2012.73 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Czarnecki PG, Shah JV. The ciliary transition zone: from morphology and molecules to medicine. Trends Cell Biol. 2012;22:201–10. 10.1016/j.tcb.2012.02.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Gilula NB, Satir P. The ciliary necklace. A ciliary membrane specialization The Journal of cell biology. 1972;53:494–509. 10.1083/jcb.53.2.494 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Sang L, Miller JJ, Corbit KC, Giles RH, Brauer MJ, et al. Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways. Cell. 2011;145:513–28. 10.1016/j.cell.2011.04.019 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Schouteden C, Serwas D, Palfy M, Dammermann A. The ciliary transition zone functions in cell adhesion but is dispensable for axoneme assembly in C. elegans. J Cell Biol. 2015;210:35–44. 10.1083/jcb.201501013 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Li C, Jensen VL, Park K, Kennedy J, Garcia-Gonzalo FR, et al. MKS5 and CEP290 Dependent Assembly Pathway of the Ciliary Transition Zone. PLoS Biol. 2016;14:e1002416 10.1371/journal.pbio.1002416 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Williams CL, Li C, Kida K, Inglis PN, Mohan S, et al. MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J Cell Biol. 2011;192:1023–41. 10.1083/jcb.201012116 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Craige B, Tsao C-C, Diener DR, Hou Y, Lechtreck K-F, et al. CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. J Cell Biol. 2010;190:927–40. 10.1083/jcb.201006105 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Basiri ML, Ha A, Chadha A, Clark NM, Polyanovsky A, et al. A migrating ciliary gate compartmentalizes the site of axoneme assembly in Drosophila spermatids. Curr Biol. 2014;24:2622–31. 10.1016/j.cub.2014.09.047 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Garcia-Gonzalo FR, Corbit KC, Sirerol-Piquer MS, Ramaswami G, Otto EA, et al. A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nat Genet. 2011;43:776–84. 10.1038/ng.891 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Coppieters F, Lefever S, Leroy BP, De Baere E. CEP290, a gene with many faces: mutation overview and presentation of CEP290base. Hum Mutat. 2010;31:1097–108. 10.1002/humu.21337 [DOI] [PubMed] [Google Scholar]
27.Jana SC, Mendonca S, Machado P, Werner S, Rocha J, et al. Differential regulation of transition zone and centriole proteins contributes to ciliary base diversity. Nat Cell Biol. 2018;20:928–41. 10.1038/s41556-018-0132-1 [DOI] [PubMed] [Google Scholar]
28.Drivas TG, Holzbaur EL, Bennett J. Disruption of CEP290 microtubule/membrane-binding domains causes retinal degeneration. J Clin Invest. 2013;123:4525–39. 10.1172/JCI69448 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Lapart J-A, Gottardo M, Cortier E, Duteyrat J-L, Augière C, et al. Dzip1 and Fam92 form a ciliary transition zone complex with cell type specific roles in Drosophila. elife. 2019;8:e49307 10.7554/eLife.49307 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Riparbelli MG, Callaini G, Megraw TL. Assembly and persistence of primary cilia in dividing Drosophila spermatocytes. Dev Cell. 2012;23:425–32. 10.1016/j.devcel.2012.05.024 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Gottardo M, Callaini G, Riparbelli MG. The cilium-like region of the Drosophila spermatocyte: an emerging flagellum? J Cell Sci. 2013;126:5441–52. 10.1242/jcs.136523 [DOI] [PubMed] [Google Scholar]
32.Lattao R, Kovács L, Glover DM. The Centrioles, Centrosomes, Basal Bodies, and Cilia of Drosophila melanogaster. Genetics. 2017;206:33–53. 10.1534/genetics.116.198168 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Jana SC, Bettencourt-Dias M, Durand B, Megraw TL. Drosophila melanogaster as a model for basal body research. Cilia. 2016;5:22–2. 10.1186/s13630-016-0041-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Vieillard J, Paschaki M, Duteyrat JL, Augiere C, Cortier E, et al. Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells. J Cell Biol. 2016;214:875–89. 10.1083/jcb.201603086 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Wang C, Low W-C, Liu A, Wang B. Centrosomal Protein DZIP1 Regulates Hedgehog Signaling by Promoting Cytoplasmic Retention of Transcription Factor GLI3 and Affecting Ciliogenesis. J Biol Chem. 2013;288:29518–29. 10.1074/jbc.M113.492066 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Wang C, Li J, Takemaru KI, Jiang X, Xu G, et al. Centrosomal protein Dzip1l binds Cby, promotes ciliary bud formation, and acts redundantly with Bromi to regulate ciliogenesis in the mouse. Development. 2018;145 (6). [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Breslow DK, Hoogendoorn S, Kopp AR, Morgens DW, Vu BK, et al. A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies. Nat Genet. 2018;50:460–71. 10.1038/s41588-018-0054-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Lu H, Galeano MCR, Ott E, Kaeslin G, Kausalya PJ, et al. Mutations in DZIP1L, which encodes a ciliary-transition-zone protein, cause autosomal recessive polycystic kidney disease. Nat Genet. 2017;49:1025–34. 10.1038/ng.3871 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Enjolras C, Thomas J, Chhin B, Cortier E, Duteyrat JL, et al. Drosophila chibby is required for basal body formation and ciliogenesis but not for Wg signaling. J Cell Biol. 2012;197:313–25. 10.1083/jcb.201109148 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Zhang B, Zhang T, Wang G, Wang G, Chi W, et al. GSK3beta-Dzip1-Rab8 cascade regulates ciliogenesis after mitosis. PLoS Biol. 2015;13:e1002129 10.1371/journal.pbio.1002129 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Nachury MV, Loktev AV, Zhang Q, Westlake CJ, Peranen J, et al. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell. 2007;129:1201–13. 10.1016/j.cell.2007.03.053 [DOI] [PubMed] [Google Scholar]
42.Sato T, Iwano T, Kunii M, Matsuda S, Mizuguchi R, et al. Rab8a and Rab8b are essential for several apical transport pathways but insufficient for ciliogenesis. J Cell Sci. 2014;127:422–31. 10.1242/jcs.136903 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Li FQ, Chen X, Fisher C, Siller SS, Zelikman K, et al. BAR Domain-Containing FAM92 Proteins Interact with Chibby1 To Facilitate Ciliogenesis. Mol Cell Biol. 2016;36:2668–80. 10.1128/MCB.00160-16 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Burke MC, Li FQ, Cyge B, Arashiro T, Brechbuhl HM, et al. Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation. J Cell Biol. 2014;207:123–37. 10.1083/jcb.201406140 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Malicki J, Avidor-Reiss T. From the cytoplasm into the cilium: bon voyage. Organogenesis. 2014;10:138–57. 10.4161/org.29055 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Rachel RA, Yamamoto EA, Dewanjee MK, May-Simera HL, Sergeev YV, et al. CEP290 alleles in mice disrupt tissue-specific cilia biogenesis and recapitulate features of syndromic ciliopathies. Hum Mol Genet. 2015;24:3775–91. 10.1093/hmg/ddv123 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Shimada H, Lu Q, Insinna-Kettenhofen C, Nagashima K, English MA, et al. In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations. Cell Rep. 2017;20:384–96. 10.1016/j.celrep.2017.06.045 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Galletta BJ, Guillen RX, Fagerstrom CJ, Brownlee CW, Lerit DA, et al. Drosophila pericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies. Mol Biol Cell. 2014;25:2682–94. 10.1091/mbc.E13-10-0617 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Roque H, Saurya S, Pratt MB, Johnson E, Raff JW. Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation. PLoS Genet. 2018;14:e1007198–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Gottardo M, Persico V, Callaini G, Riparbelli MG. The "transition zone" of the cilium-like regions in the Drosophila spermatocytes and the role of the C-tubule in axoneme assembly. Exp Cell Res. 2018;371:262–8. 10.1016/j.yexcr.2018.08.020 [DOI] [PubMed] [Google Scholar]
51.Vieillard J, Duteyrat JL, Cortier E, Durand B. Imaging cilia in Drosophila melanogaster. Methods Cell Biol. 2015;127:279–302. 10.1016/bs.mcb.2014.12.009 [DOI] [PubMed] [Google Scholar]
52.Chen JV, Kao LR, Jana SC, Sivan-Loukianova E, Mendonca S, et al. Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila. J Cell Biol. 2015;211:435–53. 10.1083/jcb.201502032 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Lapart JA, Billon A, Duteyrat JL, Thomas J, Durand B. Role of DZIP1-CBY-FAM92 transition zone complex in the basal body to membrane attachment and ciliary budding. Biochem Soc Trans. 2020;48:1067–75. 10.1042/BST20191007 [DOI] [PubMed] [Google Scholar]
54.Voronina VA, Takemaru K-I, Treuting P, Love D, Grubb BR, et al. Inactivation of Chibby affects function of motile airway cilia. J Cell Biol. 2009;185:225–33. 10.1083/jcb.200809144 [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Kobayashi T, Kim S, Lin YC, Inoue T, Dynlacht BD. The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly. J Cell Biol. 2014;204:215–29. 10.1083/jcb.201304153 [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Diggle CP, Moore DJ, Mali G, zur Lage P, Ait-Lounis A, et al. HEATR2 plays a conserved role in assembly of the ciliary motile apparatus. PLoS Genet. 2014;10:e1004577–7. 10.1371/journal.pgen.1004577 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS Biol. doi: 10.1371/journal.pbio.3001034.r001

Decision Letter 0

Ines Alvarez-Garcia

15 Jan 2020

Dear Dr Wei,

Thank you for submitting your manuscript entitled "CEP290 is essential for the initiation of transition zone assembly" for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review. Nevertheless, we will need to be convinced by the reviewers that the novelty and significance of the findings is sufficient for publication in the journal. A better characterization of the role of vesicle and membrane assembly, which is a bit confusing in the study at the moment, and which other factor(s) in adition to Rab8 might be implicated in this process might be required.

Please note that before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by Jan 17 2020 11:59PM.

During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Ines

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

Carlyle House, Carlyle Road

Cambridge, CB4 3DN

+44 1223–442810

PLoS Biol. doi: 10.1371/journal.pbio.3001034.r002

Decision Letter 1

Ines Alvarez-Garcia

3 Mar 2020

Dear Dr Wei,

Thank you very much for submitting your manuscript "CEP290 is essential for the initiation of transition zone assembly" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by four independent reviewers.

The reviews of your manuscript are appended below. As you will see, the reviewers find the work very nicely done and potentially interesting, but they also raise concerns regarding the novelty of certain aspects of the findings

They each suggests experiments to confirm and extend some of the findings, like the role of Rab8 in TZ assembly, the interaction between CEP290 and DZIP1/Rab8 or exploring thoroughtly the phenotype of the CEP290 mutants. After discussing the reviews with the academic editor, we think that in general elucidating the Rab8 axis will be necessary for us to consider the manuscript further for publication.

I regret that we cannot accept the current version of the manuscript for publication. We remain interested in your study and we would be willing to consider resubmission of a comprehensively revised version that thoroughly addresses all the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript would be sent for further evaluation by the reviewers.

We appreciate that these requests represent a great deal of extra work, and we are willing to relax our standard revision time to allow you six months to revise your manuscript.We expect to receive your revised manuscript within 6 months.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Related" file type.

*Resubmission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this resubmission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ines

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

Carlyle House, Carlyle Road

Cambridge, CB4 3DN

+44 1223–446970

---------------------------------------------------------------

Reviewers’ comments

Rev. 1:

In this manuscript, Wu et al. investigate the role of Cep290 in ciliogenesis and transition zone (TZ) assembly in Drosophila. By analyzing a novel null mutant of Cep290, they find that the Cep290 N-terminal domain is essential for TZ formation and for persistent membrane association of centrioles in spermatids. These defects are proposed to be due to a newly described interaction between Cep290-N and Dzip1 that is necessary for TZ localization of Dzip1. Lastly, the authors show that Dzip1 cooperates with Cby and Rab8 to promote ciliary membrane formation.

Overall, the data are of high quality and clearly presented. The significance of the findings is partly diminished by a number of studies in Drosophila and other organisms that have explored the roles of Cep290, Dzip1, and Cby in transition zone formation. However, the present work fills in important details (such as the novel connection between Cep290 and Dzip1) and carefully examines ciliary phenotypes in several Drosophila cell types. These results are thus likely to be of strong interest to the cilia field (especially if the points below are addressed). The conceptual advance is somewhat more limited, and thus the significance to a broader audience is less clear. However, based on the overall high quality of the data and the new molecular insights into transition zone formation, I believe a revised manuscript could be a good candidate for publication in PLoS Biology.

Major points:

1. While a variety of experimental approaches are employed to demonstrate binding between Cep290-N and Dzip1, a relatively large fragment of Cep290 is used (aa 1-650). This region has previously been shown to have various important functional properties (homodimerization; binding to Cp110, Pcm1, and Nphp2; membrane binding via a conserved amphipathic helix), and thus it is possible that the various phenotypic defects attributed to loss of Cep290-N are not due to loss of Dzip1 binding per se but due to disruption of one or more of these other functional elements (in fact the phenotypes seen in Cep290-l mutants that are not present in Cep290-deltaC could be due to any elements within aa 1-1385). This concern is partly mitigated by the similarity of phenotypes seen in Cep290-l and Dzip1 mutants, but it would be helpful if the authors can narrow Cep290's Dzip1 binding region more precisely and analyze ciliary phenotypes when Dzip1 binding is more selectively disrupted (e.g. by rescue of Cep290-l with such a targeted Cep290 mutant). At a minimum this issue should be discussed in more detail in the manuscript.

2. A key point in the manuscript is that the N-terminus of Cep290 has key roles in transition zone formation. This conclusion relies on the more severe phenotypes observed in the Cep290-l mutant than the Cep290-deltaC mutant. However, the predicted frameshift produced by the Cep290-deltaC allele is not experimentally verified, and the possibility of alternative splice forms and/or altered transcript stability is not considered. Given the key conclusions based on this mutant, it would be very helpful if the authors could better characterize precisely what form(s) of Cep290 are expressed in this mutant and at what levels relative to wildtype.

Minor points:

1. Is there any evidence from the authors or others indicating that the reported connection between Cep290 and Dzip1 might be conserved in other species? Similarly, can the pathogenic features of any disease-associated CEP290 mutants potentially be attributed to disruption of the Cep290-Dzip1 interaction? Experiments examining these points may be beyond the immediate scope of this paper but could make for an impactful addition; at minimum further discussion of these points would be helpful.

2. There are some confusingly worded sentences and grammatical errors throughout the text (particularly in the last section of the Results).

Rev. 2:

The transition zone is a particular structure of the ciliary base which plays many roles in cilia assembly and function. Defects in TZ assembly lead to life threatening ciliopathies. Cep290 is a core component of the TZ and has been shown to act upstream of the TZ assembly program in many organisms, but it's precise function in Drosophila is only partially investigated and how it recruits other TZ proteins is not clear.

In this manuscript, the authors describe the first Drosophila cep290 complete null mutant and show that Cep290 plays, like in many other organisms, a critical function in the initiation of ciliogenesis and TZ assembly. They demonstrate for the first time that Cep290 can be recruited to the basal body by its N- or C-terminus domains and that Cep290 directly interacts with Drosophila Dzip1. They show that Dzip1 interacts with Cby a key component of TZ assembly in Drosophila and also with Rab8. Their work also investigates if Cby and Rab8 cooperate to build the drosophila TZ.

Whereas the manuscript conclusions on Cep290 are well supported by the observations, the role of Rab8 in TZ assembly is not clearly demonstrated and the authors' conclusion is largely overstated. In particular, the phenotype of the rab8 mutant is weakly documented and does not support their conclusion. To be able to conclude on Rab8 function in TZ assembly in Drosophila, the authors should at least address the following points:

-show the rescue of the phenotype of cby; rab8 double mutant by Rab8-GFP and, even more demonstrating, not by Rab8T22N.

-the sole phenotype described for the cby; rab8 double mutant is a reduction in MKS1-GFP at the TZ? Is this the only phenotype observed for this double mutant? Is there any consequence on behavioral assays? What is the testis phenotype of the rab8 mutant alone and the double cby; rab8 mutant? Is it consistent with a function in TZ assembly? My opinion is that the results on rab8 are much too preliminary to support a role of Rab8 in TZ assembly and actually reduce the impact of the other conclusions that are very interesting.

The other following points also need to be addressed before publication:

Major points :

-The authors nicely show that basal bodies (BB) in cep290 mutants do not build any TZ and are, at best, just apposed to the plasma membrane. This result was already proposed by Jana et al., 2018 using RNAi. However, it is not clear why the authors' conclude that the basal bodies are docked: in particular do the authors observe a thickening of the plasma membrane and connections between the membrane and the BB, as described by Gottardo et al, 2018? How many centrioles were observed by EM? Are they all apposed to the plasma membrane ? On Figure 2G : one of the two basal body does not seem to be docked to the plasma membrane: can this be explained by the level of the section? Do the authors have other sections of this same centriole?

-The authors analyze the distribution of different Cep290 truncated fragments fused to GFP. This analysis is apparently done in WT conditions where the endogenous protein is present, what happens in cep2901 rescue conditions ? Could the difference in the distribution of Cep290 truncated fragments just reveal differences in competition behavior between the truncated and Wt protein?

Minor points :

Overall the IF images of the sensory cilia are of poor resolution. In particular the images of sensory neurons in 1B, 1C, 4F, 5B and D.

Figure 1. The authors only show one example of the distribution of the different Cep290-GFP fragments: is the distribution homogenous between centrioles? Please quantify the length on several centrioles to show the distribution.

Figure 2C : Quantify the fluorescence intensity in cep290deltaC mutant compared to WT: is there any effect of deleting only the C-terminus, as the flies are hypofertile, and taking into account that mecH alleles show defects in TZ integrity in the testes (Basiri et al., 2014).

Figure 4D: it was previously shown in Jana et al, 2018, that Cep290 radially extends from the center to the periphery of the TZ, depending on whether GFP is fused to the N-ter or C-ter of the protein. This should be taken into account in the interpretation of the high resolution imaging observations.

Page 6: the authors propose that expressing the N-ter-Cep290 fragment has a dominant negative effect : are all transgenes inserted on the same platform as this is not explained in the methods section? Thus, in all the experiments based on the expression of these truncated forms, could there be an effect of the expression levels?

Page 7: The authors propose that the C-ter and N-ter domains have an inhibitory effect on their localization and cite Drivas et al. to support their hypothesis. However this latter article shows that overexpressing the N or C-ter domain of human CEP290 leads to increased ciliogenesis in cells. This is completely different to what the authors observe here in their manuscript and is misleading.

Figure 7 and discussion: there is an apparent confusion in the text and in the figure between what the authors call dense fibers on page 20, which in the referred publications correspond to lateral connections of the proximal (daughter) centriole and the dendrite membrane and what is called "thin fibers" described in Gottardo et al. 2018. These are completely different structures that likely have completely different roles and Figure 7 must be corrected to "thin fibers" as described in Gottardo et al. The EM images of this manuscript do not show that these connections are still present in cep290 mutants. In addition, to my knowledge, ciliary vesicles have not been described on Drosophila EM images, thus parts of Figure 7 are speculative.

At many occurrences, the conclusions appear to be overstated:

Page 8, end of second paragraph :

« Probably its N-terminus alone » this is likely overstated. It shows that the N-terminus is required but not sufficient as the C-terminus truncated Cep290mecH mutants do have a TZ gating defects (Basiri et al., 2014).

Page 13: "DZIP1 did not affect the initial docking of the BB": same comment as above for the cep290 mutant. What are the arguments to say that the BB are docked and not only apposed to the plasma membrane?

Page 20: second paragraph: "suggests that they have synergistic roles in early ciliary membrane formation". The manuscript only shows that MKS1 is affected not that there are defects in early ciliary membrane formation.

Last paragraph of the result section: the last sentence is highly speculative and should be removed.

Some of the results obtained for dzip1 mutant confirm recently published results by Lapart et al, 2019. This should be stated in the result or discussion section.

As well, some observations of the cep290 mutants confirm previous published results by Basiri et al., Jana et al. and Lapart et al., and this should be recalled accordingly in the text.

Page 21 "regulating DZIP1 mediated early ciliary membrane": incomplete sentence.

Rev. 3:

In this manuscript, Wu et al. report that the N-terminal region of CEP290 plays an essential role in the initiation of transition zone assembly of the cilia in in neurons and spermatocyte of Drosophila. They demonstrate that the N-terminus of CEP290 directly interacts with DZIP1 and recruits DZIP1 to transition zone, where it recruits CBY and RAB8 to assemble the ciliary membrane. The authors propose that CEP290 coordinates the early ciliary membrane formation and transition zone assembly.

Overall, this work is of importance to the ciliogenesis field because little is known about the regulation of early ciliary assembly events. Requirements for CEP290 in transition zone assembly and function has been investigated in several model systems including Drosophila (Jana et al 2018, Li et al 2016). Importantly, CEP290 has previosuly been reported to act upstream of DZIP1 recruitment - which was not mentioned in this paper. Moreover, associations with Rab8 function have been reported and were for the most part noted in this manuscript with exception a paper in 2008 by Kim et al. Thus, the main findings from this work largely relate to the identification of interactions for CEP290-DZIP1 and DZIP1-CBY/Rab8. However, the interaction between DZIP1 and Rab8 and CBY seem rather weak compared to the input. Another concern I have is that conclusions drawn about TZ structure and protein localization may be over interpreted from the provided SIM imaging. The quality/resolution of these images seems lower than what has been published by others for the TZ in Drosophila.

Additional concerns/questions/suggestions are listed below.

1. The C-terminus of CEP290 has microtubule binding domain while the N-terminus has membrane binding motif. Here the authors show that the N-terminus alone localizes to the transition zone (Fig. 1). How might this fragment be anchored at the transition zone and basal body? Understanding this question could help enhance the mechanistic understanding of this work.

2. The 21A6 antibody recognizes two domains longitudinally in auditory neurons. Which domain is shown in Fig. 1B, 4F and 5B? What are the directions of the cilia in these figures? Showing un-cropped images may help to predict the position of the cilia in these cells.

3. What are the stages of the spermatocyte in Fig 2C? What structure is the elongated UNC signal related to when the ciliary bud formation is blocked in CEP290 null cells as shown in Fig. 2C and 2F?

4. The localization of CG6652 in cep290 null cells is very different comparing to wt and delta C cells in Fig. 2D. The authors claim that this is abnormally extended axoneme. But the distribution of CG6653 does not look like an axoneme. Electron microscopy analysis would be necessary to confirm this structure.

5. The association between basal bodies and plasma membrane was not defective in later spermatocytes and spermatids in CEP290 dC mutant cells (Fig. 2E). The transition zone can be assembled too (Fig. 2C). Are the cilia buds formed in these cells comparing to wt and null cells as in Fig. 2F and 2G?

6. The authors indicate that DZIP1 occupied the gap region between FBF1 and MKS1 in Fig. 4E and S3E. However, there is still a gap between DZIP1 and FBF1 in Fig. 4D auditory neurons. Are these representative images?

7. It would be helpful to note in the figure legends that some of the blots are antibody staining while other are are Poncuau S. stained for clarity.

8. Showing images of the whole auditory and olfactory systems can help confirm signal from background. The cropped area in images in Fig 1BCD, Fig.3C, Fig. 4F and Fig. 5AB are too small.

Rev. 4:

The manuscript Wu et al. is a very carefully crafted, well executed and powerful genetic analysis of the role of well-known transition zone factor CEP290 in assembly of different types of cilia in Drosophila. Coupling powerful fly genetics, with rapid transgenics and reporters and quantitative imaging, the authors have created an elegant study into how CEP290, a key human ciliopathy gene, is mechanistically driving assembly of the transition zone in these highly modified cilia types. Indeed, understanding distinct and overlapping properties of the transition zone of different cilia types is key towards understanding the clinical pleiotropy observed in human ciliopathy patients in terms of symptom severity and penetrance between different tissue types. As such the study by Wu et al. is timely and of general interest to an audience like that of PLoS Biology. The tangible and highly subjective aspect of novelty of this study has been recently dinged by the Lampard et al in eLife (December 2019) from the Durand lab, where they looked at the same problem in the same system from a slightly different perspective, focusing on DZIP1 and FAM92, as well as CBY and of least focus CEP290. I believe the studies are complementary, the findings by Wu et al supporting and extending on those in the Lampard study- there is something important to be said for reproduction/confirmation of findings. However, given this and in light of the ethos of PLoS Biology to publish 'outstanding scientific significance', I feel this is somewhat an editorial issue for PLoS to decide where this one falls. Indeed, journals like eLIFE have a clear policy on publishing 'scooped' results of high quality and scientific significance, even if the novelty has been compromised. My gut feeling is it is better placed there.

Scientifically I can comment- as shown below. Generally, it is a well-written paper, although many of the figures are too busy/too many panels such that details cannot be seen but this is not fatal. It is an excellent example of genetics, imaging and biochemistry to carefully phenotypically and structurally dissect cellular process- I think it is very elegant in its execution and very thorough, very strong in its analysis. There is a propensity in the discussion to overextend speculation in the mammalian system for some of the points, but I will address them below.

Major points:

1. Accessibility: Many of the figures are too complex- it is very difficult to see details/labels even at their current full page size (see all Figure 2, Figure 3C graphs, Fig4D,E graphs, Fig 7). Also as most of the panels have only two dyes/fluorophores- I would strongly urge the authors to consider a color blind palette (magenta/green, cyan/orange- see - https://imagej.nih.gov/ij/docs/guide/146-9.html- about 10% of your male audience may be colorblind). Also the dark blue/indigo of the CellMask in 2E, 4H-I and 5F is difficult to discern from the black background- consider magenta or something bright.

2. Allelism: This current study does not address any about protein levels between lines- but puts it down to differences in activities of different protein domains- which should remain an interpretation of the results, given it could be down to just protein levels. There is a big point made about the previously published fly line (Basiri et al 2014; cep290mecH) which causes a premature termination codon resulting from a frameshift, in the 5th last exon of a very large gene- not being a null allele and instead being a milder loss-of-function allele due to remaining action of the C-terminally truncated protein (lacking the final~600 aa). Showing my mammalian bias, one would think this would subject the transcript to nonsense mediated decay, instead of protein truncation. Although speculated this truncated protein species exists, it is never investigated. I know that antibodies are difficult for Drosophila studies but some RNA/semi-quantitative PCR to look at levels and altered splice variants could lead credence to this- and that the cep290l and cep290�C lines generated here by genome editing, where larger on-target 'off-target' effects like inversions or larger deletions, make a phenotype seem more severe. Similarly, the transgenic over-expression some of the GFP CEP290 variants result in defective motility where the N-terminal CEP290 'may play a dominant negative role'- if this was true then heterozygous carriers for the cep290�C allele would have a similar uncoordinated, motor phenotype which they do not. Indeed according to the Basiri paper 'cep290mecH homozygotes (cep290) and hemizygotes (cep290/−) cannot stand….cep290mecH heterozygotes (cep290/+) and rescue (Cep290 and Cep290-GFP) are normal.' The lines of argument here are contorted. A safer approach would be like the Lampard paper uses an enhancer trap line- and just says it is a strong hypomorph and has a similar phenotype- loss of FAM92 and DZIP from the transition complexes, and '76% of aberrant axonemal growth, suggesting that basal body to membrane attachment is compromised in this mutant'. Genetics is complicated and sometime a rabbit hole not worth completely going down to prove your alleles are in fact nulls. Indeed in the clinical setting, we know that generally the severity of CEP290-associated phenotypes seems to correlate with the residual amount of functional protein that a mutant allele could produce (Drivas et al., 2015; Shimada et al., 2017)- something you have not looked at here rather tie it to domains. This is a weakness for me.

Minor points:

1. Awkward and inexact: Abstract 'Cilia play critical roles in organ generation and maintenance' consider 'during embryonic development and adult homeostasis'.

2. Introduction, p3, first line- typo '…that extend from the surface of most eukaryotic…'.

3. Introduction, p4, second line- typo '…which connect proximal axonemal…'.

4. Introduction, p4, second line Long sentence consider splitting- 'to the ciliary case membrane. These have been proposed to organize… on the membrane surface. The transition zone functions…'.

5. Introduction, p4. Second paragraph, third line. Italicize gene 'CEP290' and consider rewording from list of disorders to a more informative statement for non-expert readers like '…several ciliopathies ranging from non-syndromic retinal degeneration (i.e. Leber Congenital Amaurosis (LCA) to syndromic disorders including Senior-Loken syndrome (SLS),…'

6. Introduction, p4. Second paragraph, missing text. '…diseases and phenotypes associated with CEP290 mutations highlight the multiple and critical…'. Gene names should always be italics.

7. Introduction, p.5- inconsistency. I may be missing something but the statement 'We proposed that ciliary membrane assembly is a prerequisite for TZ assembly initiation' seems to be not what it is shown in your summary figure 7- where binding of CEP290 to apical membrane and recruitment of TZ facilitators DZIP and CBY occur before recruitment of Rab8/ciliary membrane vesicles. To the model is the inverse of this 'We proposed that TZ assembly initiation is a prerequisite for ciliary membrane assembly…'

8. Results, p.5, last paragraph, missing text. '…microtubules and ciliary membranes, where its C-terminus…'. Consider referring to Figure 1A here.

9. Results, p.5/6- clarity. Consider emphasizing constructs are overexpressed in wild type flies. Consider removing dominant negative statement at end of paragraph (see above).

10. Figure 1 A-D- do you know that all these GFP constructs are expressed- the lack of staining at the basal body of cilia for CEP290-M could simply be due to unstable protein. Also- I am perplexed: Figure 1B vs 1E- why such a different look to the cilia localizations between modalities- the confocal has two closely aligned structures and the SIM data only has one a field of view that is only double the magnification of the first, according to scale bars. Maybe a statement clarifying this is needed in the legends.

11. Results, p.7, for transparence- add reference to previously published alleles for cep290. 'Similar to previously reported alleles, both cep2901 and cep290�C were uncoordinated….. (Basiri et al 2014, Lampard et al 2019).

12. Figure 2A- for clarity, consider annotating where cep290mecH falls. Also explicitly add into the figure legends that these are predicted protein sizes- both of your CRIPSR alleles (and cep290mecH) may be subject to NMD and thus have little protein of any size to speak of.

13. Results, p.9, last paragraph- clarify. '…leading to loss of cilia/TZ' as the paragraph is written these structures never form- consider 'completely blocked, such that cilia with transition zones never form'. The following sentence may need some gentle reworking for non-fly enthusiasts, '…resulting in subsequent abnormal elongation of membraneless ciliary axonemes within the cytoplasm'.

14. Results, p.10, typo, first sentence. '…the initiation of TZ assembly, spermatogenesis….'.

15. Results, p.11, typo, second sentence. '…intensities were severely reduced compared to….'.

16. Results, p.11, emphasis. Tone down final statement first paragraph- ' …in sensory neurons, the CEP290 N-terminus may be essential for initial TZ assembly, whereas TZ elongation or maturation may involve a tissue-specific requirement for the C-terminus of CEP290.'

17. Results, p.11, typo. '… we employed a yeast two hybrid….' Also- 'Drosophila DZIP1 is the ortholog of mammalian DZIP1..'

18. Results, p.11, clarification. What is the CEP290-�N construct? What is in it is never detailed anywhere and is confusing with what is outlined in figure 1A.

19. Results, p.12, clarification. 'While the role of Drosophila DZIP1 in ciliogenesis was unknown when we started this project, a very recent publication has confirmed our findings (Lampard et al 2019).'

20. Results, p.13, typo. '…completely lost but CEP290 persisted…'. Also 'Furthermore, similar to cep2901 mutants…'

21. Results, p.14, typo. '… C-terminus may have tissue-specific roles in regulating…'.

22. Results, p.17, typo. '…compensate for the loss of Rab8, as has been reported for Rab8 and Rab10 in mammalian ciliogenesis…'.

23. Results, p.17, clarify. I was confused by the statement '…then facilitate the Rab8- and its redundant player- mediated…'. Consider '…then facilitate the Rab8- and its yet-unknown compensatory mechanism regulating…'.

24. Discussion, p.18, see point 7. '…TZ assembly is completely blocked in dzip1 mutants suggests that early ciliary membrane formation is a prerequisite for TZ assembly initiation'. Again I think this is the opposite of what your data shows- TZ assembly predates early ciliary membrane formation in your analysis.

25. Discussion, p.18, careful. The discussion about CEP290 mutations in human patients- there are retinal specific isoforms for many key RP genes which are believed to be the reason for the syndromic versus non-syndromic divide for some of these clinical phenotypes. This should be referenced. Again although the genotype-phenotype correlation is challenging, in general it is believed that the severity of CEP290-associated clinical phenotypes appears to reflect the residual amount of functional protein produced by a mutant allele (Drivas et al., 2015; Shimada et al., 2017). This current study does not address any of this- but puts it down to protein domains- which should remain an interpretation of the results, given it could be down to just protein levels.

26. Discussion, p.19, typo. 'In Drosophila, the centriole…'. Also take out the '(our unpublished data)' , either you show it or don't mention it at all.

27. Discussion, p.20, typo. '…understand the mechanisms of…'.

28. Discussion, p.20, clarify (see point 23). Change '…then facilitate the Rab8- and its redundant player- mediated…'. Consider '…then facilitate the Rab8- and its yet-unknown compensatory mechanism regulating…'.

29. Discussion, p.20, nomenclature. Mouse alleles should be title case italics- Dzip1 or Dzip1l. See final paragraph too. There is also a typo here- mice is plural so should be 'have much more severe…'. Same for Cby and Fam92a. Within species, they are not orthologs, they are paralogs. Final sentence in paragraph change to '…the existence of so many paralogs complicates the study of their genetic relationship'.

30. Discussion, p.21, missing word. '….regulating DZIP1-mediated early ciliary membrane WHAT?….'.

31. Methods, p.24, add information. The microscopy and image analysis- how was the quantitative imaging done- how many flies, how many basal bodies/TZ to get traces'.

32. Acknowledgements, p.35, typo. Change to '…for their technical support'.

33. Figure Legends, 4 title, clarify. '…with the N-terminus of CEP290, and dzip1�C deletion mutants…'

34. Supplementary Figure legends, p.42. Clarify PCR is of what- genomic DNA? In figure S1E- there is a typo throughout with your driver- should be elav-GAL4 not ealv. Figure S1- add in what acronym DBB and PBB mean.

35. Supplementary Figure legend 2, p.43. There is a typo in the figure for H. Sapiens several times.

36. Supplementary Figure legend 3, p.44. Typo '…to the tip of the flagella.'

PLoS Biol. 2020 Dec 28;18(12):e3001034. doi: 10.1371/journal.pbio.3001034.r003

Author response to Decision Letter 1

15 Sep 2020

Attachment

Submitted filename: Response to Reviewers final(1).docx

Click here for additional data file.^{(550.7KB, docx)}

PLoS Biol. doi: 10.1371/journal.pbio.3001034.r004

Decision Letter 2

Ines Alvarez-Garcia

18 Nov 2020

Dear Dr Wei,

Thank you for submitting your revised Research Article entitled "CEP290 is essential for the initiation of transition zone assembly" for publication in PLOS Biology. I have now obtained advice from the original reviewers and have discussed their comments with the Academic Editor.

Based on the reviews (attached below), we will probably accept this manuscript for publication, assuming that you will modify the manuscript to address the remaining points raised by the reviewers. We would also like you to consider some suggestions to improve the title. Please consider one of these two alternatives:

"CEP290 is essential for the initiation of ciliary transition zone assembly" or

"CEP290 is essential for the initiation of transition zone assembly in cilia"

Please also make sure to address the data and other policy-related requests noted at the end of this email.

We expect to receive your revised manuscript within two weeks. Your revisions should address the specific points made by each reviewer. In addition to the remaining revisions and before we will be able to formally accept your manuscript and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable)

- a track-changes file indicating any changes that you have made to the manuscript.

*Copyediting*

Upon acceptance of your article, your final files will be copyedited and typeset into the final PDF. While you will have an opportunity to review these files as proofs, PLOS will only permit corrections to spelling or significant scientific errors. Therefore, please take this final revision time to assess and make any remaining major changes to your manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Early Version*

Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

Please do not hesitate to contact me should you have any questions.

Sincerely,

Ines

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

------------------------------------------------------------------------

DATA POLICY: IMPORTANT - PLEASE READ

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:

Fig. 1E, F; Fig. 2C; Fig. 3C; Fig. 4D, E; Fig. 6E; Fig. S1D, E, G; Fig. S5D and Fig. S6G

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

Please also ensure that figure legends in your manuscript include information on WHERE THE UNDERLYING DATA CAN BE FOUND, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

-------------------------------------------------------------------------

BLOT AND GEL REPORTING REQUIREMENTS:

For manuscripts submitted on or after 1st July 2019, we require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

--------------------------------------------------------

Reviewers’ comments

Rev. 1:

In this revised manuscript, Wu et al. largely address the concerns I raised previously. While it is unfortunate that the Cep290-Dzip1l interaction could not be dissected more precisely, the authors made a reasonable effort, and they revised the text accordingly. I would encourage the authors to include the coIP showing interaction of mammalian Dzip1l and Cep290 if possible. There are also some grammatical errors that should be corrected before publication, and parts of the Discussion are not as clearly written as the rest of the manuscript. These minor issues notwithstanding, I believe the manuscript is suitable for publication in PLoS Biology.

Rev. 2:

The authors have answered to all my initial requests and the additional data reinforce their conclusions.

In particular they have corrected all the overstatements and added interesting data showing functional interactions between Fam92 and Rab8, indicating that Rab8 and Fam92 or Cby cooperate during spermatogenesis.

Minor point:

Page 13 fourth line: MKS6 is not presented on sup Fig 4C.

page 14 last paragraph: "is still existed" check English grammar.

Rev. 3:

The revised manuscript has been improved and addressed most of my concerns. Though how the N-terminus of CEP290 is anchored to the basal body to recruit Dzip1 is not completely clear. Overall this work and model proposed for CEP290-Dzip1-Rab8 pathway in TZ assembly initiation is important to the field.

Rev. 4:

The revised work by Wu et al represents a significant genetic and biochemical study into early stages of ciliogenesis in Drosophila regulated by CEP290 through the DZIP1-CBY/Rab8 module. Using a range of imaging modalities and generating many genetic tools, the work is novel and sound- appropriate for publication in a revised manner in PLoS Biology.

1. Accessibility: I am disappointed the authors did not choose to make their data more accessible to the broad readership of the journal. 10% of all readers, including some of the corresponding authors whose work they cite here, are red-green colorblind and as such are unable to interpret the bulk of the data presented in this manuscript. Changing pseudocolors in two color panels is very easy in FIJI with plug-ins- it would take the authors a half-day tops but would be so inclusive, improving pick up and citation of their work. (On a side note, I do wish PLoS would make this policy and mandate, not be optional).

2. Allelism and expression: as I wrote, we know from human genetics that levels of variant expression are key for CEP290 phenotypes- this is ultimately at the protein level, which the authors are unable to look at. I think this statement needs to be made explicitly as a caveat to their interpretation in the discussion. Something akin to 'Without a robust antibody to survey expression to validate levels of CEP290 protein expression within our alleles, we cannot fully unpick how much of our phenotypes are due solely to reduced levels versus requirement for specific domains. We suspect it is likely a combination of both'. Reviewer 1 (Question 2) also raises issues of protein levels, as does reviewer 2 who wrote '…all the experiments based on the expression of these truncated forms, could there be an effect of the expression levels?' Their response 'To ensure that CEP290 and its truncations were expressed at a comparable level, all these transgenes were inserted o the same integration platform (the attP site of 25C6 locus on chromosome 2), and they were driven by the same ubiquitin promoter. Therefore, it is unlikely that the different defective phenotypes with expressing various CEP290 truncations are due to their different expression levels'. Again, this reflect only transcript level and not stability of the protein- this is emphasized in Supp Fig 8 where there is no GFP expressed for CEP290-M suggesting it is not stable. Without a GFP blot to show similar levels of protein expression, untangling protein instability/levels versus changes in localization cannot be unpicked.

Minor points:

1. A comment: My point about was about on-target 'off-targets' in their crispant alleles- larger or complex rearrangements at the locus that because they are at the locus and cannot be 'eliminated by outcrossing 3 times to dilute any potential off targeted lesion'. Large long read sequencing is necessary and/or southern blots to really confirm, but I suspect were not done.

2. Last paragraph of the introduction- it is all past tense, but work you describe in the paper- present tense is more apropos.

3. Figure 1C legend: Sequencing of what gDNA or cDNA? Also in this figure why are the numbers different at the bottom of the chromatograms- consider removing as they are confusing, without adding value.

4. p7 'This is consistent with the transcription level of cep290 in cep290ΔC mutant' should read ' consistent with the transcript level'- you are assaying only transcript level (result of production and turnover).

5. P8 Protein DILA should be all caps.

6. Top of p12 typo : 'Drosophila DIZP1 is' should read 'Drosophila DZIP1 is'

7. Page 12 Overexpression in a cell line does not constitute in vivo 'GFP-tagged CEP290 and HA-tagged DZIP1 in cultured Drosophila S2 cells'- removing 'also occurs in vivo,' without losing emphasis.

8. Top of p16- typo 'cby or fam92 single mutants'

9. Top of p18- typo single or plural- clarify 'Then we created a fam92 deletion mutants'

PLoS Biol. 2020 Dec 28;18(12):e3001034. doi: 10.1371/journal.pbio.3001034.r005

Author response to Decision Letter 2

10 Dec 2020

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(55.9KB, docx)}

PLoS Biol. doi: 10.1371/journal.pbio.3001034.r006

Decision Letter 3

Ines Alvarez-Garcia

16 Dec 2020

Dear Dr. Wei,

I am writing concerning your manuscript submitted to PLOS Biology, entitled “CEP290 is essential for the initiation of ciliary transition zone assembly.”

We have now completed our final technical checks and have approved your submission for publication. You will shortly receive a letter of formal acceptance from the editor.

Kind regards,

PLOS Biology

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Identification and phenotype analysis of cep290 mutants.

(TIF)

Click here for additional data file.^{(31.8MB, tif)}

S2 Fig. DZIP1 is the ortholog of mammalian DZIP1 and DZIP1L in Drosophila.

(TIF)

Click here for additional data file.^{(26.1MB, tif)}

S3 Fig. CEP290–DZIP1 interaction analysis.

(TIF)

Click here for additional data file.^{(31.4MB, tif)}

S4 Fig. The subcellular localization of DZIP1 in Drosophila.

(TIF)

Click here for additional data file.^{(27.4MB, tif)}

S5 Fig. Identification and phenotype analysis of dzip1 mutants.

(TIF)

Click here for additional data file.^{(31.2MB, tif)}

S6 Fig. Subcellular localization of Rab8 in sensory cilia and phenotype of rab8 mutants.

(TIF)

Click here for additional data file.^{(27.4MB, tif)}

S7 Fig. Human CEP290 N-terminal was coimmunoprecipitated with DZIP1L.

(TIF)

Click here for additional data file.^{(25.5MB, tif)}

S1 Table. Primers were used in this paper.

(TIF)

Click here for additional data file.^{(25.5MB, tif)}

S1 Data. Excel file of numerical data.

(XLSX)

Click here for additional data file.^{(83.7KB, xlsx)}

S1 Raw Image. Uncropped images of immunofluorescence staining of sensory cilia.

(TIF)

Click here for additional data file.^{(28.5MB, tif)}

S2 Raw Image. Full scans of western blots.

(TIF)

Click here for additional data file.^{(26.6MB, tif)}

Attachment

Submitted filename: Response to Reviewers final(1).docx

Click here for additional data file.^{(550.7KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(55.9KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pbio.3001034.ref001] 1.Mitchison HM, Valente EM. Motile and non-motile cilia in human pathology: from function to phenotypes. J Pathol. 2017;241:294–309. 10.1002/path.4843 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref002] 2.Anvarian Z, Mykytyn K, Mukhopadhyay S, Pedersen LB, Christensen ST. Cellular signalling by primary cilia in development, organ function and disease. Nat Rev Nephrol. 2019;15:199–219. 10.1038/s41581-019-0116-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref003] 3.Nachury MV, Mick DU. Establishing and regulating the composition of cilia for signal transduction. Nat Rev Mol Cell Biol. 2019;20:389–405. 10.1038/s41580-019-0116-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref004] 4.Shinohara K, Hamada H. Cilia in Left-Right Symmetry Breaking. Cold Spring Harb Perspect Biol. 2017;9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref005] 5.Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. N Engl J Med. 2011;364:1533–43. 10.1056/NEJMra1010172 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref006] 6.Reiter JF, Leroux MR. Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol. 2017;18:533–47. 10.1038/nrm.2017.60 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref007] 7.Sorokin S. Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Biol. 1962;15:363–77. 10.1083/jcb.15.2.363 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref008] 8.Sorokin SP. Reconstructions of Centriole Formation and Ciliogenesis in Mammalian Lungs. J Cell Sci. 1968;3:207 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref009] 9.Wu CT, Chen HY, Tang TK. Myosin-Va is required for preciliary vesicle transportation to the mother centriole during ciliogenesis. Nat Cell Biol. 2018;20:175–85. 10.1038/s41556-017-0018-7 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref010] 10.Lu Q, Insinna C, Ott C, Stauffer J, Pintado PA, Rahajeng J, et al. Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nat Cell Biol. 2015;17:228–40. 10.1038/ncb3109 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref011] 11.Knödler A, Feng S, Zhang J, Zhang X, Das A, et al. Coordination of Rab8 and Rab11 in primary ciliogenesis. Proc Natl Acad Sci U S A. 2010;107:6346–51. 10.1073/pnas.1002401107 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref012] 12.Westlake CJ, Baye LM, Nachury MV, Wright KJ, Ervin KE, et al. Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome. Proc Natl Acad Sci U S A. 2011;108:2759–64. 10.1073/pnas.1018823108 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref013] 13.Gonçalves J, Pelletier L. The Ciliary Transition Zone: Finding the Pieces and Assembling the Gate. Mol Cells. 2017;40:243–53. 10.14348/molcells.2017.0054 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref014] 14.Garcia-Gonzalo FR, Reiter JF. Open Sesame: How Transition Fibers and the Transition Zone Control Ciliary Composition. Cold Spring Harb Perspect Biol. 2017;9:a028134 10.1101/cshperspect.a028134 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref015] 15.Ishikawa H, Marshall WF. Ciliogenesis: building the cell's antenna. Nat Rev Mol Cell Biol. 2011;12:222–34. 10.1038/nrm3085 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref016] 16.Reiter JF, Blacque OE, Leroux MR. The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO Rep. 2012;13:608–18. 10.1038/embor.2012.73 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref017] 17.Czarnecki PG, Shah JV. The ciliary transition zone: from morphology and molecules to medicine. Trends Cell Biol. 2012;22:201–10. 10.1016/j.tcb.2012.02.001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref018] 18.Gilula NB, Satir P. The ciliary necklace. A ciliary membrane specialization The Journal of cell biology. 1972;53:494–509. 10.1083/jcb.53.2.494 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref019] 19.Sang L, Miller JJ, Corbit KC, Giles RH, Brauer MJ, et al. Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways. Cell. 2011;145:513–28. 10.1016/j.cell.2011.04.019 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref020] 20.Schouteden C, Serwas D, Palfy M, Dammermann A. The ciliary transition zone functions in cell adhesion but is dispensable for axoneme assembly in C. elegans. J Cell Biol. 2015;210:35–44. 10.1083/jcb.201501013 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref021] 21.Li C, Jensen VL, Park K, Kennedy J, Garcia-Gonzalo FR, et al. MKS5 and CEP290 Dependent Assembly Pathway of the Ciliary Transition Zone. PLoS Biol. 2016;14:e1002416 10.1371/journal.pbio.1002416 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref022] 22.Williams CL, Li C, Kida K, Inglis PN, Mohan S, et al. MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J Cell Biol. 2011;192:1023–41. 10.1083/jcb.201012116 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref023] 23.Craige B, Tsao C-C, Diener DR, Hou Y, Lechtreck K-F, et al. CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. J Cell Biol. 2010;190:927–40. 10.1083/jcb.201006105 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref024] 24.Basiri ML, Ha A, Chadha A, Clark NM, Polyanovsky A, et al. A migrating ciliary gate compartmentalizes the site of axoneme assembly in Drosophila spermatids. Curr Biol. 2014;24:2622–31. 10.1016/j.cub.2014.09.047 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref025] 25.Garcia-Gonzalo FR, Corbit KC, Sirerol-Piquer MS, Ramaswami G, Otto EA, et al. A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nat Genet. 2011;43:776–84. 10.1038/ng.891 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref026] 26.Coppieters F, Lefever S, Leroy BP, De Baere E. CEP290, a gene with many faces: mutation overview and presentation of CEP290base. Hum Mutat. 2010;31:1097–108. 10.1002/humu.21337 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref027] 27.Jana SC, Mendonca S, Machado P, Werner S, Rocha J, et al. Differential regulation of transition zone and centriole proteins contributes to ciliary base diversity. Nat Cell Biol. 2018;20:928–41. 10.1038/s41556-018-0132-1 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref028] 28.Drivas TG, Holzbaur EL, Bennett J. Disruption of CEP290 microtubule/membrane-binding domains causes retinal degeneration. J Clin Invest. 2013;123:4525–39. 10.1172/JCI69448 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref029] 29.Lapart J-A, Gottardo M, Cortier E, Duteyrat J-L, Augière C, et al. Dzip1 and Fam92 form a ciliary transition zone complex with cell type specific roles in Drosophila. elife. 2019;8:e49307 10.7554/eLife.49307 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref030] 30.Riparbelli MG, Callaini G, Megraw TL. Assembly and persistence of primary cilia in dividing Drosophila spermatocytes. Dev Cell. 2012;23:425–32. 10.1016/j.devcel.2012.05.024 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref031] 31.Gottardo M, Callaini G, Riparbelli MG. The cilium-like region of the Drosophila spermatocyte: an emerging flagellum? J Cell Sci. 2013;126:5441–52. 10.1242/jcs.136523 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref032] 32.Lattao R, Kovács L, Glover DM. The Centrioles, Centrosomes, Basal Bodies, and Cilia of Drosophila melanogaster. Genetics. 2017;206:33–53. 10.1534/genetics.116.198168 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref033] 33.Jana SC, Bettencourt-Dias M, Durand B, Megraw TL. Drosophila melanogaster as a model for basal body research. Cilia. 2016;5:22–2. 10.1186/s13630-016-0041-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref034] 34.Vieillard J, Paschaki M, Duteyrat JL, Augiere C, Cortier E, et al. Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells. J Cell Biol. 2016;214:875–89. 10.1083/jcb.201603086 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref035] 35.Wang C, Low W-C, Liu A, Wang B. Centrosomal Protein DZIP1 Regulates Hedgehog Signaling by Promoting Cytoplasmic Retention of Transcription Factor GLI3 and Affecting Ciliogenesis. J Biol Chem. 2013;288:29518–29. 10.1074/jbc.M113.492066 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref036] 36.Wang C, Li J, Takemaru KI, Jiang X, Xu G, et al. Centrosomal protein Dzip1l binds Cby, promotes ciliary bud formation, and acts redundantly with Bromi to regulate ciliogenesis in the mouse. Development. 2018;145 (6). [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref037] 37.Breslow DK, Hoogendoorn S, Kopp AR, Morgens DW, Vu BK, et al. A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies. Nat Genet. 2018;50:460–71. 10.1038/s41588-018-0054-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref038] 38.Lu H, Galeano MCR, Ott E, Kaeslin G, Kausalya PJ, et al. Mutations in DZIP1L, which encodes a ciliary-transition-zone protein, cause autosomal recessive polycystic kidney disease. Nat Genet. 2017;49:1025–34. 10.1038/ng.3871 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref039] 39.Enjolras C, Thomas J, Chhin B, Cortier E, Duteyrat JL, et al. Drosophila chibby is required for basal body formation and ciliogenesis but not for Wg signaling. J Cell Biol. 2012;197:313–25. 10.1083/jcb.201109148 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref040] 40.Zhang B, Zhang T, Wang G, Wang G, Chi W, et al. GSK3beta-Dzip1-Rab8 cascade regulates ciliogenesis after mitosis. PLoS Biol. 2015;13:e1002129 10.1371/journal.pbio.1002129 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref041] 41.Nachury MV, Loktev AV, Zhang Q, Westlake CJ, Peranen J, et al. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell. 2007;129:1201–13. 10.1016/j.cell.2007.03.053 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref042] 42.Sato T, Iwano T, Kunii M, Matsuda S, Mizuguchi R, et al. Rab8a and Rab8b are essential for several apical transport pathways but insufficient for ciliogenesis. J Cell Sci. 2014;127:422–31. 10.1242/jcs.136903 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref043] 43.Li FQ, Chen X, Fisher C, Siller SS, Zelikman K, et al. BAR Domain-Containing FAM92 Proteins Interact with Chibby1 To Facilitate Ciliogenesis. Mol Cell Biol. 2016;36:2668–80. 10.1128/MCB.00160-16 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref044] 44.Burke MC, Li FQ, Cyge B, Arashiro T, Brechbuhl HM, et al. Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation. J Cell Biol. 2014;207:123–37. 10.1083/jcb.201406140 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref045] 45.Malicki J, Avidor-Reiss T. From the cytoplasm into the cilium: bon voyage. Organogenesis. 2014;10:138–57. 10.4161/org.29055 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref046] 46.Rachel RA, Yamamoto EA, Dewanjee MK, May-Simera HL, Sergeev YV, et al. CEP290 alleles in mice disrupt tissue-specific cilia biogenesis and recapitulate features of syndromic ciliopathies. Hum Mol Genet. 2015;24:3775–91. 10.1093/hmg/ddv123 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref047] 47.Shimada H, Lu Q, Insinna-Kettenhofen C, Nagashima K, English MA, et al. In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations. Cell Rep. 2017;20:384–96. 10.1016/j.celrep.2017.06.045 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref048] 48.Galletta BJ, Guillen RX, Fagerstrom CJ, Brownlee CW, Lerit DA, et al. Drosophila pericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies. Mol Biol Cell. 2014;25:2682–94. 10.1091/mbc.E13-10-0617 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref049] 49.Roque H, Saurya S, Pratt MB, Johnson E, Raff JW. Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation. PLoS Genet. 2018;14:e1007198–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref050] 50.Gottardo M, Persico V, Callaini G, Riparbelli MG. The "transition zone" of the cilium-like regions in the Drosophila spermatocytes and the role of the C-tubule in axoneme assembly. Exp Cell Res. 2018;371:262–8. 10.1016/j.yexcr.2018.08.020 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref051] 51.Vieillard J, Duteyrat JL, Cortier E, Durand B. Imaging cilia in Drosophila melanogaster. Methods Cell Biol. 2015;127:279–302. 10.1016/bs.mcb.2014.12.009 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref052] 52.Chen JV, Kao LR, Jana SC, Sivan-Loukianova E, Mendonca S, et al. Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila. J Cell Biol. 2015;211:435–53. 10.1083/jcb.201502032 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref053] 53.Lapart JA, Billon A, Duteyrat JL, Thomas J, Durand B. Role of DZIP1-CBY-FAM92 transition zone complex in the basal body to membrane attachment and ciliary budding. Biochem Soc Trans. 2020;48:1067–75. 10.1042/BST20191007 [DOI] [PubMed] [Google Scholar]

[pbio.3001034.ref054] 54.Voronina VA, Takemaru K-I, Treuting P, Love D, Grubb BR, et al. Inactivation of Chibby affects function of motile airway cilia. J Cell Biol. 2009;185:225–33. 10.1083/jcb.200809144 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref055] 55.Kobayashi T, Kim S, Lin YC, Inoue T, Dynlacht BD. The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly. J Cell Biol. 2014;204:215–29. 10.1083/jcb.201304153 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pbio.3001034.ref056] 56.Diggle CP, Moore DJ, Mali G, zur Lage P, Ait-Lounis A, et al. HEATR2 plays a conserved role in assembly of the ciliary motile apparatus. PLoS Genet. 2014;10:e1004577–7. 10.1371/journal.pgen.1004577 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

CEP290 is essential for the initiation of ciliary transition zone assembly

Zhimao Wu

Nan Pang

Yingying Zhang

Huicheng Chen

Ying Peng

Jingyan Fu

Qing Wei

Roles

Abstract

Introduction

Results

Both N-terminal and C-terminal truncation mutants of CEP290 localize to the TZ in Drosophila

Fig 1. Both the N-terminal and C-terminal truncation mutants of CEP290 localized to TZs in Drosophila.

CEP290 is essential for the initiation of TZ assembly in spermatocytes

Fig 2. CEP290 is essential for TZ assembly initiation in spermatocytes.

CEP290 is critical for the initiation of TZ assembly in sensory neurons

Fig 3. CEP290 is critical for TZ assembly initiation in sensory neurons.

DZIP1 is a TZ protein interacting with the N-terminus of CEP290

Fig 4. DZIP1 interacts with the N-terminus of CEP290, and DZIP1 deletion mutants mimic the phenotype of cep2901 mutants.

dzip1 deletion mutants mimic the phenotype of cep290 mutants

CEP290 recruits DZIP1 to regulate TZ assembly

Fig 5. The N-terminus of CEP290 recruits DZIP1 to regulate TZ assembly.

DZIP1 regulates TZ assembly upstream of CBY and Rab8

Fig 6. DZIP1 recruits CBY and Rab8 to promote TZ assembly.

Discussion

Fig 7. Model for the initiation of TZ assembly in Drosophila.

Methods

Fly stocks

Generation of transgenic flies

Generation of deletion mutants in Drosophila

Immunofluorescence

Cell membrane staining

Antibodies

Microscopy and image analysis

Transmission electron microscopy

Male fertility test

Negative geotaxis assay

Larval touch assay

Larval hearing assay

Yeast two-hybrid assay

Immunoprecipitation

GST pull-down assay

Statistics

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Ines Alvarez-Garcia

Roles

Decision Letter 1

Ines Alvarez-Garcia

Roles

Author response to Decision Letter 1

Decision Letter 2

Ines Alvarez-Garcia

Roles

Author response to Decision Letter 2

Decision Letter 3

Ines Alvarez-Garcia

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Fig 4. DZIP1 interacts with the N-terminus of CEP290, and DZIP1 deletion mutants mimic the phenotype of cep290¹ mutants.