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Ciliary central apparatus structure reveals mechanisms of microtubule patterning

Nature Structural & Molecular Biology volume 29, pages 483–492 (2022)Cite this article

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Abstract

A pair of extensively modified microtubules form the central apparatus (CA) of the axoneme of most motile cilia, where they regulate ciliary motility. The external surfaces of both CA microtubules are patterned asymmetrically with large protein complexes that repeat every 16 or 32 nm. The composition of these projections and the mechanisms that establish asymmetry and longitudinal periodicity are unknown. Here, by determining cryo-EM structures of the CA microtubules, we identify 48 different CA-associated proteins, which in turn reveal mechanisms for asymmetric and periodic protein binding to microtubules. We identify arc-MIPs, a novel class of microtubule inner protein, that bind laterally across protofilaments and remodel tubulin structure and lattice contacts. The binding mechanisms utilized by CA proteins may be generalizable to other microtubule-associated proteins. These structures establish a foundation to elucidate the contributions of individual CA proteins to ciliary motility and ciliopathies.

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Fig. 1: Cryo-EM structures of central apparatus C1 and C2 microtubules.
Fig. 2: Microtubule inner proteins of the central apparatus.
Fig. 3: PF16 spirals are determinants of projection asymmetry and periodicity.
Fig. 4: ASH proteins recognize different PF16:protofilament combinations.
Fig. 5: Atomic models of the CA projections.
Fig. 6: The C1b projection contains a cluster of kinase domains.

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Data availability

The composite cryo-EM maps of the C1 and C2 microtubules are deposited in the Electron Microscopy Data Bank (EMDB: https://www.ebi.ac.uk/pdbe/emdb/) with accession codes EMD-25381 and EMD-25361. The original unsharpened maps from cryoSPARC are associated with these depositions as additional files. The atomic models for the C1 and C2 microtubules are deposited in the Protein Data Bank (PDB: https://www.rcsb.org/) with accession codes 7SQC and 7SOM.

Code availability

Code used to obtain initial alignment parameters by EMAN1 and FREALIGN is available at https://github.com/rui--zhang/Microtubule.

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Acknowledgements

Cryo-EM data were collected at the Washington University in St. Louis Center for Cellular Imaging (WUCCI) and Case Western Research University (CWRU). We thank M. Rau and J. Fitzpatrick at WUCCI and W. Huang and K. Li at CWRU for microscopy support, J. Anderson for help with domain recognition and M. Bao for comments. M.G. is supported by a Charles A. King Trust Postdoctoral Research Fellowship. S.K.D. is supported by NIGMS grant R35GM131909 and R01HL128370. A.B. is supported by NIGMS grant 1R01GM141109, the Smith Family Foundation and the Pew Charitable Trusts. R.Z. is supported by NIGMS grant 1R01GM138854.

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Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Miao Gui & Alan Brown

  2. Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, School of Medicine, St. Louis, MO, USA

    Xiangli Wang & Rui Zhang

  3. Department of Genetics, Washington University in St. Louis, St Louis, MO, USA

    Susan K. Dutcher

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  1. Miao Gui
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  2. Xiangli Wang
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Contributions

X.W. prepared the sample for cryo-EM. X.W. and R.Z. collected and processed cryo-EM data. M.G. and A.B. built the atomic models. S.K.D. generated Chlamydomonas mutant strains. M.G., A.B. and R.Z. analyzed results and generated figures. R.Z. and A.B. supervised the research and wrote the manuscript with input from all authors.

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Correspondence to Alan Brown or Rui Zhang.

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Nature Structural & Molecular Biology thanks Antonina Roll-Mecak and Gaia Pigino for their contribution to the peer review of this work. Primary Handling Editors: Beth Moorefield and Sara Osman, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Data processing.

a,b, Two representative micrographs of the total 9271 micrographs showing a mixture of CA microtubules (C1 and C2) and doublet microtubules (DMT), including loosely associated pairs of C1 and C2. c, Wedge masks used to reconstruct the ‘core’ of the C1 microtubule. Each of the seven wedge masks covering 2 protofilaments (PFs) is uniquely colored. The choice of where two 2-PF wedge masks overlap (necessary because of the odd number of protofilaments in the C1 or C2 microtubule) was arbitrary, for example, PF7 for C1 and PF2 for C2. To make the final composite map, we multiplied one of the overlapping reconstructions with a smaller wedge mask covering just a single protofilament (PF8 for C1 and PF1 for C2). d, Shell masks used to reconstruct the ‘outer shell’ of the C1 microtubule. The nominal resolution of each of subregion, defined by the masks, is given in the table. e, Wedge masks used to reconstruct the core of the C2 microtubule with nominal resolutions of each subregion. In panels c-e, the protofilaments are numbered according to33. f, Shape masks were used to improve the local resolution of the projections. As exemplified here for the C2a projection, three different shape masks were used that correspond to the microtubule-proximal, middle, and distal regions of the projection.

Extended Data Fig. 2 Protein identification strategies.

a, A flow diagram showing the strategies used to identify proteins in the C1 and C2 maps. If no matches were found in the lists of candidate proteins from mass spectrometry analysis11,12, the search was expanded to include the entire Chlamydomonas proteome. b, An example showing how DeepTracer70 was used to identify FAP213. c, Examples of density for proteins that are resolved at the side chain level (FAP388 and FAP147) and those that have regions that are only resolved at the domain level (FAP101, Hydin and PF16). Extended Data Table 1 lists the built regions of each identified protein and whether there is side chain information.

Extended Data Fig. 3 Locations of proteins within the central apparatus (CA).

a, Cross-section of the Chlamydomonas CA (as in Fig. 1b) annotated with the relative positions of the proteins identified within the cryo-EM maps. Proteins listed twice appear in more than one projection. b, The catalytic subunit of Protein phosphatase 1 (PP1c) occurs in two locations in the CA: bound to FAP81 in the C1e projection (left) and bound to PF16 in the C1d projection (right). c, Proteins identified within the CA and its projections by mass spectrometry11,12 that have not been identified in the cryo-EM maps.

Extended Data Fig. 4 Domain organization of central apparatus (CA) proteins.

CA proteins are divided into two groups based on their sequence length and listed by alphabetical order within these groups. Domains are annotated based on the experimentally determined structures and AlphaFold232 predictions. Globular domains that could not be confidently classified are indicated as white boxes. Abbreviations used in the key: ARM, armadillo repeat-containing domain; ASH, ASPM, SPD-2, Hydin domain; C2, a calcium-binding immunoglobulin-like domain; CH, calponin homology domain; HSP70, heat shock protein 70; Ig-like, immunoglobulin-like domain; IQ, IQ calmodulin-binding motif; MORN, Membrane Occupation and Recognition Nexus repeat; TPR, tetratricopeptide repeat-containing domain. Some of the ASH domains appear larger than others due to insertions. Most notably, Hydin has a guanylate kinase domain inserted in one of its ASH domains.

Extended Data Fig. 5 Protein-microtubule interactions.

a, FAP20 is a component of C2 microtubules and doublet microtubules. Left, FAP20 interacts with FAP65 and FAP147 on the surface of the C2 microtubule and makes only weak interactions with β-tubulin. Right, FAP20 links the A and B tubules of doublet microtubules at the inner junction (PDB 6U42)2 and interacts extensively with both α- and β-tubulin. b, Calponin homology (CH) domains prototypically interact with microtubules by binding the interface between four tubulin molecules in locations other than the seam, as exemplified by end-binding protein 3 (EB3; PDB 3JAR)61. Our structures show that CH domains are versatile and can interact with various microtubule lateral interfaces including the microtubule seam. The seam-binding ability of FAP178 is important for directing PF20 to the seam of the C2 microtubule.

Extended Data Fig. 6 Structural and functional analyses of Chlamydomonas mutants.

a, Visualization of the luminal surface of the C1 microtubule from wild-type Chlamydomonas (top) and a fap236 mutant (bottom). The fap236 mutant lacks SAXO density on protofilament 4 (red arrow). b, Visualization of the luminal surface of the C2 microtubule from wild-type Chlamydomonas (top) and a fap236 mutant (bottom). The fap236 mutant lacks SAXO density on protofilament 11 (red arrow). In panels a and b, map densities corresponding to α,β-tubulin and SAXO proteins are colored as indicated, the remaining densities including arc-MIPs are colored in light grey. c, Swimming velocities of CA MIP mutants. The numbers after the hyphen (for example, fap105-940) are the last three digits of the unique code for the CLiP mutant strain. Box plots showing the swimming velocities of each mutant strain, backcrossed with the wild-type CC-4402 strain, as judged by light microscopy. CC-4533 is the parental strain of the CLiP collection. Colors indicate biological replicates (different progeny from the backcross) for each strain. Number of cells analyzed: 188 (fap105-940), 36 (fap105-283), 94 (fap275-435), 90 (fap275-824), 67 (fap196-348), 108 (fap213-992), 84 (fap239-434), 94 (fap424-848), 540 (CC-4533), 170 (CC-4402). Data are presented as mean values + /- SD. The boxes indicate fifty percent. The minima and maxima used in the box plot is defined as Q1-1.5*IQR and Q3 + 1.5*IQR, respectively (Q1 is first quartile, Q3 is third quartile, IQR is Interquartile Range). Statistical analysis was performed by ANOVA on ranks.

Extended Data Fig. 7 Interactions between the K40 loop of α-tubulin and microtubule inner proteins (MIPs).

a, Interaction between the αK40 loop on C1 protofilament (PF) 8 and the arc-MIP FAP275. b, Interaction between the αK40 loop on C1 PF10 and the arc-MIP FAP275. c, Interaction between the αK40 loop on C2 PF5 and two MIPs (FAP388 and FAP196). The two MIPs form an inter-molecular β-sheet (magenta arrow). d, Interaction between the αK40 loop on C2 PF13 and the arc-MIP FAP388. e, Remodeling of the H1’-S2 loop (residues 47–64) of α-tubulin into an α-helix by the arc-MIP FAP225 at the lateral interface between C2 PF1 and PF2. f, The αK40 loop is disordered (and therefore invisible) in the cryo-EM density map of an in vitro assembled and undecorated microtubule (MT) (EMD-7974 and PDB 6DPV)23. In b and d, the red arrows point to MIPs occupying the taxol-binding pocket of β-tubulin. In panels a-f, the αK40 loops (residues 37 to 47) are colored orange (and drawn as a dashed line if invisible). Residue αK40 is colored red.

Extended Data Fig. 8 Details of the PF16 spiral.

a, Overview showing spirals of PF16 decorating the C1 microtubule. Each spiral is shown in a different color, as is FAP194 which caps the spirals. b, Molecular environment showing the capping of a PF16 spiral by FAP194. FAP194 alternates every 16 nm with PF16. c, Molecular environment at the beginning of the PF16 spiral. PF6 and FAP81 prevent the spiral from initiating earlier.

Extended Data Fig. 9 Analysis of microtubule curvature.

The interprotofilament angle is defined as the relative rotation (in conjugation with necessary translation) from a tubulin heterodimer on one protofilament to the neighboring tubulin heterodimer on a second protofilament. The red dashed line at 27.7° is the theoretical interprotofilament angle for a perfectly symmetric 13-protofilament microtubule (360° divided by 13). The seam locations for each microtubule are indicated with asterisks. Note for in vitro assembled GDP microtubule, the largest interprotofilament angle occurs at the seam as previously reported23.

Extended Data Fig. 10 Projection surfaces colored by electrostatic potential.

a, Three projections for which we have near-complete atomic models (C1a, C1d, and C1b) colored by surface electrostatic potential. The schematic on the left-hand side shows the angle from which the projection surfaces are viewed. b, Unmodeled surface loops (indicated with dashed lines) within the projections contain charged residues. The sequences of these loops are shown on the right. Within these sequences, negatively charged residues are colored red and positively charged residues are colored blue.

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Gui, M., Wang, X., Dutcher, S.K. et al. Ciliary central apparatus structure reveals mechanisms of microtubule patterning. Nat Struct Mol Biol 29, 483–492 (2022). https://doi.org/10.1038/s41594-022-00770-2

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