Structural basis of the 9-fold symmetry of centrioles

Abstract

The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles.

Graphical abstract

Figure 1

Domain Organization and Molecular Model of C. elegans SAS-6 (A) Schematic representation of C. elegans SAS-6. N, N-terminal domain; CC, coiled coil; C, C terminus. Numbers above the schematic correspond to amino acids. (B) Rotary metal shadowing electron micrographs of ceFL, ceN-CC, and ceCC specimens. Arrowheads indicate globular domains. Scale bar, 50 nm. (C and D) CD spectrum (C) and thermal unfolding profile recorded by CD at 222 nm (D) of the ceCC fragment. The data support the formation of a highly helical structure with moderate thermal stability. (E) MALS analysis of the ceCC fragment. The UV absorbance profile of size exclusion chromatography (black line) is overlaid with the molecular weight (50 kDa) estimation by MALS (gray line). (F) ceCC dilution series monitored by CD at 222 nm. The gray solid line represents the fit to the data (open circles) using a monomer-dimer model. (G) SDS-PAGE of the ceCC fragment run under reduced (+βMe) and nonreducing (−βMe) conditions. Arrowheads point to protein bands corresponding to monomeric (M) and disulfide-linked dimeric (D) forms of ceCC. (H) Molecular model of SAS-6 homodimer. Each monomeric subunit is composed of a globular N-terminal domain, a coiled-coil domain that forms a parallel dimer, and a poorly structured C-terminal part. See also Figure S1.

Figure 2

Structural Analysis of C. elegans SAS-6 N-Terminal Domain (A) Two overall views of the ceN-dimer structure seen in the asymmetric unit of the crystal 90° apart. Monomers A and B (in cartoon representation) are colored in light gray and yellow, respectively. Secondary structure elements and the N and C termini are assigned. Loop α2-β5, which is unique to C. elegans, is not seen in the electron density presumably due to disorder and is indicated by a dashed line. Each monomer displays two α helices that cap the end of a two-stranded β sheet sandwich. The PISA motif spans region β3 to α2, with evolutionarily conserved residues in this region contributing to the protein core as well as to a predominantly hydrophobic cavity between α1 and α2 (see also Figure S2). The locations of loops β6-β7 are indicated by arrows. (B) Structure of the ceN-dimer, with monomer A shown as surface representation. Highly conserved residues are colored dark green, and mostly conserved residues are colored bright green. I154 of monomer B is depicted as stick representation. (C) Close-up views of the interaction network observed at the dimer interface in cartoon (main chains) and stick (contacting residues) representations. Oxygen and nitrogen atoms are colored in red and blue, respectively, and carbon atoms are colored in light gray (monomer A) or yellow (monomer B). (D) Sedimentation velocity AUC analysis of ceN (red) and ceN[I154E] (blue) fragments. The peak labeled “Monomer” corresponds to a molecular weight of ∼20 kDa, which is consistent with the molecular weight of the ceN[I154E] monomer. The peak labeled “Dimer” corresponds to a molecular weight of ∼40 kDa. Protein concentration was 300 μM. (E) Dissociation isotherm obtained by ITC for ceN. A 1.6 mM ceN solution was injected stepwise into buffer. Shown are the integrated heat changes upon dilution. The solid red line represents the fit to the data (open circles) assuming dissociation of ceN dimers into monomers. (F) Sedimentation velocity AUC analysis of ceN-CC (red) and ceN-CC[I154E] (blue) fragments. The peak labeled “Dimer” corresponds to a molecular weight of ∼90 kDa, which is consistent with the formation of ceN-CC[I154E] dimers. The broad profile observed for ceN-CC (labeled “Higher-order oligomers”) suggests formation of higher-order oligomers beyond dimers. Protein concentration was 200 μM. See also Figure S2 and Figure S3.

Figure 3

Functional Analysis of SAS-6 in C. elegans and Human Cells (A)–(F) Anterior is to the left and scale bar is 10 μm. (A–C) Images at the end of the second cell cycle from representative DIC recordings of embryos treated with sas-6(RNAi) and expressing GFP-SAS-6RR (A), GFP-SAS-6RR[I154E] (B), or GFP-SAS-6RR[I154G] (C). Elapsed time after pronuclear meeting is indicated in minutes and seconds; arrowheads indicate centrosomes. (D–F) Embryos during mitosis of the second cell cycle treated with sas-6(RNAi) and expressing GFP-SAS-6RR (D), GFP-SAS-6RR[I154E] (E), or GFP-SAS-6RR[I154G] (F) stained with antibodies against α-tubulin (green) and SAS-4 (red); DNA in blue. Insets show an ∼2.5-fold magnified view of one MTOC. Note that GFP-SAS-6RR[I154E] and GFP-SAS-6RR[I154G] are not present at centrioles (data not shown), presumably because they fail to be incorporated as a result from the lack of oligomerization. (G) Quantification of experiments illustrated in (A)–(C). The percentages of embryos with four cells at the end of the second cell cycle are indicated (n = 31 for wild-type, n = 37 for GFP-SAS-6RR, n = 50 for GFP-SAS-6RR[I154E], and n = 35 for GFP-SAS-6RR[I154G]). Shown are the mean percentages ± SEM from two independent experiments. (H) Western blot analysis of GFP-SAS-6RR, GFP-SAS-6[I154E], or GFP-SAS-6RR[I154G] embryonic extracts probed with SAS-6 antibodies to reveal both endogenous protein (filled arrowhead) and GFP fusions (open arrowhead). (I–K) Metaphase U2OS, iU2OS:HsSAS-6-GFP, and iU2OS:HsSAS-6[F131E]-GFP cells transfected with siRNAs targeting the 3′UTR of endogenous HsSAS-6 (siHsSAS-6-3′UTR), induced concomitantly with doxycycline, fixed after 48 hr, and stained with antibodies against centrin (red) and GFP (green); DNA in blue. Scale bar, 10 μm. Insets show magnified view of the delineated regions; scale bar in insets, 1 μm. Whereas the vast majority of mitotic cells expressing HsSAS-6[F131E]-GFP did not exhibit centriolar GFP (see C), a centriolar signal was detected earlier during the cell cycle in most cells (data not shown), suggestive of a failure in stable incorporation as a result of the lack of oligomerization. (L) Percentage of cells in mitosis (prophase to metaphase) with four or more centrioles after 48 hr treatment with Stealth RNAi Low GC negative control or siHsSAS-6-3′UTR (n = 135 for U2OS + control siRNA, n = 236 for U2OS + 3′UTR siRNA, n = 226 for U2OS + 3′UTR siRNA + HsSAS-6-GFP, and n = 160 for U2OS + 3′UTR siRNA + HsSAS-6[F131E]-GFP). Data from at least three independent experiments (≥50 cells/experiment) are shown; error bar indicates SEM. See also Figure S3, Movies S1, Movie S2, and Movie S3.

Figure 4

Structural Analysis of C. reinhardtii Bld12p (A) Two overall views of the crN-dimer structure 90° apart and superimposed onto the ceN-dimer structure. Monomers A and B are depicted in cartoon representations and colored in dark gray and red (crN-dimer) and light gray and yellow (ce-N dimer), respectively. The global superimposition yielded a root-mean-square deviation of 1.6 Å for 217 backbone atoms. (B) Two overall views of the crCC-dimer structure 90° apart. Monomers A and B are colored in magenta and light pink, respectively. (C) Close-up views of the interaction network seen at the crCC-dimer interface in cartoon (main chains) and stick (contacting residues) representations. Key secondary structure elements are assigned. Oxygen and nitrogen atoms are colored in red and blue, respectively. Carbon atoms are colored in magenta and light pink. (D) Superimposition of monomer B of the crN-dimer onto monomer A of the crCC-dimer. The resulting assembly was used as a template for building the Bld12p ring structure shown in Figure 5. See also Figure S2 and Figure S4.

Figure 5

Structural Model of C. reinhardtii Bld12p Ring Oligomer (A) Two views of the crCC-dimer building block 90° apart. (B) Nine crCC-dimers were associated such that their N-terminal domains interact as observed in the crN-dimer (Figure 4D). The resulting 9-fold symmetric ring oligomer exhibits a diameter of ∼23 nm and a thickness of ∼3.5 × 5 nm. The long axis of the coiled-coil domains are in plane with and radiate out from the ring. See also Figure S5.

Figure 6

Electron Microscopy of C. reinhardtii Bld12p (A–D) Rotary metal shadowing electron micrographs of crN-CC specimens. Schematic interpretations of the specimens are indicated. Note that not all nine spokes represented in the schematic of (D) are unambiguously discerned in the electron micrographs, presumably because some of them are perturbed during sample preparation. The arrow in (A) highlights the head-like moieties of crN-CC. Scale bars, 50 nm. (E) Histogram representation of angles measured between the two legs of the V-shaped crN-CC specimens shown in (B) (n = 51). (F) Histogram representation of mean diameters measured from crN-CC ring oligomers shown in (D) (n = 73). The majority of rings possess a diameter of 22 nm, which is in good agreement with the 23 nm diameter determined from the 9-fold symmetric structural ring model of Bld12p (Figure 5). Note also that a minor fraction of crN-CC rings displayed a lower than 22 nm diameter, which probably correlates with a different number of crN-CC dimers. See also Figure S6.

Figure 7

Structural Model of the SAS-6-Based Cartwheel in the Context of the Centriole In this model, viewed from the proximal end, the SAS-6 coiled-coil domains would contribute substantially to the formation of the spokes of the cartwheel and possibly connect to the pinheads. The coiled-coil domains were extended to the size expected from structure prediction, and their lengths fit well with the ∼40 nm length measured from electron micrographs of crN-CC specimens (Figure 6A). However, we note that the protein Bld10p from C. reinhardtii, which localizes to the pinhead of the cartwheel, also contributes to the formation of the spokes (Hiraki et al., 2007). Scale bar, 50 nm.

Figure S1

C. elegans SAS-6 Characterization, Related to Figure 1 (A) Schematic representation of C. elegans SAS-6 and fragments used in this study. ceFL: full-length (amino acids 1–492), ceN-CC: N-terminus plus coiled coil (amino acids 1–414), ceCC: coiled coil (amino acids 181–408); ceN: N-terminus (amino acids 1–168). (B) Sections of reducing and Coomassie-stained SDS-PAGE showing final purification products for the indicated recombinant proteins. From left to right: SAS-6 full-length (ceFL), ceN-CC (residues 1–414), ceCC (residues 181–408) and ceN (residues 1–168). Approximate molecular weights from in-gel markers are shown. (C) Helical wheel representation of the SAS-6 coiled-coil domain in the vicinity of Cys204 in a two-stranded parallel configuration. The predicted heptad repeat (denoted a to g) and the residues occupying its position are indicated. (D) Relative location of the Cys204 sulfur group on ceCC for a parallel or antiparallel coiled coil configuration. Efficient disulphide bridge formation is possible only in the parallel in-register coiled coil configuration.

Figure S2

Structure-Based Sequence Alignment of SAS-6 Orthologs, Related to Figure 2 and Figure 4 Highly conserved and conserved residues are highlighted in dark and light gray, respectively. Secondary structure assignments based on the crystal structures of C. elegans SAS-6 and Bld12p (Figures 2 and 4) are shown on top of the alignment. Interacting residues seen in the CC-dimer are indicated in magenta; the ones seen in the N-dimer are indicated in red. The PISA domain characteristic of SAS-6 proteins is indicated by a dashed black line at the bottom of the alignment. Species identifiers are: ce, Caenorhabditis elegans; hs, Homo sapiens; gg, Gallus gallus; xl, Xenopus laevis; dr, Dario rerio; dm, Drosophila melanogaster; cr, Chlamydomonas reinhardtii. UniProtKB/Swiss-Prot sequence accession identifiers are as follows: ceSAS-6, SAS6_CAEEL; Bld12p (crSAS-6), A9CQL4_CHLRE; hsSAS-6, SAS6_HUMAN; ggSAS-6, SAS6_CHICK; xlSAS-6, SAS6_XENLA; drSAS-6, SAS6_DANRE; dmSAS-6, SAS6_DROME.

Figure S3

Characterization of C. elegans SAS-6 Protein Fragments and Depletion of Endogenous HsSAS-6 Using siHsSAS-6-3′UTR, Related to Figure 2 and Figure 3 (A) ITC of the C. elegans N-N interaction. Top panel: raw data representing the response to injections of ceN at high concentration into sample buffer. Bottom panel: integrated heat change (closed squares) and associated curve fit (black solid line). (B) CD spectrum of ceN (open circles) or ceN[I154E] (closed circles) fragments. (C) SDS-PAGE showing final purification products for AUC of ceN and ceN[I154E] recombinant proteins. (D) SDS-PAGE showing final purification products for AUC of ceN-CC and ceN-CC[I154E] recombinant proteins. (E) Cells left untreated (-), transfected with LO negative control siRNA (LO) or siHsSAS-6-3′UTR (si) for 48h before Western blot analysis with HsSAS-6 antibody; tubulin served as loading control. Arrows point to endogenous HsSAS-6 or HsSAS-6-GFP proteins. Dots indicate endogenous HsSAS-6 bands, stars unspecific bands.

Figure S4

Characterization of C. reinhardtii Bld12p, Related to Figure 4 (A) Schematic representation of Bld12p and fragments generated in this study. crN, N-terminal domain; crN-6HR, N-terminal domains extended by 6 heptad repeats of the adjacent coiled coil; crN-CC; N-terminal domains extended by the full adjacent coiled coil. Numbers correspond to Bld12p amino acids. (B) Coomassie-stained SDS-PAGE sections showing final purification products of the indicated recombinant proteins. Approximate molecular weights from in-gel markers are shown. (C) Close up view of the interaction network seen at the crN-dimer interface in cartoon (main chains) and stick (contacting residues) representations. Monomers A and B are colored in dark gray and orange, respectively. (D) Dissociation isotherm obtained by ITC for crN. Top panel: raw data representing the response to injections of crN at high concentration into sample buffer. Bottom panel: integrated heat change (closed squares) and associated curve fit (red solid line). (E) Sedimentation velocity analysis of the crN-6HR (red) and crN-6HR[F145E] (blue). Protein concentration was 150 μM for both samples. The peak labeled with ‘Dimer’ corresponds to a molecular weight of ∼50 kDa, which is consistent with the formation of dimers. The region of S values highlighted with ‘Higher order oligomers’ is indicative of higher order oligomer formation beyond dimers. (F) MALS analysis of crN-6HR[F145E]. The UV absorbance profile of size exclusion chromatography (black line) is overlaid with the molecular weight estimation by multi-angle light scattering (gray line). The determined molecular weight of 48.3 kDa is consistent with the formation of a stable dimer. Molecular weight of the crN-6HR[F145E] monomer: 25.8 kDa. (G) crN-6HR[F145E] dilution series monitored by CD at 222 nm. The red solid line represents the fit to the data (closed squares) using a monomer-dimer model.

Figure S5

Modeling of C. reinhardtii Bld12p Oligomers, Related to Figure 5 Only small differences are observed between crN-dimers (yellow, chains C and D are shown) and the corresponding dimer resulting from modeling of the idealized 9-fold symmetric ring using crCC-dimers (light gray). Note the close fit in the position of the β3-β4 loop (close-up), which is critically involved in determining the CC-N interface.

Figure S6

Electron Microscopy of C. reinhardtii crN-CC and crN-6HR, Related to Figure 6 (A, B) Electron micrographs of crN-CC (A) and crN-CC[F145E] (B) after glycerol spraying and rotary metal shadowing. Protein concentration was 1 mg/ml for both samples. Scale bars are indicated. Arrows in panel (A) highlight the ring oligomers only obtained with crN-CC. (C) Electron micrographs of crN-6HR ring oligomers after glycerol spraying and rotary metal shadowing. Scale bar, 50 nm. (D) Histogram representation of mean diameters measured from crN-6HR ring oligomers shown in (C). Note that the observed mean ring diameter and their distribution are very similar to those observed with crN-CC (Figure 6F).