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.
Cilia play critical roles in cell signal transduction and organ development. This study shows that the Cep131-Cep162 module near the axoneme and the Cby-Fam92 module near the membrane work together to regulate the localization of the highly conserved ciliopathy protein Cep290 at the basal body to initiate ciliogenesis.
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 [[
The structure of cilia remains highly conserved across evolution [[
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 [[
Although speculations have accumulated that ciliary bud formation must be a coordinated event matching the TZ assembly and the ciliary membrane formation [[
Centrosome protein 131 (Cep131) localizes to both centrosome centriolar satellites and ciliary TZ in mammals [[
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.
To understand the function of the Cep131 in ciliogenesis, we generated the cep131
Graph: (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.
Cep290 is the most upstream protein known in the initiation of ciliogenesis in Drosophila [[
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.
Next, we wondered whether Cep131 directly interacts with Cep290. However, no interaction between Cep131 and Cep290 was observed in our yeast two-hybrid (Y2H) assay (S2A Fig), suggesting that additional proteins might mediate the functional interaction between Cep131 and Cep290. In mammalian cells, it has been reported that Cep162 localizes to the centriole distal end and interacts with Cep290 to promote its association with microtubules [[
Graph: (A, B) Cep131 directly interacts with Cep162. (A) Schematic representation of full-length (Cep162-FL) or truncated (Cep162-N, Cep162-C) Cep162 proteins used for interaction assays in B. (B) Cep131 interacts with Cep162-FL and Cep162-C, but not Cep162-N in the Y2H assay. The upper panel shows the presence of Y2H plasmids as evidenced by colony growth on SD-Leu-Trp plates. The lower panel shows the positive interaction between Cep131 and Cep162 as evidenced by colony growth on SD-Ade-Leu-Trp-His plates. (C) Immunostaining of Cep162:GFP (green) in spermatocyte cilia in cep131 mutants. Quantification of Cep162 signal intensity at the ciliary base is shown on the right. γ-Tubulin (red) labels the centriole/basal body. The error bars represent the mean ± SD, n = 30. (D) Immunostaining of Cep162:GFP (green), Actin (red), and 21A6 (blue) in auditory cilia or olfactory cilia in WT or cep131 mutants. Quantification of Cep162 signal intensity at the ciliary base is shown on the right. The error bars represent the mean ± SD, n = 30. (E) The subcellular localization of exogenous Cep162:GFP during spermatogenesis. From spermatogonia to late spermatocytes, Cep162 is localized at the tip of centriole/basal body. In round spermatids, as flagella elongates and the ciliary cap moves away from the BBs, Cep162 migrates with the ring centriole labeled by γ-Tubulin (arrowhead) and no signal is maintained at the BBs (arrow). During subsequent spermatid flagellar elongation, Cep162 signal disappears from the ciliary cap base labeled with Fbf1 (a transition fiber protein, red). γ-Tubulin (red) labels the centriole/basal body, axoneme is marked with Ac-Tub (magenta) and nuclei are marked with DAPI (blue). (F) The localization of GFP-tagged Cep162 N-terminus (1–447 aa) and C-terminus (448–897 aa) in spermatocytes. γ-Tubulin was used to label the BBs (red). (G) 3D-SIM images of Cep131:GFP, Cep162:GFP, Cep290-C:GFP, or Cep290-N:GFP co-immunostained with antibody against Dzip1 (red). The plots of the signal intensity are shown on the right, respectively. (H) Graph showing the radial diameter of Cep131, Cep162, Cep290-N, and Cep290-C. (I) Schematic diagrams of localization pattern of Cep131 and Cep162 in the cross section of TZ. Scale bars: 5 μm (C, D), 2 μm (F), 500 nm (G), Zoom, 1 μm (D, E). The data underlying this figure can be found in S1 Data. BB, basal body; TZ, transition zone; WT, wild type.
The function of CG42699/Cep162 in fly is unknown yet. We constructed a transgenic fly strain expressing Cep162:GFP under the control of its endogenous promoter to examine its subcellular localization in testis and ciliated sensory neurons. We observed that Cep162 was localized to the BB in all types of cilia (Fig 2C–2E), indicating that the subcellular localization of Cep162 in Drosophila is conserved. Interestingly, we found that the BB signal of Cep162 was completely lost in cep131 mutants (Fig 2C and 2D), indicating that Cep131 plays a critical role in recruiting Cep162. Notably, using transgenic flies expressing a GFP-tagged C-terminal fragment of Cep162 (Cep162-C:GFP) encompassing amino acids 448–897, we observed that Cep162 C-terminus alone was able to target to the BB (Fig 2F). Conversely, the GFP-tagged N-terminal fragment Cep162-N:GFP comprising amino acids 1–447 failed to localize to the BB. These results indicate that the C-terminus of Cep162 is critical for BB targeting. This characteristic is conserved in mammals, as it has been reported previously that the centrosome localization of mammalian Cep162 also depends on its C-terminus [[
In spermatocyte cilia, Cep162:GFP was localized at the tips of the basal bodies (Fig 2E). To more accurately determine the localization of Cep162, we performed 3D-SIM and examined its spatial distribution with respect to other BB proteins. As shown in Fig 2G, both Cep131:GFP and Cep162:GFP were surrounded by the TZ protein Dzip1. Notably, the plot profile of fluorescence showed that the distribution of Cep162 was bimodal, while the distribution of Cep131 has a single peak. Consistent with this observation, the average radial diameter of Cep162 signal was larger than that formed by Cep131 (Fig 2G and 2H), suggesting that Cep162 surrounds Cep131 (Fig 2I). Furthermore, we calculated the average radial diameters of Cep290-C:GFP and Cep290-N:GFP signals, and found that Cep290-C:GFP was close to Cep162, and formed a smaller diameter domain than that formed by Cep290-N (Fig 2G–2I). Taken together, our 3D-SIM spatial distribution data support the role of Cep162 in mediating the connection between Cep131 and Cep290 C-terminus at the TZ.
In round spermatids, the TZ starts migrating along the growing axoneme. Cep162:GFP also migrated away from the BB with the ring centriole labeled by γ-tubulin, but its signal was gradually decreased and eventually completely disappeared from the ciliary cap base (labeled by Fbf1) in elongating spermatids (Fig 2E). This behavior is similar to that of Cep131 but different from other TZ proteins [[
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, cep162
Graph: (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.
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.
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 [[
Graph: (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.
Since ciliogenesis initiation was completely abolished in cep131 and cby double mutants [[
Graph: (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.
As Cby and Fam92 function together in a module to regulate ciliogenesis [[
The synthetic defects observed in Cep162/Cep131 and Cby/Fam92 double mutants could likely be attributed to the complete loss of Cep290 signal at the BB. Considering that Cep131-Cep162 module is required for the localization of Cep290 C-terminus, and the localization pattern of Cep290 N-terminus is similar to that of Cby near the membrane, we speculated that Cby-Fam92 module might play a role in promoting the BB localization of Cep290 N-terminus. To exclude the effect of Cep290 C-terminus on our analysis, we combined deletions of Cep290 C-terminus (cep290
Graph: (A) Immunostaining of the exogenous Cep290-N:GFP (green) in spermatocyte cilia of WT, cby, cep290ΔC, cby; cep290ΔC testis and the quantification of corresponding relative fluorescence intensities is shown on the right. The signal of Cep290-C:GFP is almost lost at the tips of centrioles in cby; cep290ΔC double mutants. Centriole/basal body is marked with γ-Tubulin (red). The error bars represent the mean ± SD, n = 60. (B) Immunostaining of the exogenous Cep290-C:GFP (green) in spermatocyte cilia of WT and cby; cep290ΔC testis. Cep290-C:GFP was localized to the axoneme in cby; cep290ΔC mutants. Centriole/basal body is marked with γ-Tubulin (red). (C) Immunostaining of the endogenous Cep290 in WT, cby, cep290ΔC, and cby; cep290ΔC testis and the quantification of corresponding relative fluorescence intensities were shown on the right. The C-terminal truncated Cep290 form fails to locate to the tips of centrioles in cby; cep290ΔC spermatocytes. Centriole/basal body is marked with γ-Tubulin (red). The error bars represent the mean ± SD, n = 30. (D) The endogenous Cep290 C-terminal truncated form fails to locate to the cilia base in antennal auditory neurons of cby; cep290ΔC antenna. Right panel shows the quantification of corresponding relative fluorescence intensities. 21A6 (blue) marks the cilia base; Actin (red) marks the ciliated region. The error bars represent the mean ± SD, n = 60. (E) In cby; cep290ΔC mutants, the percentage of spermatocyte cilia with aberrant extension of CG6652 signal reaches about 79.8%. Axoneme is marked with CG6652. Centriole/basal body is marked with γ-Tubulin (red). The error bars represent the mean ± SD, n = 30. (F) Dzip1 and Mks1 are absent from the TZ in spermatocyte cilia of cby; cep290ΔC mutants. Centriole/basal body is marked with γ-Tubulin (red). The error bars represent the mean ± SD, n = 30. (G) Dzip1 and Mks1 fail to locate to the cilia base in antennal auditory neurons of cby; cep290ΔC antenna. 21A6 (blue) marks the cilia base; Actin (red) marks the ciliated region. Scale bars, 2 μm (A, C, F), 5 μm (B, D, E, G), Zoom, 1 μm (D, G). The data underlying this figure can be found in S1 Data. BB, basal body; TZ, transition zone; WT, wild type.
Given that Cby affects the localization of Cep290 N-terminus, we reasoned that endogenous truncated Cep290 may not be able to target to TZ in cby; cep290
Since Cep290 was completely lost from BBs in cby; cep290
The TZ protein Cep290 bridges the ciliary membrane and the axonemal microtubules with its N-terminus close to the membrane and its C-terminus close to the axonemal microtubules [[
Graph: (A) Model for ciliogenesis initiation in Drosophila. ① In spermatogonia, Cep131 localizes to the distal end of centriole and recruits Cep162. ② When the centriole starts to grow cilia in spermatocytes, Cep162 recruits Cep290 and promotes the binding of Cep290-C to the axoneme. ③ Subsequently, the conformation of Cep290 changes from a closed to an open state, and the N-terminus of Cep290 recruits Dzip1-Cby-Fam92 module; ④ and then start early ciliary membrane formation and ciliary bud formation. (B) The cooperative model for Cep290 localization. Cep131-Cep162 module together with Cby-Fam92 module regulates the localization of Cep290 at the TZ. Mechanistically, Cep131 recruits Cep162 which mediates the association of C-terminus of Cep290 to microtubule. The N-terminus of Cep290 recruits Dzip1-Cby-Fam92 module to start early ciliary membrane formation and ciliary bud formation, whereas Cby-Fam92-mediated ciliary membrane formation has a positive feedback effect on promoting the association of Cep290 N-terminus with the ciliary membrane. TZ, transition zone.
Previously, Drivas and colleagues have proposed a conformation change model of Cep290 during ciliogenesis [[
Notably, Cep290 has its own microtubule-binding domain and membrane-binding amphipathic α-helix motif [[
Our work not only uncovers the molecular mechanism of the synthetic interaction between Cby-Fam92 module and Cep131-Cep162 module in Cep290 recruitment and ciliogenesis initiation, but also reveals a novel molecular function of Cep131 in ciliogenesis, which recruits Cep162 to promote the binding of the C-terminus of Cep290 to the axoneme. We demonstrated that the Cep131-Cep162 module promotes Cep290 C-terminus binding to the axoneme, whereas Cby-Fam92 module promotes and stabilizes the localization of Cep290 N-terminus on the membrane, therefore, the concurrent mutation of these 2 modules collectively leads to the failure of Cep290 to localize to the TZ and blocks the initiation of ciliogenesis. Cep290 is an intriguing ciliopathy gene. More than 130 mutations have been identified, but their associated phenotypes can be dramatically different, suggesting that there may be genetic modifiers involved in the development of their phenotypes [[
Transgenic flies of Cby:GFP, Cep131:GFP, Cep290-N:GFP, Cep290-C:GFP, CG6652:GFP, Mks1:GFP, Mks6:GFP, and nompC-Gal4;UAS:GFP were previously reported [[
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Generation of cep131 and cep162 mutants were performed as previously reported [[
Primers used to construct gRNA vector:
Cep162 g1 F: 5′-GTCGCTCTGAAGGCGAACGGGTTTTAGAGCTAGAAATAGC-3′;
Cep162 g1 R: 5′-CCGTTCGCCTTCAGAGCGACCGACGTTAAATTGAAAATAGG-3′;
Cep162 g2 F: 5′-TCAGTATGCGCTCCATCTCGGTTTTAGAGCTAGAAATAGC-3′; Cep162 g2 R: 5′-CGAGATGGAGCGCATACTGACGACGTTAAATTGAAAATAGG-3′; Cep131 g1 F: 5′-CAAGCACAAGCCAGGACTGG GTTTTAGAGCTAGAAATAGC-3′: Cep131 g1 R: 5′-CCAGTCCTGGCTTGTGCTTGCGACGTTAAATTGAAAATAGG-3′: Cep131 g2 F: 5′-CTCCCTCTGCGAGAAGGTGGGTTTTAGAGCTAGAAATAGC-3′: Cep131 g2 R: 5′-CCACCTTCTCGCAGAGGGAGCGACGTTAAATTGAAAATAGG-3′.
Primers used to identify mutants:
Cep162 F: 5′-ATATTTTCGCGAGCTGAGGACAC-3′;
Cep162 R: 5′-GCGATGTGAGTCTCATATTTGGC-3′;
Cep131 F: 5′-CATCAGTGTGGGCAGCCTACG-3′;
Cep131 R: 5′-GCGAATGCTAGTCTCGATCTGC-3′.
For IF staining of antennae or testes, 36 to 48 h after puparium formation (APF), pupae were collected and their antennae or testes were dissected with forceps in PBS. Antennae or testes were transferred to the center of a coverslip, and then gently covered by a slide over the coverslip. The slide was dipped into liquid nitrogen for 30 s, and the coverslip was immediately removed with a blade. The specimens were then fixed using methanol (−20°C) for 15 min, followed by acetone (−20°C) for 10 min. To block nonspecific binding, the specimens were incubated for 1 h in blocking buffer (0.1% Triton X-100, 3% bovine serum albumen in PBS). The primary antibodies were applied overnight in a moisture chamber at 4°C, and then the secondary antibodies were applied for 3 h at room temperature.
The primary antibodies used were as follows: Rabbit anti-Dzip1 (aa 451–737), Rabbit anti-Cep290 (aa 292–541), and Rabbit anti-Fbf1 (aa 1–336) antibodies were generated at YOUKE Biotech, rabbit anti-GFP (1:500, ab290, Abcam), mouse anti-GFP (1:200, 11814460001, Roche), mouse anti-Ac-tub (1:500, T6973, Sigma-Aldrich), mouse anti-γ-Tubulin (1:500, T5326, Sigma-Aldrich), and mouse anti-21A6 (1:200, AB528449, DSHB). The following secondary antibodies were used: goat anti-mouse Alexa Fluor 488 (1:1,000, A-11001, Invitrogen), goat anti-rabbit Alexa Fluor 594 (1:1,000, A1000701, Invitrogen), goat anti-rabbit Alexa Fluor 488 (1:1,000, A-11006, Invitrogen), goat anti-mouse IgG1 Alexa Fluor 488 (1:1,000, A-21121, Invitrogen), goat anti-mouse Alexa Fluor 594 (1:1,000, A-11007, Invitrogen), goat anti-mouse Alexa Fluor 647 (1:1,000, A-21242, Invitrogen).
For IF staining, images were taken on a fluorescence microscope (Nikon Ti) with a 100× (1.4 NA) oil-immersion objective, or a confocal microscope (Leica Stellaris 5) with a 63× (1.4 NA) oil-immersion objective, or the Delta Vision OMX SR (GE Healthcare) with a 60× (1.42 NA) oil-immersion objective. Confocal images were acquired as Z-stacks (0.5 to 0.8 μm for Z-step size and 3 to 5 for number of steps) using xzy scan pattern. The sections of 3D-SIM images were acquired at 0.125 μm Z-steps (20 steps) and the raw data were reconstructed by using softWoRx software (GE Healthcare). Images were quantified for the pixel density using ImageJ (National Institutes of Health). For quantification of the pixel density, images were taken using equal microscopy settings. The pixel density values were calculated by the sum pixel density values in a defined region subtracting the sum pixel density values in an area close to the defined region. All images assembled into figures using Photoshop (CS5, Adobe).
Samples were prepared for electron microscopy as previously described [[
Virgin flies were collected and cultured in fresh medium for 3 to 5 days. A total of 50 flies were sorted into 5 measuring vials of 10 each, and then tapping flies to the bottom of the vials and counting the number of flies that climbed over the 8 cm high bar within 10 s. Each group was repeated 3 times.
Third instar larvae were divided into 5 groups of 5, placed on an agar plate above the speaker, and stimulated with 1k Hz sound every 30 s, the number of larvae with contractile responds on the head or body within 1 s after stimulation were counted. Each group was repeated 5 times.
Cep131-FL (1–1114 aa), Cep131-N1 (1–480 aa), Cep131-M1 (481–781 aa), Cep131-C1 (782–1114 aa), Cep162-N (1–447 aa), Cep162-C (448–897 aa), Cep290-N (1–887 aa), and Cep290-ΔN (888–1978 aa) were introduced into either pGBKT7 or pGADT7 vector. Clones in pGADT7 and pGBKT7 were transformed into yeast strain AH109 (Takara Bio). The yeasts were grown on SD-Leu-Trp plates at 30°C. After 3 to 4 days incubation, 2 independent positive colonies were picked and diluted with TE buffer (10 mM Tris-HCl, 1 mM EDTA (pH 7.5)), and then transferred to SD-Ade-Leu-Trp-His or SD-Leu-Trp-His plates with 3-amino-1,2,4-triazole and incubated for 5 days at 30°C.
To generate bacterial expression plasmids for His-Cep162-N (1–447 aa), His-Cep162-C (448–897 aa), His-Cep131-N (1–549 aa), His-Cep131-C (550–1114 aa), the cDNA fragments encoding the indicated amino acids were amplified by PCR and subcloned into the pET28a or pGEX-4 T-1 vector. The proteins were expressed using BL21 (DE3) E. coli. strain with IPTG induction and purified with Glutathione-agarose beads or Ni-resin (Yeasen). Purified His-Cep131 truncations were incubated with immobilized GST or GST-Cep162 truncations in the binding buffer (25 mM Tris-HCl at pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM dithiothreitol, 10% glycerol, and protease inhibitors) at 4°C for 4 h. After incubation, the beads were washed 3 times with washing buffer (binding buffer with 20 mM imidazole) and then boiled for 10 min in 1 × SDS loading buffer. The protein samples were then separated by SDS-PAGE gels and transferred to the PVDF membrane for either immunoblotting with His antibody (Invitrogen) or staining with Ponceau S. Primary antibodies were used at a dilution of 1/1,000, and secondary antibodies were used at a dilution of 1/2,000. All uncropped images can be found in S1 Raw Image.
Data were analyzed and graphed using Microsoft Excel or Graphpad Prism. Unless otherwise indicated, all error bars represent the standard deviation (SD) of the mean, and the statistical significance between data was assessed with an unpaired two-tailed Student's T tests. Differences between data were considered statistically significant when P ≤ 0.05.
S1 Fig
Identification and phenotype analysis of cep131 mutants.
(A) Diagram showing of the generation of cep131 mutants. Schematics show the genomic (upper panel) and protein (lower panel) structures of Cep131, along with the predict protein product of Cep131
S2 Fig
Yeast two-hybrid assay of the interaction between Cep290 and Cep162 or Cep131.
(A) In Y2H assay, Cep290 interacts with Cep162 (CG42699), but not Cep131 in Drosophila. LW: Selective media SD-Leu-Trp plates. LWH: Selective media SD-Leu-Trp-His plates. LWHA: SD-Ade-Leu-Trp-His plates. (B) The GST pull-down assay confirmed the direct interaction between Cep131 and Cep162 in vitro. GST, GST-Cep162-N, and GST-Cep162-C recombinant proteins were pulled down with His-Cep131-N or His-Cep131-C proteins. (C) In Y2H assay, Cep162 interacts with Cep131-N1 and Cep131-C1, but not Cep131-M1. LW: Selective media SD-Leu-Trp plates. LWH: Selective media SD-Leu-Trp-His plates. LWHA: SD-Ade-Leu-Trp-His plates.(TIF)
S3 Fig
Protein sequence alignment of the C-terminus of human Cep162, mice Cep162, and Drosophila CG42699.
CG42699 is the only one significant alignment sequence when searching for homology of human or mice Cep162 protein in Drosophila melanogaster.(TIF)
S4 Fig
Cep162 is required for BBs docking to the plasma membrane in spermatocytes.
(A) Sequence confirmation of the deletion in cep162 mutant. Primers used for sequence are marked with orange frames. The locations of 2 gRNAs used for mutant generation are underlined with black. Red and blue frames label the boundary of deletion cep162 in mutant. (B) Genotyping of cep162 flies using PCR. The amplification products were 881 bp long for WT and 555 bp long for mutants. (C) Immunostaining of CG6652 in spermatocyte cilia of WT or cep162
S5 Fig
Synthetic defects in Cep290 localization in cep162; fam92 or fam92; cep290ΔC mutants.
Cep290 are absent from the TZ in the spermatocyte of cep162; fam92 or fam92; cep290
S1 Raw Image
Full scans of western blots.
(TIF)
S1 Data
Raw data underlies the figures.
(XLSX)
Alvarez-Garcia Ines Senior Editor
6 Sep 2023
Dear Dr Wei,
Thank you for submitting your manuscript entitled "Cep290 is cooperatively maintained at basal body by Cep131-Cep162 and Cby-Fam92 modules" for consideration as a Research Article by PLOS Biology.
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Ines Alvarez-Garcia, PhD
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Alvarez-Garcia Ines Senior Editor
12 Oct 2023
Dear Dr Wei,
Thank you for your patience while your manuscript entitled "Cep290 is cooperatively maintained at basal body by Cep131-Cep162 and Cby-Fam92 modules" went through peer-review at PLOS Biology as an Update Article. Your manuscript has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by two independent reviewers.
The reviews are attached below. You will see that the reviewers find the conclusions interesting and recommend pursuing the paper for publication after addressing several issues. Reviewer 1 would like you to clarify several points regarding your previous paper, such as the relationship between Cep290 and the Cby-Fam92 module during ciliogenesis, among other issues. Reviewer 2 only raises several minor points that should be easy to address.
In light of the reviews, we are pleased to offer you the opportunity to address the comments from the reviewers in a revision that we anticipate should not take you very long. We will then assess your revised manuscript and your response to the reviewers' comments with our Academic Editor aiming to avoid further rounds of peer-review, although might need to consult with the reviewers, depending on the nature of the revisions.
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Sincerely,
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Senior Editor
PLOS Biology
ialvarez-garcia@plos.org
----------------------------------------------------------------
Reviewers' comments
Rev. 1:
This paper by Wu et al. discusses the regulation of CEP290 localization during ciliogenesis in Drosophila. The authors demonstrate that the Cep131-Cep162 module near the axoneme and the Cby-Fam92 module at the membrane cooperate to control the basal body localization of Cep290, facilitating transition zone assembly during the initiation of ciliogenesis. Previous research (Vieillard et al., 2016) has already shown that Cep131 and Cby collaborate to recruit Cep290 and build the transition zone. However, this study adds a novel contribution by highlighting the essential role of Cep162, which bridges Cep131 and the C-terminus of Cep290 in transition zone formation. Additionally, the authors provide evidence that Dzip1-Cby-Fam92 interacts with the N-terminus of Cep290, playing a role in Cep290 recruitment. In summary, the paper proposes a model in which three central molecular pathway modules—Cep131-Cep162, Cep290, and Dzip1-Cby-Fam92—cooperatively regulate the initiation of cilium assembly. This work contributes to our understanding of the critical role of Cep290 in ciliogenesis and its regulation by other ciliogenesis modules, shedding light on related ciliopathies. I do have some questions/concerns that should be addressed.
Major Points:
Rev. 2:
CEP290 is a major structural element of the ciliary transition zone that was shown initially in Chlamydomonas to link the axoneme to the ciliary membrane. It has a central role in gating ciliary transport. Human CEP290 mutations are associated with a range of ciliopathy syndromes including but limited to MKS, JBTS, BBS, LCA, and SLS. This work builds on previous work showing that Cep290 integration into the transition zone requires Cep131 and Cby, and on prior work indicating that Cep290 associates with the axoneme through the C-terminal domain, and with the ciliary membrane through the N-terminal domain.
The authors provide strong evidence in Drosophila that Cep290 is integrated into the transition zone through association by its C-terminal region with a Cep131-Cep162 module at the axoneme, and with the ciliary membrane by its N-terminal region by association with the Cby-Fam92 module. They examine this molecular organization in spermatocyte cilia/spermatid flagella and in sensory cilia.
The authors show that Cep131 and Cep162 interact directly and generated deletion alleles of both genes. They additionally showed that Cep162 associates with Cep290 at its C-terminal region, providing a link between Cep290 and its association with the axoneme. They further show that the N-terminal region of Cep290 associates with the Cby-Fam92 module at the ciliary membrane. Mutation of either Cep131 or Cep162, together with either Cby or Fam92 produces a severe loss of cilia similar to the phenotype of loss of Cep290.
Overall, the work advances our understanding of how Cep290 integrates into the transition zone of cilia. A model depicting an epistatic ordering of the assembly of the components is supported by the findings and presented in Figure 7. The work is clearly communicated, and the experiments are well-presented and support the major findings of the work.
I have some minor points to address:
Additional points:
Manuscript needs page numbers.
Please run a spellcheck on the ms, there are numerous typos.
Typos in Introduction: "strucutres", "Chibbly", "foramtion", "seemly", "cordinating", "stablize". In Results: "Collectivley". In Discussion: "accessbile". In legends: "Trpplates"
21 Nov 2023
Attachment
Submitted filename: Response to Reviewers.docx
Alvarez-Garcia Ines Senior Editor
11 Dec 2023
Dear Dr Wei,
Thank you for your patience while we considered your revised manuscript entitled "Cep290 is cooperatively maintained at basal body by Cep131-Cep162 and Cby-Fam92 modules" for publication as a Update Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors and the Academic Editor.
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"Cep131-Cep162 and Cby-Fam92 complexes cooperatively maintain Cep290 at the basal body and contribute to ciliogenesis initiation"
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Sincerely,
Ines
--
Ines Alvarez-Garcia, PhD
Senior Editor
PLOS Biology
ialvarez-garcia@plos.org
------------------------------------------------------------------------
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22 Dec 2023
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Submitted filename: Response to Reviewers.docx
Alvarez-Garcia Ines Senior Editor
31 Jan 2024
Dear Dr Wei,
Thank you for the submission of your revised Update Article entitled "Cep131-Cep162 and Cby-Fam92 complexes cooperatively maintain Cep290 at the basal body and contribute to ciliogenesis initiation" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Renata Basto, I am delighted to let you know that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.
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Sincerely,
Ines
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Ines Alvarez-Garcia, PhD
Senior Editor
PLOS Biology
ialvarez-garcia@plos.org
We thank Dr. Wei Zhang from Tsinghua University for strains. We thank core facilities of Drosophila Resource and Technology (SIBCB, CAS), confocal imaging core facilities (SIAT, CAS), and EM core facilities (SIPPE, CAS) for their technical support.
• APF
- after puparium formation
• BB
- basal body
• BBS
- Bardet–Biedl syndrome
• IFT
- intraflagellar transport
• JBTS
- Joubert syndrome
• LCA
- Leber congenital amaurosis
• MKS
- Meckel–Gruber syndrome
• SLS
- Senior–Loken syndrome
• TZ
- transition zone
• WT
- wild type
By Zhimao Wu; Huicheng Chen; Yingying Zhang; Yaru Wang; Qiaoling Wang; Céline Augière; Yanan Hou; Yuejun Fu; Ying Peng; Bénédicte Durand and Qing Wei
Reported by Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author