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Cep131-Cep162 and Cby-Fam92 complexes cooperatively maintain Cep290 at the basal body and contribute to ciliogenesis initiation [1]

['Zhimao Wu', 'Center For Energy Metabolism', 'Reproduction', 'Institute Of Biomedicine', 'Biotechnology', 'Shenzhen Institutes Of Advanced Technology', 'Chinese Academy Of Sciences', 'Cas', 'Shenzhen', 'Huicheng Chen']

Date: 2024-03

Cilia play critical roles in cell signal transduction and organ development. Defects in cilia function result in a variety of genetic disorders. Cep290 is an evolutionarily conserved ciliopathy protein that bridges the ciliary membrane and axoneme at the basal body (BB) and plays critical roles in the initiation of ciliogenesis and TZ assembly. How Cep290 is maintained at BB and whether axonemal and ciliary membrane localized cues converge to determine the localization of Cep290 remain unknown. Here, we report that the Cep131-Cep162 module near the axoneme and the Cby-Fam92 module close to the membrane synergistically control the BB localization of Cep290 and the subsequent initiation of ciliogenesis in Drosophila. Concurrent deletion of any protein of the Cep131-Cep162 module and of the Cby-Fam92 module leads to a complete loss of Cep290 from BB and blocks ciliogenesis at its initiation stage. Our results reveal that the first step of ciliogenesis strictly depends on cooperative and retroactive interactions between Cep131-Cep162, Cby-Fam92 and Cep290, which may contribute to the complex pathogenesis of Cep290-related ciliopathies.

Here, we report that Cep131 recruits Cep162 to regulate the TZ localization of Cep290 C-terminus and promote ciliogenesis. We show that Cep162 is a Cep131-interacting protein and acts downstream of Cep131 to mediate the association of Cep290 C-terminus with the axonemal microtubules. In addition, we demonstrate that Cby-Fam92 module regulates the TZ localization of the N-terminus of Cep290. Both modules cooperate to recruit and stabilize Cep290 at the TZ, as combined loss of either module (Cep131-Cep162 or Cby-Fam92) results in complete failure of Cep290 localization to the TZ and ultimately prevents the initiation of ciliogenesis. Our results reveal the crucial molecular function of Cep131 in ciliogenesis and unveil a cooperative and orderly assembly of Cep290 facilitated by Cep131-Cep162 and Cby-Fam92 modules during the initiation of ciliogenesis. Thus, our work defines a central molecular pathway composed of 3 modules: Cep131-Cep162, Cep290, and Dzip1-Cby-Fam92, which cooperatively regulate the initiation of cilium assembly.

Centrosome protein 131 (Cep131) localizes to both centrosome centriolar satellites and ciliary TZ in mammals [ 35 – 37 ] and has been demonstrated to be required for ciliogenesis, centriole amplification, genome stability, and cancer [ 35 , 36 , 38 – 40 ]. Studies on the model organisms Drosophila and zebrafish revealed that Cep131 is an evolutionarily conserved BB protein [ 41 , 42 ], and that deletion of Cep131 results in abnormal cilia formation, suggesting that Cep131 has an evolutionarily conserved role in ciliogenesis. But the precise mechanism by which Cep131 regulates ciliogenesis remains largely unknown. In Drosophila, Cep131 (also called dilatory in flies) has been shown to localize to the lumen of the distal BB and TZ, playing a role in the initiation of ciliogenesis [ 42 , 43 ]. Interestingly, although both Cep131 and Cby single mutants display mild defects in cilium assembly, the initiation of ciliogenesis is completely blocked and Cep290 is totally absent from basal bodies in cep131; cby double mutant [ 43 ]. However, the molecular function of Cep131 in the initiation of ciliogenesis is still largely unknown, and the underlying mechanism by which Cep131 genetically interacts with Cby to regulate Cep290 localization remains unclear.

Although speculations have accumulated that ciliary bud formation must be a coordinated event matching the TZ assembly and the ciliary membrane formation [ 27 , 29 ], a detailed molecular linkage between these 2 seemingly independent processes remains largely elusive. Using Drosophila model, we provide evidence that the core TZ protein Cep290 play a pivotal role in coordinating ciliary membrane formation and TZ assembly [ 29 ]. We demonstrated that, in addition to its classical role in TZ assembly, Cep290 acts upstream of Dzip1, playing an essential role in ciliogenesis initiation and ciliary bud formation [ 29 ]. Cep290 is an intriguing cilia gene associated with several ciliopathies [ 32 ], including Leber congenital amaurosis (LCA), Senior–Loken syndrome (SLS), Joubert syndrome (JBTS), MKS, and Bardet–Biedl syndrome (BBS). More than 100 ciliopathy mutations have been identified in Cep290 [ 32 ]. The broad spectrum of diseases highlights the critical roles of Cep290 in cilia. We and others have demonstrated that the N-terminus of Cep290 associates with the ciliary membrane, while its C-terminus connects with the ciliary axoneme, both being critical for ciliogenesis [ 29 , 33 , 34 ]. Nonetheless, how Cep290 is targeted to the TZ and whether axoneme derived signal and ciliary membrane localized cues converge to determine the localization and stability of Cep290 remain unknown.

The formation of cilia involves 2 main processes: ciliogenesis initiation and axoneme elongation. The elongation of axoneme relies on the intraflagellar transport (IFT), a evolutionarily conserved transport machinery within cilia [ 13 , 14 ]. The initiation of ciliogenesis involves the membrane docking of BB and the formation of ciliary bud [ 10 , 15 , 16 ]. In mammals, the membrane docking of BB is mediated by centriole distal appendages/ciliary transition fibers [ 17 – 21 ]. However in invertebrate model organism Drosophila, transition fiber proteins are dispensable for BB membrane docking [ 22 ], suggesting the presence of alternative mechanisms. The ciliary bud consists of the TZ and its surrounding membrane [ 10 , 23 , 24 ]. Dozens of proteins have been identified as components of TZ, and mutations in most of them lead to ciliopathy [ 5 ]. Studies have categorized TZ proteins into 3 functional modules: Meckel–Gruber syndrome (MKS) module, Nephronophthisis (NPHP) module, and Cep290 [ 25 ]. The formation of ciliary membrane relies on the membrane transport regulator small GTPase Rab8 related signaling cascade [ 26 – 28 ]. Dzip1 (DAZ interacting zinc finger protein 1) and its close paralog Dzip1L have recently been found to play a role in ciliary bud formation in both Drosophila and mammals [ 24 , 29 , 30 ]. Downstream of Dzip1/1L, Rab8, and Cby (Chibby)-Fam92 (family with sequence similarity 92) module work together to regulate ciliary membrane formation [ 24 , 29 , 31 ].

The structure of cilia remains highly conserved across evolution [ 8 ]. A cilium comprises a basal body (BB), a transition zone (TZ), an axoneme and its overlying membrane. The BB originates from the mother centriole and attaches to the membrane through transition fibers that come from the distal appendages of the mother centriole [ 9 – 11 ]. The TZ is situated just above transition fibers [ 12 ] and is characterized by Y-linker structures that connect the axoneme and ciliary membrane, serving as a diffusion barrier to control ciliary protein entry [ 9 ]. The axoneme forms the cilium skeleton and consists of a 9-fold array of doublet microtubules, which are templated from the BB microtubules and surrounded by the ciliary membrane [ 5 , 8 ].

Cilia are microtubule-based organelles that extend from the surface of many cell types and are widely present in eukaryotes. They play crucial roles in the development and maintenance of various organs in humans [ 1 – 4 ], and their dysfunction has been linked to a wide range of human genetics diseases known as ciliopathies [ 5 – 7 ].

Results

Cep131 is required for the basal body localization of Cep290 C-terminus To understand the function of the Cep131 in ciliogenesis, we generated the cep1311 (C terminal deletion) mutant flies using the CRISPR-Cas9 system (S1A–S1C Fig). Similar to reported cep131 mutant [42], our cep1311 flies exhibited typical symptoms related to ciliary defects, and were severely uncoordinated during walking and flying. Consistent with previous reports that cep131 single mutant fly has mild defects in the initiation of ciliogenesis [42], we observed that approximately 33.6% of cep1311 spermatocyte centrioles showed abnormal elongation of the microtubules labeled by CG6652::GFP (S1D Fig), a Drosophila spermatocyte-specific phenotype associated with defects in BB docking and TZ membrane cap formation. Consistent with this observation, the signal of the ciliogenesis initiation regulators Cep290, Dzip1, and Cby, as well as the TZ marker Mks1 were notably reduced at the basal bodies of spermatocytes of cep1311 mutants, although they were still detectable (Fig 1A). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Cep131 is required for the BB localization of Cep290-C terminus. (A) Localization of various TZ proteins in spermatocyte cilia of WT flies and cep131 mutants and quantifications of corresponding relative fluorescence intensities. In cep131 mutants, the signals of Cep290, Dzip1, Cby::GFP, and Mks1::GFP are significantly reduced compared to WT. Importantly, Cep290-C::GFP signal is almost completely lost in cep131 mutants. The BB is labeled with γ-Tubulin (red). The error bars represent the mean ± SD, n = 30. (B) The localization of various TZ proteins in auditory cilia of WT flies and cep131 mutants. Similar to what we observed in spermatocyte cilia, the signals of Cep290, Dzip1, and Cby::GFP are significantly decreased, and Cep290-C::GFP is completely lost at the base of the sensory cilia. 21A6 (blue) marks the cilia base, Actin (red) marks the ciliated region. The error bars represent the mean ± SD, n = 30. Scale bars: 2 μm (A), 5 μm (B, full-scale images on the left), 1 μm (B, insets or zoomed in areas on the right). The data underlying this figure can be found in S1 Data. BB, basal body; TZ, transition zone; WT, wild type. https://doi.org/10.1371/journal.pbio.3002330.g001 Cep290 is the most upstream protein known in the initiation of ciliogenesis in Drosophila [29,30]. Works in fly and mammalian cells have suggested that Cep290 bridges the ciliary axoneme and the membrane, with its C-terminus associated with microtubule doublets and N-terminus associated with the membrane [29,33,34]. Previously, we have demonstrated that both N-terminal truncation (Cep290-N, aa 1–650) and C-terminal truncation (Cep290-C, aa 1385 to the end at 1978) of Drosophila Cep290 are capable of localizing to the TZ, independently of each other [29]. 3D-SIM microscopy revealed that Cep290-C::GFP localizes close to the axoneme, whereas Cep290-N::GFP localizes close to the membrane, displaying a noticeably larger diameter [29]. Given that Cep131 localizes to the lumen of TZ [43], we hypothesized that Cep131 might have a role in regulating the localization of Cep290 C-terminus. As expected, we found that the signal of Cep290-C::GFP signal was nearly completely lost in spermatocyte cilia of cep131 mutants, whereas the signal intensity of Cep290-N::GFP was similar to that in wild-type (WT) (Fig 1A). Interestingly, we noticed that both the diameter of endogenous Cep290 N-terminus (labeled by anti-Cep290 antibody against aa 292–541 in its N-terminus) and Cep290-N::GFP in cep131 mutants were significantly smaller than that in WT, suggesting a potential alternation in the conformation of Cep290 in cep131 mutants (Figs 1A and S1E). Collectively, our results indicate that Cep131 specifically regulates the localization of Cep290 C-terminus in spermatocyte cilia. To determine whether our observation is specific to spermatocyte cilia, we focused on sensory cilia, another type of cilia in Drosophila. Similar to our observation in spermatocytes, the signal intensities of Cep290, Cep290-N, Dzip1, Cby, and Mks1 were mildly affected, whereas Cep290-C::GFP was almost completely lost from the basal bodies in auditory cilia of cep131 mutants (Fig 1B). Hence, Cep131 also promotes the binding of Cep290 C-terminus to the axoneme in sensory cilia.

Cep162 acts downstream of Cep131 to regulate ciliogenesis To elucidate the role of Cep162 in fly ciliogenesis, we designed 2 gRNA to knockout Cep162 using the CRISPR-Cas9 system. We obtained a deletion mutant line, cep1621 (c.981-1306Del), in which the C-terminus of Cep162 was lost due to reading frame shift caused by the deletion (Figs 3A, S4A and S4B). cep1621 mutants were viable, but showed defects in cilia-related behaviors such as movement and hearing, which could be effectively rescued by expression of Cep162 (Fig 3B). Examination of the cilia morphology in auditory organ showed that about 20.2% of cilia were missing or very short in cep162 mutants (Fig 3C). In addition, TEM analysis showed that missing spermatids were frequently observed in the cysts of cep162 testes (Fig 3D). Therefore, Cep162 is indeed a key component for ciliogenesis in Drosophila. Notably, our TEM images revealed evident mitochondrial abnormalities in cep162 spermatids, with some axonemes displaying very small or entirely lost mitochondrial derivatives (highlighted in Fig 3D). Additionally, the sizes of mitochondrial derivatives in the cep162 mutant vary, contrasting with the overall even size observed in WT. How Cep162 affects mitochondria dynamics remain unclear. Interestingly, a recent study by Bauerly and colleagues reported the impact of cilia-related gene on mitochondria dynamics during Drosophila spermatogenesis [45]. Mutants in dynein-related ciliary genes exhibited the absence or reduced size of minor mitochondrial derivatives. Therefore, the influence of cilia-related genes on mitochondria is not a singular occurrence. While the underlying mechanism is presently unclear, further investigating is needed. PPT PowerPoint slide

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TIFF original image Download: Fig 3. cep162 mutant mimics the phenotype of cep131 mutant. (A) Generation of cep162 deletion mutants. Schematics show the genomic (upper panel) and protein (lower panel) structures of Cep162, along with the predicted protein products of cep1621 mutant (Cep1621_p.(Glu327_Met436delfsTer24)). Arrows point to 2 gRNA targeting sites. cep1621 mutant has a deletion in cDNA from nt 981 to 1306, resulting in a reading frame shift and C-terminus loss. (B) Analysis of hearing and negative geotaxis of cep162 mutants. cep1621 flies show mild hearing defects. The retraction index indicates the larval response to a 1k Hz tone. The box shows the median and interquartile range; n = 25. The percentage of cep1621 flies passing the 8 cm high scale was significantly lower than that of WT flies. The error bars represent the mean ± SD, n = 50. (C) Living images of cilia morphology in antennal auditory organ of WT fly and cep162 mutant pupae. Sensory neurons were labeled by nompC-Gal4/UAS::GFP (green), cilia are localized at the tip of dendrites. Sensory cilia are lost in partial sensory neurons (white asterisks) of cep1621 mutant. The graph on the right shows the percentage of sensory neurons with ciliary defects. (D) Representative TEM images of elongating spermatid cysts in WT and cep162 mutants. There are 64 spermatids per cyst in WT, whereas the number of spermatids per cyst is reduced in cep162 mutant. (E) Immunostaining of Cep131, Cep290, Dzip1, Cby, Mks1, and Mks6 in WT or cep1621 testis. The quantification of the TZ protein intensities is shown on the lower panel. Unlike other TZ proteins, the localization of Cep131 in the TZ is normal. The error bars represent the mean ± SD, n = 30. Scale bars, 5 μm (C), 1 μm (D), 2 μm (E). The data underlying this figure can be found in S1 Data. TZ, transition zone; WT, wild type. https://doi.org/10.1371/journal.pbio.3002330.g003 In spermatocyte cilia of cep162 mutants, similar to cep131 mutants, there was a significantly reduction in the signal intensities of Cep290, Dzip1, Cby, and TZ proteins Mks1 and Mks6 (Fig 3E). Additionally, we observed abnormally extended CG6652::GFP signals in 12.6% of spermatocyte centrioles (S4C Fig), indicating impaired BB docking in cep162 mutants. Live imaging of the connection between the BB and the plasma membrane further confirmed the defective BB docking in some round spermatids (S4D Fig). Importantly, we found that the BB localization of Cep131 was normal in cep162 mutants (Fig 3E). Collectively, our data indicate that cep162 and cep131 mutants exhibit similar phenotypes, with Cep131 being necessary for recruiting Cep162, whereas the reverse is not true.

Cep162 is required for the correct localization of C-terminus of Cep290 We then asked whether Cep162 is the downstream protein of Cep131 responsible for regulating the localization of Cep290 C-terminus to the TZ. In fact, the signal of overexpressed Cep290-C::GFP in cep162 mutant spermatocytes was significantly reduced compared to WT (Fig 4A), but a certain amount of signal could still be observed. Notably, such residual Cep290-C::GFP signal was much stronger than that observed in cep131 mutants (Figs 1A and 3E). Mammalian Cep290 C-terminal fragment was previously shown to interact with itself or to Cep290 N-terminal fragment, forming homodimers or heterodimers [46]. As part of endogenous Cep290 was still able to localize to the TZ in cep162 mutants, we therefore speculated that overexpressed Cep290-C::GFP might bind to remaining endogenous Cep290. To exclude this possibility, we generated the cep162 and cep2901 double mutant. Previously, we have shown that the TZ assembly is completely blocked and that centriole/basal body microtubules extend abnormally in cep2901 mutant [29]. Interestingly, we observed that both Cep162-FL::GFP and Cep162-C::GFP were localized along the abnormally extended microtubules in cep2901 single mutant (Fig 4B), suggesting that Cep162 can recognize microtubules independently of Cep290. Intriguingly, in cep2901 single mutants, Cep290-C::GFP showed a similar localization pattern as Cep162::GFP along the abnormally extended microtubules (Fig 4A), demonstrating that Cep290-C::GFP does not need full length Cep290 to be targeted to the axoneme. Furthermore, the signal of Cep290-C::GFP was significantly reduced in spermatocytes of cep162; cep2901 double mutants (Fig 4A), despite the persistence of abnormal microtubule extensions indicated by Ac-tub (S4E Fig) in these double mutants. This observation suggests that Cep162 regulates the association between Cep290 C-terminus with microtubules in spermatocyte cilia. Collectively, our results indicate that the localization of Cep290-C to the axoneme is mediated by Cep162, while it can still be retained by endogenous Cep290 in cep162 mutants. Similar results were also observed in sensory cilia, where the deletion of Cep290 did not affect the targeting of Cep162-FL or Cep162-C to the ciliary base in sensory neurons (Fig 4C), but Cep290-C::GFP was missing from the TZ in cep162; cep2901 sensory neurons (Fig 4D). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Cep162 is required for the BB localization of Cep290 C-terminus. (A) Immunostaining of Cep290-C::GFP (green) in spermatocyte cilia of WT, cep162, cep2901, and cep162; cep2901 flies, and the quantification of their corresponding relative fluorescence intensities is shown on the right. Cep290-C::GFP completely lose TZ localization in the cep162 and cep2901 double mutant spermatocytes. Centriole/basal body is marked with γ-Tubulin (red). The error bars represent the mean ± SD, n = 50. (B) Immunostaining of Cep162-FL::GFP (green) and Cep162-C::GFP (green) in WT or cep2901 spermatocyte cilia. The cartoon shows Cep162 signals in WT or Cep2901. Centriole/basal body is marked with γ-Tubulin (red). (C) Cep162 signals are grossly normal in cep2901 mutant antennae. 21A6 (blue) marks the cilia base; Actin (red) marks the ciliated region. (D) Immunostaining of Cep290-C::GFP (green) in WT, cep162, cep2901, and cep162; cep2901 antennae, and the quantification of their corresponding relative fluorescence intensities is shown on the right. Notably, Cep290-C::GFP signal is significantly reduced in cep162; cep2901 double mutants. 21A6 (blue) marks the ciliary base; Actin (red) marks the cilia region. The error bars represent the mean ± SD, n = 50. Scale bars, 4 μm (A, B), 5 μm (C, D), Zoom, 1 μm (C, D). The data underlying this figure can be found in S1 Data. BB, basal body; WT, wild type. https://doi.org/10.1371/journal.pbio.3002330.g004

Cep162 genetically interacts with Cby-Fam92 module to initiate ciliogenesis Since ciliogenesis initiation was completely abolished in cep131 and cby double mutants [43], we speculated that cep162 and cby double mutants should have a similar phenotype. Indeed, cep162; cby flies showed much more severe cilia-related defects than either single mutant. The cep162; cby flies were severely uncoordinated, unable to walk and fly. Hearing assay indicated that hearing was completely lost in double mutants (Fig 5A). Morphological examination of auditory cilia revealed a failure to form cilia in auditory organ (Fig 5B). In spermatocytes, the percentage of abnormal extensions of the centriole microtubules, labeled by CG6652, increased from 12.6% in cep162 single mutants, or 47.6% in cby single mutants to 81.1% in cep162; cby double mutants, indicating a strong synthetic defect in the initiation of ciliogenesis (Fig 5C). In addition, similar to cep131; cby double mutants, sperm flagella in cep162; cby spermatids were severely affected, with almost no axoneme observed in TEM analysis (Fig 5D). Consistent with this observation, the signals of Cep290, Dzip1, Mks1, and Mks6 were completely lost from the tips of BBs in the double mutants (Fig 5E and 5F). All these results indicate that the cep162; cby mutants mimic the phenotype of cep131; cby double mutants previously reported. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Cep162 genetically interacts with Cby-Fam92 module to initiate ciliogenesis. (A) cep162; cby flies completely lose their hearing and negative geotaxis. (B) Living images of cilia morphology in pupal antennal auditory neurons in WT flies and cep162; cby mutants. Cilia are completely lost in cep162; cby mutants. Sensory neurons are labeled by nompC-Gal4/UAS::GFP (green), and cilia are localized at the tip of dendrites. (C) Compared to WT or cby single mutants, in cep162; cby mutants, the percentage of spermatocyte cilia with aberrant extensions of CG6652 signal is significantly increased. Basal bodies are marked with γ-Tubulin (red). (D) Compared to WT which has 64 flagella in each spermatid cyst, few normal flagella are observed in spermatid cysts of cep162; cby mutants. (E) Cep290, Dzip1, Cby, and Mks1 are absent from the TZ in cep162; cby mutant spermatocytes. Right panel show the quantification of corresponding relative fluorescence intensities. Centriole/basal body is marked with γ-Tubulin (red). The error bars represent the mean ± SD, n = 30. (F) Cep290, Dzip1, Cby, and Mks1 are absent from the TZ in cep162; cby mutant sensory neuron. Right panel show the quantification of corresponding relative fluorescence intensities. 21A6 (blue) marks the ciliary base; Actin (red) marks the ciliated region. The error bars represent the mean ± SD, n = 30. Scale bars, 5 μm (B, C, F), 2 μm (D, E), Zoom, 1 μm (F). The data underlying this figure can be found in S1 Data. TZ, transition zone; WT, wild type. https://doi.org/10.1371/journal.pbio.3002330.g005 As Cby and Fam92 function together in a module to regulate ciliogenesis [30,47,48], we speculated that the combined mutation of Cep162 with Fam92 might also lead to synthetic ciliary defects. Indeed, as expected, the cep162; fam92 flies showed much more severe defects in walk and fly than either single mutant alone, and Cep290 was also completely lost in spermatocyte cilia of the double mutants (S5 Fig).

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002330

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