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Structure of the radial spoke head and insights into its role in mechanoregulation of ciliary beating

Nature Structural & Molecular Biology volume 28, pages 20–28 (2021)Cite this article

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Abstract

Motile cilia power cell locomotion and drive extracellular fluid flow by propagating bending waves from their base to tip. The coordinated bending of cilia requires mechanoregulation by the radial spoke (RS) protein complexes and the microtubule central pair (CP). Despite their importance for ciliary motility across eukaryotes, the molecular function of the RSs is unknown. Here, we reconstituted the Chlamydomonas reinhardtii RS head that abuts the CP and determined its structure using single-particle cryo-EM to 3.1-Å resolution, revealing a flat, negatively charged surface supported by a rigid core of tightly intertwined proteins. Mutations in this core, corresponding to those involved in human ciliopathies, compromised the stability of the recombinant complex, providing a molecular basis for disease. Partially reversing the negative charge on the RS surface impaired motility in C. reinhardtii. We propose that the RS-head architecture is well-suited for mechanoregulation of ciliary beating through physical collisions with the CP.

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Fig. 1: Reconstitution and cryo-EM structure of the RS head and neck.
Fig. 2: Architecture of the RS head.
Fig. 3: Characterization of ciliopathy-based RS-head mutants.
Fig. 4: Intertwined proteins connect the RS head and neck.
Fig. 5: The RS head surface is composed of conserved acidic loops.
Fig. 6: Perturbing the acidic RS head surface affects Chlamydomonas motility.

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Data availability

The 3D cryo-EM density maps and atomic coordinates have been deposited in the Electron Microscopy Data Bank and Worldwide Protein Data Bank, respectively, under accession numbers EMD-22444 and PDB 7JR9 (minimal head complex); EMD-22446 and PDB 7JRJ (head–neck complex). Uncropped gel images are available in Supplementary Fig. 1. Source data for Chlamydomonas swimming analysis are available online. Source data are provided with this paper.

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Acknowledgements

We thank S. Ramundo and H. Ishikawa for advice on handling C. reinhardtii strains, T. Wu for assistance with thermal denaturation experiments and D. Ekiert for advice on model building. We thank D. Bulkley for assistance in the UCSF EM core facility. Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (349247), NYSTAR and the NIH National Institute of General Medical Sciences (GM103310). We thank L. Yen, H. Wei and E. Eng for their assistance there. EM data processing has utilized computing resources at the HPC Facility at New York University, and we thank the HPC team for high-performance computing support. We thank L. Kohlstaedt form the Vincent J. Proteomics/Mass Spectrometry Laboratory at the University of California, Berkeley, supported in part by NIH S10 Instrumentation Grant S10RR025622, for performing MS analysis. We are grateful to members of the Vale laboratory for discussions and critical reading of the manuscript and to N. Stuurman for assistance with Chlamydomonas swimming analysis. I.G.-H. was supported by the Helen Hay Whitney foundation. G.B. received funding from NIH/NIGMS R00GM112982 and Damon Runyon Cancer Research Foundation DFS‐20‐16. R.D.V. received funding from NIH R35GM118106.

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Authors and Affiliations

  1. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA

    Iris Grossman-Haham & Nan Zhang

  2. Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA

    Nicolas Coudray & Gira Bhabha

  3. Applied Bioinformatics Laboratories, New York University School of Medicine, New York, NY, USA

    Nicolas Coudray

  4. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA

    Zanlin Yu & Feng Wang

  5. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Ronald D. Vale

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Contributions

I.G.-H. and R.D.V. conceptualized research. N.Z. and I.G.-H. cloned constructs. Z.Y. and F.W. prepared functionalized grids. I.G.-H., N.C. and Z.Y. collected cryo-EM data. I.G.-H. processed EM data with guidance from N.C. and G.B. and performed all other experiments. I.G.-H., G.B. and R.D.V. analyzed the data and wrote the manuscript, with comments from all authors.

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Correspondence to Ronald D. Vale.

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Extended data

Extended Data Fig. 1 Cryo-EM data processing workflow for the RS head-neck complex.

Overall schematic for 3D classification, masking and refinement is shown. Examples of picked particles are indicated with green circles on the micrograph. Masks used for focused refinement are shown in blue. Red boxes indicate the class that was chosen for the next step. Yellow boxes indicate maps used for model building and submitted to EMDB. See Methods for more details.

Extended Data Fig. 2 Overview of cryo-EM density map quality.

a, Density maps for the RS head-neck complex, colored by local resolution, estimated using RELION. b, Angular orientation distribution of all particles used in the final 3D reconstruction of each map. c, Fourier Shell Coefficient (FSC) curves measured by the Gold-standard method using RELION. d, Examples of density in various regions of the composite map for the RS head-neck complex.

Extended Data Fig. 3 Dimerization of the radial spoke head-neck complex.

Representative negative-stain EM class averages for the head-neck complex purified with a GST tag, cleaved (left), or retained (right). Box size is 4 nm.

Extended Data Fig. 4 Organization of the radial spoke head tetrameric core.

a, Topology diagram of the fold common to RSP9 and the middle domains of RSP4 and RSP6. The core of the fold is indicated with a grey background. Segments outside the core vary between the three proteins and are shown as they appear in RSP9. b, Three-dimensional structure of RSP9 colored according to the diagram in a. c, A helix-loop-helix motif found at the N-terminus of the RS head fold is used by all chains to dimerize with a neighboring subunit (RSP4 with RSP6 and RSP9 with itself, creating a homodimer). The RSP4-RSP6 dimer is shown as an example.

Extended Data Fig. 5 Structure of the radial spoke head minimal complex (RSP4, 6, 9 and 10).

a, Coomassie-stained SDS-PAGE of the recombinant RS head minimal complex. Uncropped gel image is shown in Supplementary Fig. 1. b, Cryo-EM map (‘map 7’) of the RS head minimal complex colored according to its constituent chains. c, Model built de novo for the RS head minimal complex according to the map in b. d, The positions of the RS head minimal complex within the head region of a C. reinhardtii RS subtomogram- averaged map in grey mesh (EMD-1941)8 are shown. e, Cryo-EM data processing workflow for 3D classification, masking and refinement is shown. Examples of picked particles are indicated with green circles on the micrograph. Red boxes indicate the class that was chosen for the next step. The yellow box indicates the map used for model building and submitted to EMDB. See Methods for more details. f, Cryo-EM map colored by local resolution, estimated using RELION. g, Angular orientation distribution of all particles used in the final 3D reconstruction of the map. h, Fourier Shell Coefficient (FSC) curve measured by the Gold- standard method using RELION. i, Alignment of the RS head core from our two models (RS head minimal complex (RSP4, 6, 9 and 10) colored; RS head-neck complex (grey) with an r.m.s.d. of 0.6 Å, indicating the RS head tetrameric core is a rigid unit.

Extended Data Fig. 6 RSP5 supports the radial spoke head.

a, RS head surface (viewed from the CP as shown at the bottom left) highlighting the RSP2, 3 and 5 protrusions that are linked to the core through the GAF domain of RSP2 and through RSP5. b, RSP5 has an aldo-keto reductase (AKR) fold and an extra coiled-coil positioned at the head surface (dashed oval in a). c, Expression of the head-neck proteins except for RSP5 resulted in purification of two complexes as shown by the two SEC peaks. The peak eluting earlier from the column (1) contains primarily RSP2, 3 and 23, whereas the second peak contains primarily RSP1, RSP4, RSP6, RSP9 and RSP10, as shown at the bottom by Coomassie-stained SDS-PAGE. d, Each of the complexes in c was expressed and purified separately. Top, Coomassie-stained SDS-PAGE of a complex composed of RSP2, RSP3 and RSP23 purified on an anion-exchange column. The three proteins, corresponding to most of the neck, are shown on the right as they appear in the head-neck structure. Bottom, Coomassie-stained SDS-PAGE of a complex composed of RSP1, RSP4, RSP6, RSP9 and RSP10 purified on a SEC column. The five proteins, corresponding to the center of the RS head, are shown on the right as they appear in the head-neck structure. Uncropped gel images are shown in Supplementary Fig. 1.

Extended Data Fig. 7 Characterization of recombinant RS head complexes with point mutations causing PCD.

a, Sequence alignment of C. reinhardtii and human RSPs, showing the regions of PCD causal point-mutations (indicated with a colored box) are conserved. Residues are colored according to identity (identical – dark purple; positive – light purple). Alignment was done with T-Coffee43 and the figure was prepared in Jalview44. b-f, Amino acids in the Chlamydomonas RS head, corresponding to residues in human orthologs that when mutated cause PCD, are shown (Table 2). b, Phe170 (purple) is in the middle of a beta sheet in RSP4 (grey). When mutated to Pro it is expected to disrupt the sheet. c, A region from RSP4 colored according to residue conservation65, highlighting Gly251 (spheres) is conserved and buried between two loops. Mutation to Glu is expected to disrupt the loop arrangement. d, MORN repeats (dashed boxes) from RSP1 colored according to residue conservation65, indicating a conserved Gly residue in the same position for each repeat. Mutation of Gly636 to Glu is expected to disrupt the MORN repeat. e, The C’ terminal residues 262–269 in one copy of RSP9 (dark grey spheres) are positioned in a cleft created between RSP6 and RSP10 and thus in-frame deletion of the residue Arg261 (blue spheres) may affect the interaction between these three proteins. f, Tyr 244 (green) is on a beta strand in RSP9 (grey), interacting with a neighboring loop. g, Representative negative-stain EM micrographs for the RSP4 mutants, demonstrating the purified complexes are small and inhomogeneous. A micrograph of wild-type particles is shown for comparison.

Extended Data Fig. 8 Acidic loops compose the center of the radial spoke head surface.

a, Loops at the head surface shown in the top right (some were removed for clarity) were modeled into a clear density observed in the cryo-EM map (‘map 1’), indicating the loops are structured. Asterisks indicate loops shown in b. b, Beta hairpins in RSP4 and RSP6, located at the head surface, are stabilized by hydrogen bonds (dashed lines). Residues that participate in these hydrogen bonds are indicated. c, Zoom into box in a, showing stabilizing lateral interactions (hydrogen bonds in dashed lines) between four different loops. The loops at the RS head surface are also supported by the tightly-packed folds that compose the RS head scaffold (Fig. 2). d, Electrostatic volume of the head surface generated in PyMol. Dashed red lines indicate regions not modeled, suggesting that the RS head surface is even more acidic than visualized by the electrostatic map. Number of missing residues and their percentage of D and E residues are listed on the right for each region.

Extended Data Fig. 9 Acidic loops in the RS head are important for efficient cilia motility.

a, Side view of the RS head, such that the CP is on the right. Acidic loops from RSP6 that point towards the CP, colored red and indicated with boxes, were chosen for mutagenesis. Boxes show a closer view of the residues mutated. b, PCR analyses of the Chlamydomonas RSP6-deficient strain (LMJ.RY0402.248886) that was used for genetic complementation, confirming the gene was disrupted by the insertion cassette. RSP6-specific primers (lane 1) amplified the genomic fragment in wild-type but not in the mutant. Cassette fragments on both ends (lanes 2 and 3) were amplified in the mutant, but not in wild-type. c, Immunofluorescence of HA tag and the flagellar marker acetylated tubulin in the RSP6-deficient strain (top), complemented with wild-type RSP6 (middle) and with the RSP6 charge-mutant (bottom). d, Average beat frequencies measured for Chlamydomonas wild-type cells (48 ± 1 Hz; n = 42), two clones of RSP6-deficient strain rescued with wild-type RSP6 (47 ± 1 Hz; n = 30 and 52 ± 1 Hz; n = 46), and two clones of the RSP6 charge mutant (44 ± 1 Hz; n = 42 and 41 ± 2; n = 39). Data are shown as means and s.e.m. Statistical significance was determined by a one-way ANOVA with Holm-Sidak test. ns denotes not significant, ***p < 0.001; ****p < 0.0001. Source data for graphs are available online. e, Mutations made in RSP6 acidic loops. Residues that differ from the wild-type sequence are in bold letters.

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Supplementary Tables 1–3, Supplementary Note 1 and Supplementary Figure 1.

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Supplementary Video 1

Architecture of the radial spoke head and neck.

Supplementary Video 2

Swimming of C. reinhardtii strains. Frame rate is 27 frames per s. For strains prepared in this study (RSP6-deficient + WT RSP6 and RSP-deficient + charge mutant), two panels are shown.

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Source Data Extended Data Fig. 9

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Grossman-Haham, I., Coudray, N., Yu, Z. et al. Structure of the radial spoke head and insights into its role in mechanoregulation of ciliary beating. Nat Struct Mol Biol 28, 20–28 (2021). https://doi.org/10.1038/s41594-020-00519-9

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