Structural insights into SSNA1 self-assembly and its microtubule binding for centriole maintenance

Abstract

SSNA1 is a fibrillar protein involved in dynamic microtubule remodeling, including nucleation, co-polymerization, and microtubule branching. The underlying molecular mechanism has remained unclear due to a lack of structural information. Here, we determine the cryo-EM structure of C.elegans SSNA-1 at 4.55-Å resolution and evaluate its role in embryonic development. We find that SSNA-1 forms an anti-parallel coiled-coil, with self-assembly facilitated by an overhang of 16 C-terminal residues that form a triple-stranded helical junction. The microtubule-binding region is within the triple-stranded junction, suggesting that self-assembly of SSNA-1 creates hubs for effective microtubule interaction. Genetical analysis elucidates that SSNA-1 deletion significantly reduces embryonic viability, and causes multipolar spindles during cell division. Interestingly, impairing SSNA-1 self-assembly has a comparable effect on embryonic viability as the knockout strain. Our study provides molecular insights into SSNA-1's self-assembly and its role in microtubule binding and cell division regulation through centriole stability.

Fig. 1. C. elegans SSNA-1 is required for embryonic development.

A Schematic of CRISPR/Cas-9-mediated genome editing of C. elegans and quantification of embryonic viability. Parts of this figure were created with BioRender.com. https://BioRender.com/7xupr26. This content is not covered by the article’s Creative Commons license. B Embryonic viability of wild-type, ssna-1(Δ), and ssna-1::C-tag strains at 20˚C. Source data are provided as a Source Data file. Ssna-1(wt): n = 29, ssna-1(Δ): n = 11, ssna-1::C-tag: n = 9. Bars depict mean and standard deviation. C Representative images of the gross phenotypes of wild-type and ssna-1(Δ) strains. Animals homozygous for ssna-1(Δ) presented either as wild-type in appearance (left), displayed vulva defects (center) or displayed body length anomalies (right). Scale bar 100 μm. D Representative time-lapse images from 2-cell to 8-cell stages of either wild-type or ssna-1(Δ) embryos expressing GFP::histone, mCherry::β-tubulin, and GFP::SPD-2. Arrowheads indicate poles of multipolar spindles that form in ssna-1(Δ) embryos, with each color representing a different cell. Scale bars 10 μm. The corresponding movies can be found as Supplementary Movies 1 and 2, respectively. E Immunofluorescent images of embryos at early anaphase of the zygote from either zyg-1::spot (left) or zyg-1::spot, ssna-1::C-tag (right) embryos probed with SPOT and C-tag nanobodies. SSNA-1::C-tag co-localized with the centriolar protein ZYG-1::SPOT. Scale bar 10 μm.

Fig. 2. Cryo-EM analysis of SSNA-1(3E).

A Frontal and inner view (longitudinal section) of the SSNA-1(3E) reconstruction. B Cross-section view of SSNA-1(3E) filaments at two different slices. Top: Two-stranded coiled-coil region with protomers arranged in 8 fibrils. Bottom: Three-stranded helical junction region with two protomers stacking onto each other. Additional densities extend into the disordered inner lumen (green circle). The boxes indicate regions highlighted in panels (C) and (D). The colored asterisks indicate different helices of protomers. C Frontal, inner, and side view of a single SSNA-1(3E) fibril unit (protomer) showing a periodicity of 112 Å. D Left: Frontal view of the density representing the core of the protomer (panel B slice 1). Center: inner view of the density representing the connection of two different protomers within the same fibril (three-stranded junction, panel B slice 2). Right: Side view of a three-stranded junction where the density extends to the disordered part (panel B slice 2). The colored asterisks indicate different helices of protomers. E Frontal view of densities connecting two different SSNA-1 fibrils.

Fig. 3. Refined structural model of SSNA-1(3E).

A Ribbon representation of the SSNA-1(3E) structure. Individual fibrils are formed by antiparallel two-stranded coiled-coils that are connected through three-stranded helical junctions. The visible part of the protomers comprises residues 7-105. Individual helices are highlighted in different colors with each N- and C-terminus labeled. B Areas connecting individual fibrils near the three-stranded helical junctions. One protofibril is multi-colored and another one is in gray. Additional densities are observed near residues Q98E, Y105 in one fibril and R24, S28 in another. C Three-stranded helical junction with two protomers stacking onto each other. Key positions and interactions are highlighted. The point mutations R18E/R20E/Q98E were introduced to obtain the structurally amenable form SSNA-1(3E), forming thin filaments. D Exploded view of the two terminal interaction interfaces of the three-stranded helical junctions. Key residues are highlighted with yellow circles for hydrophobic and green circles for charged residues. E Exploded view of the third interaction interface of the three-stranded helical junction created by antiparallelly oriented C-termini of two different protomers. Colors are as in (D). F The two-stranded coiled-coil regions are stabilized by a canonical hydrophobic core (yellow circles) between residues L19 and G71, respectively.

Fig. 4. Biochemical characterization of SSNA-1.

A Schematic of SSNA-1 constructs generated to assess functional regions involved in the self-assembly process. B DLS autocorrelation curves (cumulative fits) of SSNA-1 constructs and C the corresponding decay values of the autocorrelation functions. The decay threshold is considered at an intensity autocorrelation equal to 1. D–M Top: Distribution of hydrodynamic radii of SSNA-1 particles calculated from DLS autocorrelation curves represented in B. Data from three individual experiments are plotted. Bottom: Representative negative-staining EM images of the respective SSNA-1 constructs. Scale bars: 100 nm. Source data are provided as a Source Data file.

Fig. 5. Characterization of the microtubule-binding and -branching activity of SSNA-1 variants.

A Quantification of co-sedimentation assays of pre-polymerized microtubules and different SSNA-1 constructs. The total concentration of each protein was 20 μM (1:1 ratio). The data is represented as whisker plots, with the central line indicating the mean value. Whiskers extend from the minimum to the maximum value. Data points represent results from three independent experiments. B Representative SDS-PAGE images of the experiments quantified in panel (A). SSNA-1 controls are on the right, respectively. C–K Representative negative-staining EM images of different SSNA-1 variants co-polymerized with tubulin at room temperature. The yellow asterisk represents observed microtubule-branching. L Summary of the characterization of SSNA-1 variants. Source data are provided as a Source Data file.

Fig. 6. SSNA-1’s ability to form fibrils dictates C. elegans viability during its embryonic development.

A Embryonic viability of strains homozygous for different ssna-1 alleles. Each data point shows the percentage of viable embryos from a single hermaphrodite. Bars depict mean and standard deviation. The number (N) of counted hermaphrodites for each allele is given on the top of the graph. B Comparison of the viability of C. elegans mutants in vivo with the characterization of different SSNA-1 constructs in vitro. Fibril formation is assessed as in Fig. 5L. Microtubule-binding ability was taken from Fig. 5A, B. C Model for the molecular function of SSNA-1 in stabilizing centrioles in C. elegans. Top: SSNA-1 stabilizes centrioles only when it is able to form filaments and bind to microtubules. Filaments (coiled-coils) are depicted as pink lines with red dots indicating the position of key residue R18. Bottom: When residue R18 is mutated (indicated by a black x), fibril- and oligomer formation is impaired, and worms show low viability as centrioles lose their integrity. The location of SSNA-1 within centrioles is based on the expansion microscopy results shown in ref. . Details of in vivo function of SSNA-1 are available in ref. . Source data are provided as a Source Data file.