Ciliary central apparatus structure reveals mechanisms of microtubule patterning

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.

Extended Data Fig. 1 |

Data processing.

Extended Data Fig. 2 |

Protein identification strategies.

Extended Data Fig. 3 |

Locations of proteins within the central apparatus (CA).

Extended Data Fig. 4 |

Domain organization of central apparatus (CA) proteins.

Extended Data Fig. 5 |

Protein-microtubule interactions.

Extended Data Fig. 6 |

Structural and functional analyses of Chlamydomonas mutants.

Extended Data Fig. 7 |

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

Extended Data Fig. 8 |

Details of the PF16 spiral.

Extended Data Fig. 9 |

Analysis of microtubule curvature.

Extended Data Fig. 10 |

Projection surfaces colored by electrostatic potential.

Fig. 1 |. Cryo-EM structures of central apparatus C1 and C2 microtubules.

a, Schematic representation of the cross section of the Chlamydomonas axoneme. The major axonemal complexes are colored and labeled. b, Cross section of the composite cryo-EM maps of the C1 and C2 microtubule, determined in this study, positioned within the subtomogram average of the Chlamydomonas CA (EMD-31143). The projections are numbered according to^, and are uniquely colored. The seams of the C1 and C2 microtubules, located at the bridge, are indicated with yellow asterisks. c, Two longitudinal views of the C1 microtubule. d, Two longitudinal views of the C2 microtubule. In panels c and d, the minus (−) and plus (+) ends of the microtubules are indicated at the ends of the scale bar.

Fig. 2 |. Microtubule inner proteins (MIPs) of the central apparatus (CA).

a, Cross section (left) and two longitudinal views (middle and right) of the C1 microtubule showing the positioning and atomic models of its MIPs. b, Cross section (left) and two longitudinal views (middle and right) of the C2 microtubule showing the positioning and atomic models of its MIPs. In panels a and b, the minus (−) and plus (+) ends of the microtubules are indicated at the ends of the scale bar. c, Stabilization of the K40 loop of α-tubulin by FAP388 in the C2 microtubule. d, Comparison of a typical lateral interface (above) with one remodeled by FAP225 (below). FAP225 substitutes part of the H1’-S2 loop. e, Two examples of MIPs (FAP275 and FAP213) that insert loops into the taxol-binding pocket. In the 16-nm repeat of the C2 microtubule, 23/26 taxol-binding pockets are occupied. In the 32-nm repeat of the C1 microtubule, 14/52 are occupied. f, Interactions between MIPs and external proteins of the C1 microtubule. g, Interactions between MIPs and external proteins of the C2 microtubule. In panels f and g, MIPs are labeled within the microtubule lumen and external proteins are labeled outside the microtubule. In all panels, protofilaments are numbered according to Ref..

Fig. 3 |. PF16 spirals are determinants of projection asymmetry and periodicity.

a, Two longitudinal views showing how the noncontinuous spirals of PF16 form an imperfect right-handed triple helix around the C1 microtubule. Each spiral is capped after 10 homodimers of PF16 by a homodimer of FAP194. b, Cross section of the PF16 spirals on the C1 microtubule showing a distinctive flowerhead arrangement with a single mismatch site on protofilament 9. c, Two longitudinal views of the C1 microtubule showing the positions of its ASH proteins relative to the PF16 spirals. The ASH repeats form strings of domains. d, Positions of the ASH proteins when viewed in cross section. Each ASH protein interacts with a specific PF16:protofilament combination. In all panels, other CA proteins are hidden for clarity. e, Composite cryo-EM map of the CA colored by periodicity. The map is supplemented in the C2b region with segmented density (shown in light purple) from the subtomogram average of the Chlamydomonas CA (EMD-31143). The pattern of periodicity of the C1 microtubule matches the periodicity of the PF16 spirals (inset). f, The β-propeller domain of PF20 and the calponin homology (CH) domain of FAP178 bind the seam of the C2 microtubule, following the 8-nm periodicity of tubulin. Neighboring copies of PF20 form a coiled coil at the base of the C2a projection, and thus transitions from 8- to 16-nm periodicity. The seam of the microtubule is marked with an asterisk.

Fig. 4 |. ASH proteins recognize different PF16:protofilament combinations.

a-e, Cross sections and 90° rotations showing the binding of ASH-domain containing proteins to different PF16:protofilament combinations on the C1 microtubule. Tubulin and other microtubule-associated proteins are omitted from the longitudinal views for clarity. For FAP74 and FAP81, the interface involves contributions from their microtubule-binding N-termini. For FAP221, Hydin, and FAP47, the interface involves a third protein (FAP108, CPC1, and FAP219, respectively).

Fig. 5 |. Atomic models of the CA projections.

A schematic of the Chlamydomonas CA colored by projection is shown in the top left for orientation. a, Domain organization of the C1a/e projection. MOT17 at the base of the C1a projection and FAP253 at the base of radial spoke 1 (RS1; inset) both have a long vertical α-helix that contains calmodulin-bound IQ motifs. The top view (right) shows the helical connections between the distal regions of the C1a and C1e projections and PF6 and the FAP7 helical array that faces the C2a projection. b, Domain organization of the C1b projection. Each projection contains two copies of CPC1. c, Domain organization of the C1d/f projection. d, Domain organization of the C2a projection. The base of the C2a projection has a coiled-coil homodimer of PF20. The stalk contains two ASH proteins (FAP65 and FAP147, highlighted with an asterisk).

Fig. 6 |. The C1b projection contains a cluster of kinase domains.

a, Orthogonal views of the distal region of the C1b projection with a schematic of the CA cross section to aid orientation. Each C1b projection contains two adenylate kinase (ADK) domains (one each from FAP42 and CPC1), five guanylate kinase (GUK) domains from FAP42, and a homodimer of enolase. In the longitudinal view, FAP42 can be seen to interact with other copies of itself from neighboring projections. The regions boxed are shown in panels b-e. b, Model of the CPC1 ADK domain superposed with yeast adenylate kinase bound to an ATP analog (PDB: 1DVR) . Density (transparent pink, contoured at 0.1) consistent with a nucleotide is observed in the ATP binding pocket of CPC1. The AMP binding site is occupied by a loop of FAP246. c, Model of the FAP42 ADK domain superposed with yeast adenylate kinase bound to an ATP analog (PDB: 1DVR) . Density (transparent pink, contoured at 0.1) consistent with a nucleotide is observed in the ATP binding pocket. The AMP binding site is occupied by a loop from FAP42 of the adjacent C1b projection. d, Model of a FAP42 GUK domain superposed with mouse guanylate kinase in complex with ADP and GMP (PDB: 1LVG) . Density (transparent pink, contoured at 0.1) consistent with a nucleotide is present in the ADP binding site. e, Model of an enolase monomer superposed with yeast Enolase (PDB: 1ONE) . Density (transparent pink, contoured at 0.1) consistent with a small molecule is bound in the active site.