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Differential regulation of transition zone and centriole proteins contributes to ciliary base diversity

Nature Cell Biology volume 20, pages 928–941 (2018)Cite this article

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Abstract

Cilia are evolutionarily conserved structures with many sensory and motility-related functions. The ciliary base, composed of the basal body and the transition zone, is critical for cilia assembly and function, but its contribution to cilia diversity remains unknown. Hence, we generated a high-resolution structural and biochemical atlas of the ciliary base of four functionally distinct neuronal and sperm cilia types within an organism, Drosophila melanogaster. We uncovered a common scaffold and diverse structures associated with different localization of 15 evolutionarily conserved components. Furthermore, CEP290 (also known as NPHP6) is involved in the formation of highly diverse transition zone links. In addition, the cartwheel components SAS6 and ANA2 (also known as STIL) have an underappreciated role in basal body elongation, which depends on BLD10 (also known as CEP135). The differential expression of these cartwheel components contributes to diversity in basal body length. Our results offer a plausible explanation to how mutations in conserved ciliary base components lead to tissue-specific diseases.

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Fig. 1: Micron-scale organizational map of the ciliary base shows diverse global organization.
Fig. 2: BB nanoscale structure and composition vary between cell types.
Fig. 3: The TZ nanoscale structure and composition vary in different cilia types.
Fig. 4: CEP290 is required to form diverse non-MT structures in different types of TZs, but not the hooks in spermatocytes.
Fig. 5: SAS6 is essential for centriole assembly, but is not required for BB function in neurons.
Fig. 6: Cartwheel components, SAS6 and ANA2, are both required for BLD10 localization to the sperm BB and for its elongation.
Fig. 7: SAS6 and ANA2 cooperate to elicit ectopic BB elongation in olfactory neurons, leading to reduced smelling ability.
Fig. 8: The ciliary structure and composition are distinct in different cell types.

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Acknowledgements

We thank T. Avidor-Reiss, B. Durand, T. Megraw and J. Raff for reagents. We thank B. Durand, P. Bastin, A. Dammermann, L. Saúde, J. Shah, G. Marteil, M. Lince Faria, S. Zitouni and MBD Lab members for reviewing the manuscript and providing helpful discussions on the manuscript. We thank the Gurdon Institute Imaging Facility (Cambridge, UK), the Bacterial Cell Biology Lab (M. Pinho, ITQB, Portugal), IGC Advance Imaging (and its Head, G G. Martins), Histopathology and Electron Microscopy units (A. L. Sousa, S. Bonucci and E. Tranfield) for helping with sample preparation and image acquisition, and the IGC fly facility for fly husbandry. S.C.J. and S.W. are supported by the FCT (Fundação Portuguesa para a Ciência e Tecnologia) Fellowships SFRH/BPD/87479/2012 and SFRH/BD/52176/2013, respectively. The laboratory of H.M. is supported by the ERC (ERC-681443-CODECHECK) and FLAD Life Science 2020. M.B.-D. and her laboratory are supported by the Fundação Calouste Gulbenkian/Instituto Gulbenkian de Ciência, an EMBO installation grant and ERC grants (ERC-261344-CentrioleStructNumb and ERC-683258-CentrioleBirthDeath).

Author contributions

S.C.J., S.M. and P.M. performed most of the experiments with assistance from J.R., S.W. and A.P. S.W. cloned the CEP290 from the fly, while S.C.J. characterized the transgenic flies. S.C.J. performed the walking behaviour experiments and STED experiments with help from J.R., and A.P. and H.M., respectively. S.C.J. and M.B.-D. designed all of the experiments with input from S.M., P.M., S.W., A.P. and H.M. S.C.J. and M.B.-D. wrote the manuscript and all authors commented on the manuscript.

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Author notes

  1. Susana Mendonça

    Present address: Portugal and Instituto de Investigação e Inovação em Saúde—i3S, Universidade do Porto, Porto, Portugal

  2. Pedro Machado

    Present address: European Molecular Biology Laboratory, Electron Microscopy Core Facility, Heidelberg, Germany

  3. Sascha Werner

    Present address: Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany

  4. Jaqueline Rocha

    Present address: Centro de Biotecnologia e Química Fina, Universidade Católica Portuguesa, Porto, Portugal

Authors and Affiliations

  1. Instituto Gulbenkian de Ciência, Oeiras, Portugal

    Swadhin Chandra Jana, Susana Mendonça, Pedro Machado, Sascha Werner, Jaqueline Rocha & Mónica Bettencourt-Dias

  2. Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal

    António Pereira & Helder Maiato

  3. Instituto de Investigação e Inovação em Saúde—i3S, Universidade do Porto, Porto, Portugal

    António Pereira & Helder Maiato

  4. Department of Biomedicine, Faculdade de Medicina, Universidade do Porto, Porto, Portugal

    Helder Maiato

  5. Instituto de Patologia e Imunologia Molecular (IPATIMUP), Universidade do Porto, Porto, Portugal

    Susana Mendonça

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Supplementary Figure 1 Quantification of the localisation patterns of ciliary base components in different ciliated cells in adult Drosophila.

a, b) Scheme of different Drosophila ciliated cells that were studied in this work. a) Large basiconic olfactory and auditory neurons located in the marked regions of the third and second segment of the adult antenna, respectively. Primary cilia-like structure in spermatocytes and long flagella in early elongating spermatid were also studied. Schemes show longitudinal views and representative electron micrograph cross-sections of the marked regions. b) The scheme represents basal body (BB) elongation and diverse cilia assembly during Drosophila spermatogenesis. c, d) Example wide-field fluorescence and 3D-SIM micrographs of olfactory and spermatocyte cilia. Scheme of a set of three olfactory neurons innervating sensillum basiconica (c) and spermatocyte (d) showing BBs and cilia. Representative wide-field pictures (ii) and SIM pictures (iii) showing acetylated α-tubulin and PACT, a BB marker, in a set of three olfactory neurons. e) Representative wide-field pictures of olfactory (i) and auditory (ii) cilia showing rootletin (red), SAS4 (green) and DPLP (blue), a PCM marker. Arrow heads marks the rootlet region at the ciliary base. The scale bars in a, c, d and e are 100 nm, 10 µm, 10 µm and 1 µm, respectively. f-m) Quantification of the localisation patterns of BB, PCM and TZ components in neurons and sperm cells obtained using 3D-SIM. i) Scheme shows the olfactory, auditory, spermatocyte and spermatid ciliary bases with PCM and BBs (f-i) and TZs (j-m). ii-iii) Schemes show the method for quantification of proteins and different parameters. iv) Length (with mean ± S.D.), diameter (mean ± S.D.) and other variables (mean ± S.D.) of the defined zones are mentioned in the table. All values mentioned in the table are in nanometer (nm). n = 16 samples. The schemes (in left) representing the localisation patterns of the proteins are drawn based on the quantification shown in the right. The experiments presented in a, c, d, and f were repeated independently thrice, while the experiments in e were repeated independently twice. Notably, we used both TEM of ciliary bases, and 3D-SIM of a common BB marker, PACT (the C-terminal domain of pericentrin-like protein (PLP)) to estimate BB size independently, and obtained concordant results (see Fig. 1a–d, Supplementary Fig. 1F-I,2).

Supplementary Figure 2 Different ciliary bases show both similar and variable elements.

a) Left: Longitudinal tomogram stills of the ciliary base in olfactory neurons showing a proximal BB (pBB-white arrowhead), a distal BB (dBB- red arrowhead) and the transition zone (TZ). Right: Model based on the tomogram data. Model of the BB and ciliary microtubules (light green), cytoplasmic MTs (orange) that nucleate from the BBs, MTs that nucleate from the proximal BB and extend into the cilia (brown), non-MT electron densities around BBs (dark blue), the electron densities of rootlet (cyan), vesicles at the ciliary base (magenta), connections between dBB-cell membrane (golden) and the cell/ciliary membranes (black) (see also Fig. 1 and Supplementary Video V1). Based on features at the ciliary base, olfactory neurons can be divided into two types: Type-1) where singlet MTs are absent in the lumen of both BB and TZ (example 1) and Type-2) where one or more singlet MTs (orange) are present in the lumen of BBs and TZ (example 2). ~70% of olfactory neurons are of Type-1, while ~30% of them are of Type-2 (quantification not shown). b) Longitudinal tomogram stills of the ciliary base in auditory neurons showing pBB (white arrowhead), dBB (red arrowhead) and TZ. Right: Model based on the tomogram data. For auditory neurons, we modelled all objects described in a, electron density around the MTs in TZ (dark blue), and the rootlet striations (magenta). Notably, in the example 2 of olfactory cilia (a) we observed singlet microtubules (white arrows) that are bent in olfactory neurons, and the connections between the dBB and the cell membrane are less obvious in single sections of both types of neurons, justifying the importance of collecting and analysing tomograms to model these ciliary bases. c) Electron micrographs of different types of bases showing cytoplasmic MTs around BB and rootlets. The insets present regions marked with dotted squares. d, e) Representative electron micrographs of longitudinal (i) and sets of serial cross sections of marked regions (ii) of BBs in olfactory (d) and auditory neurons (e). For the cross-section series analysis, 70 nm serial sections were collected. Note that the number of singlets and the relative position of singlets and doublets in the pBB varies between individual auditory neurons. All electron micrographs in c-e represent features observed in 3 samples (the experiments presented in this figure were repeated independently twice with similar results). Scale bars on the longitudinal (a, b, c, di and fi) and cross (dii and fii) section micrographs are 500 nm and 100 nm, respectively.

Supplementary Figure 3 Both length and non-MT based electron-dense structures of the TZ vary between different cilia types.

Representative electron micrographs show longitudinal sections (i) and sets of serial cross sections (ii) of the marked regions in the TZs of olfactory neurons (a), auditory neurons (b), spermatocytes (c) and early elongating spermatids (d). For cross-section series analysis, 70 nm serial sections were collected. Arrowheads mark the transition fibres (similar to distal appendage) that connect the distal tip of the BB to the ciliary membrane. The white arrow marks the single MT in the lumen of the spermatid BB in d. The cross section images in a, c and d are from serial sections of a TZ of olfactory, spermatocyte and spermatid, respectively. The cross section images in b were compiled from three different sets of serial sections. Notably, here the area that is distal to the BB and shows electron density on and around the MTs in longitudinal sections of ciliary bases is considered TZ. The region distal to the TZ is considered ciliary shaft (axoneme). All electron micrographs shown here represent features that were observed in 3 samples (the experiments presented in this figure were repeated independently twice with similar results). Scale bars on the longitudinal (a-di) and cross (a-dii) section micrographs are 500 nm and 100 nm, respectively (see also Fig. 3 and Supplementary Videos V7,8).

Supplementary Figure 4 CEP290 localises to the TZ in 9-fold symmetric manner and is required to form cilia in all Drosophila ciliated cells.

a-c) Localisation analysis of GFP tagged CEP290 proteins at different TZs. a) Representative SIM images of the ciliary bases of olfactory neurons marked using acetylated α-tubulin (red) and ectopically expressing CEP290::GFP (Gal4cha19b/UAS-CEP290::GFP) or GFP::CEP290 (Gal4cha19b/UAS-GFP::CEP290). SIM analysis shows CEP290::GFP localises towards the lumen and on the MTs and GFP::CEP290 localises towards the ciliary membrane in the olfactory TZ. b) Schemes show the method of measuring the distance (d) between the MT or the hook and ciliary membrane at the different TZs. c) (i) Schemes show methods of measuring the inter-distance between the outer (do) and inner (di) tips of adjacent MT-membrane linkers. Representative STED micrographs of GFP::CEP290 in the cross-section of the TZs of olfactory (i: do = 99 ± 23 nm; n = 194), auditory (ii: do = 100 ± 24 nm; n = 59) and spermatocyte (iii: do = 90 ± 23 nm; n = 112) cilia. While the white arrowheads mark the resolved GFP foci, empty arrowheads (with dotted border) indicate the postulated missing foci´s position. d-h) CEP290 is required for all cilia assembly in the fly. d) i) The scheme shows the odour repulsion test using the T-tube to measure the ability of adult flies to detect a repulsive odour (Benzaldehyde). ii) Quantification of the percentage of flies that are in the compartment with repulsive odor. A null mutant of Orco, a co-receptor essential for olfaction, was used as a positive control (control1, n = 60 and Orcomutant, n = 60: **p < 0.0001, two-tail Mann-Whitney Test; control2, n = 80 and CEP290RNAi1, n = 90: **p < 0.0001, two-tail Mann-Whitney Test). e) i) Scheme depicts the bang assay and the vertical tube used to test the gravitaxis ability of adult flies. ii) Quantification of the time taken by ≥80% of the flies to successfully climb the half height mark of the tube (18 cm long) (control1, n = 70, control2, n = 60 and CEP290RNAi1, n = 60: **p < 0.0001, two-tail Mann-Whitney Test). iii) Representative kymographs of ten flies with respective genotypes followed for the first 5 seconds after the bang. f) i) Representative pictures of olfactory cilia (marked using Acetylated α-Tubulin) in flies with different genotypes. ii) Representative images of different types of olfactory shafts. iii) Quantification of ciliary defects in flies with different genotypes (control1, n = 36, Cep290mutant, n = 27, and CEP290RNAi1, n = 25). g) i) Representative electron micrographs of cross sections of scolopale in second antennal segments of flies with different genotypes. ii) Quantification of percentage of scolopale with two or more cilia in flies with different genotypes (control1, n = 48, and CEP290RNAi1, n = 34). h) i) Quantification of number of progeny produced per male with different genotypes (control1, n = 10, control3, n = 11, and CEP290RNAi2, n = 11: **p < 0.0001, two-tail Mann-Whitney Test). ii) Representative pictures of the BBs in spermatids marked using BLD10, a centriolar protein, in flies with different genotypes. DNA, BB and sperm flagella are marked by DAPI (blue), BLD10 (green) and acetylated tubulin (red), respectively. iii) Quantification of the length of BBs marked using BLD10 as shown in (ii) (control3, n = 153, and CEP290RNAi2, n = 90: **p < 0.0001, two-tail Mann-Whitney Test). iv) Representative cross-section micrographs show the axoneme bundle of the elongating flagella in different flies. Notably, while 9 + 2 arrangement of the MTs was normal in control3 flies, the MT arrangement was defective in CEP290RNAi2 flies (see insets). All experiments were repeated independently with similar results: a (thrice), c (twice), d (thrice), e (thrice), f (twice), g (twice), h (twice). Scale bars in a, c, eiii, f, g, hii and Hiv represent 1 µm, 100 nm, 1 cm, 10 µm, 500 nm, 10 µm and 500 nm, respectively. In each Tukey-box plot, centre line indicates median and error bars indicate full range of variation (from minimum-to-maximum) and dots are outliers. For different variables of each Tukey-box plot and fly genotypes see Supplementary Table 4 and Supplementary Tables 1,3, respectively.

Supplementary Figure 5 Controls for the specificity of RNAi tools used in this manuscript.

Tukey-Box plot of the number of progeny produced per male (i), length of BBs (ii) and total GFP intensity of candidate proteins at the spermatocyte ciliary base: CEP290 (a), SAS6 (b), ANA2 (c) and BLD10 (d). Here, for each candidate (X) gene S1, S2, S3 and S4 represent the flies with ectopic-expression of UAS-mCD8GFP (Control 1X), RNAi (Knock downX), UAS-X-GFP in RNAi background (RescueX) and UAS-X-GFP in wild type background (Control 2X(cOE)). cOE indicates conditional over-expression of the given candidate in the testes. While for a-di the total number of males used for each genotype is n ≥ 10, for a-dii and b-diii, the number of BBs quantified for each genotype is n ≥ 63 and n ≥ 60 (30 pairs of BBs), respectively. For Aiii, the number of BB quantified for each genotype is n ≥ 42. NA indicates not applicable. Note that we rescued the knock down phenotypes of all candidate molecules (SAS6, ANA2, CEP290, and BLD10) both for BB length (ii) and male fertility (i). We further quantified the protein depletion (iii) in RNAi experiments in sperm cells for all candidates. Altogether, this analysis shows the specificity of the tools we used. All experiments presented in a-d were repeated independently twice with similar results. In each Tukey-box plot, centre line indicates median and error bars indicate full range of variation (from minimum-to-maximum) and dots are outliers. For exact sample size, different variables of each Tukey-box plot, statistical tests used, exact p values and fly genotypes see Supplementary Table 4 and Supplementary Tables 1,3, respectively.

Supplementary Figure 6 SAS6 is differentially required in neurons and sperm cells.

a, b) SAS6 is essential for centriole assembly in neurons, but is not required for neuronal cilia function. a) Representative electron micrographs show longitudinal and cross sections through the centrioles in olfactory neurons (before ciliogenesis: at 24 h APF) in wild type flies. Note that those centrioles are close to the cell membrane and have the cartwheel. b) i) The experimental setting used to reduce/remove SAS6 during centriole and cilia biogenesis in neurons. ii) Representative images show olfactory and auditory neurons in flies with different genotypes. Cilia in olfactory and auditory neurons were studied using anti-acetylated tubulin (green) and anti-glutamylated tubulin (green) antibody, respectively. PLP (red, centrosomes) and DAPI (blue, DNA). Arrowheads mark BBs and arrows mark cilia. c) Both SAS6 and ANA2 are required for sperm BB elongation, being important for male fertility. i) Schematic representation of the experimental setting used to reduce/remove SAS6 before and after centriole biogenesis in sperm cells. ii) Tukey-Box plot of the number of progeny produced per male with different genotypes (control1, n = 10, control5, n = 10, and SAS6RNAi2, n = 11: **p < 0.001, two-tail Mann-Whitney Test; control3, n = 11, SAS6RNAi3, n = 10 **p < 0.01, ANA2RNAi1, n = 10 *p < 0.05, and BLD10RNAi1, n = 10: **p < 0.01, Kruskal-Wallis Test). iii) Tukey-Box plot of the number of BBs per cell in mature spermatocytes (control1, n = 32, and SAS6mutant, n = 42: **p < 0.0001, two-tail Mann-Whitney Test; control3, n = 62, SAS6RNAi3, n = 56, ANA2RNAi1, n = 40, and BLD10RNAi1, n = 63). iv) Representative images of mature spermatocyte BBs of flies with different genotypes (control3, SAS6RNAi3 and ANA2RNAi1). RFP::PACT (red) marks BBs and Anti-SAS6 antibody (green) stains the proximal part of the centriole. Insets show SAS6 (green) close to the arrowhead (in grey scale). v) Tukey-Box plot of the total amount of SAS6 at the mature spermatocyte BBs of the different genotypes. For v (control3, n = 60, SAS6RNAi3, n = 42 **p < 0.0001, and ANA2RNAi1, n = 40, **p < 0.0001: Kruskal-Wallis Test; n represent the number of BB pairs). All experiments in a, cii, ciii were repeated independently twice, while in b, civ-v were repeated independently thrice with similar results. Scale bars in a, bii,iii and civ represent 100 nm, 5 µm and 1 µm, respectively. In each Tukey-box plot, centre line indicates median and error bars indicate full range of variation (from minimum-to-maximum) and dots are outliers. For different variables of each Tukey-box plot and fly genotypes see Supplementary Table 4 and Supplementary Tables 1,3, respectively.

Supplementary Figure 7 SAS6 and ANA2 cooperate to elicit ectopic neuronal BB elongation, leading to defects in sensory behaviour.

a) Representative electron micrographs of longitudinal sections through the ciliary base of olfactory neurons in flies with different genotypes. Empty arrow head marks the proximal region of the dBB without cartwheel. b) Representative electron micrographs of the dBB cross sections of olfactory neurons in flies with either no or simultaneous ectopic expression of SAS6 and ANA2. c) Quantification of the time taken by ≥80% of the flies to successfully climb the half way mark of the 18 cm long tube (SAS6::GFP/GFP::ANA2: −/−, n = 70, +/−, n = 80: (ns-not significant)p > 0.05, −/+, n = 70: (ns)p > 0.05, and +/+, n = 60: **p < 0.0001, Kruskal-Wallis Test). d) The method of measuring the dBB length (LBB). e) Representative SIM images present the longitudinal view of the BBs in flies with different genotypes. PACT (red) marks the neuronal BBs. Insets show PACT (in grey scale) close to the arrowhead. Quantification of BB length (ii), olfactory reception (iii) and gravitaxis (iv) behaviour in flies with either no or ectopic expression of both SAS6 and ANA2 using a driver that only expresses in pre-neuronal cells (Gal4neur). In ii (SAS6::GFP/GFP::ANA2: −/−, n = 47, and +/+, n = 45: **p < 0.0001, two-tail Mann-Whitney Test), iii (SAS6::GFP/GFP::ANA2: −/−, n = 70, and +/+, n = 60: **p < 0.0001, two-tail Mann-Whitney Test) and iv (SAS6::GFP/GFP::ANA2: −/−, n = 60, and +/+, n = 60: **p < 0.0001, two-tail Mann-Whitney Test). Note that upon ectopic expression of both SAS6 and ANA2 in pre-neuronal cells, we observed one cilium per cell, in most of the olfactory neurons (98%: shown in figure), as observed in controls. In the remainder olfactory neurons (not shown), we observed that cells were forming two cilia, each with one BB. (v) Representative electron micrographs show longitudinal sections through the ciliary base and the distal BB cross sections of olfactory neurons in flies with different genotypes. The empty arrow head marks the proximal region of the dBB without cartwheel. Electron micrographs of the dBB cross sections present the features (including 9-fold symmetric doublet MTs) of n ≥ 7 neurons for each genotype. vi) Tukey-Box plot of dBB length in olfactory neurons (SAS6::GFP/GFP::ANA2: −/−, n = 7, and +/+, n = 8: **p < 0.0001, two-tail Mann-Whitney Test). EM (a, b, dv-vi), SIM (ei-ii), and behaviour (c, eiii-iv) experiments were repeated independently twice, thrice and thrice with similar results, respectively. Scale bars in a, b, ei, and ev represent 0.5, 0.2, 1 and 0.1 µm, respectively. In each Tukey-box plot, centre line indicates median and error bars indicate full range of variation (from minimum-to-maximum). For different variables of each Tukey-box plot and fly genotypes see Supplementary Table 4 and Supplementary Tables 1,3, respectively.

Supplementary Figure 8 SAS6 and ANA2 recruit BLD10/CEP135.

a) Schematic representation of the experimental setting used to ectopically express SAS6 and ANA2 after BB docking in the neurons. b) Representative images of neuronal BBs and cell bodies of flies with different genotypes. We analysed RFP::PACT (red) that marks BBs and some centrosomal components, such as Anti-BLD10 and ANA1 antibodies (blue). Insets marked 1 show the region around BBs, while insets marked 2 show cell bodies with accumulation of GFP (either SAS6 or ANA2 or both of them). These experiments were repeated independently thrice displaying similar results. Scale bars represent 1 µm.

Supplementary information

Supplementary Information

Supplementary Figures 1–8, Supplementary Table and Supplementary Video legends, and Supplementary References

Reporting Summary

Supplementary Table 1

Detailed information about the flies used in this study

Supplementary Table 2

List of antibodies and their dilutions used in this study

Supplementary Table 3

Description of the genotypes of the flies that were used in this study

Supplementary Table 4

Source data

Supplementary Video 1

Structures of the ciliary base in olfactory neurons

Supplementary Video 2

Structures of the auditory ciliary base

Supplementary Video 3

The proximal basal body of olfactory neurons is composed of radially symmetric nine MT doublets

Supplementary Video 4

Nine radially symmetric MT doublets are found in the olfactory distal basal body

Supplementary Video 5

The proximal basal body of auditory neurons is composed of a mixture of nine MT singlets and doublets

Supplementary Video 6

Nano-structures in the distal basal body of auditory neurons are nine-fold symmetric

Supplementary Video 7

Nine-fold symmetric nano-structures are found in the olfactory transition zone

Supplementary Video 8

The transition zone of auditory neurons is composed of nine radially symmetric MT doublets and nano-structures

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Jana, S.C., Mendonça, S., Machado, P. et al. Differential regulation of transition zone and centriole proteins contributes to ciliary base diversity. Nat Cell Biol 20, 928–941 (2018). https://doi.org/10.1038/s41556-018-0132-1

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