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The central apparatus plays a crucial role in regulating the motility of sperm flagella by coordinating dynein activity and waveform symmetry. A recent study published in Cell Research resolves the in situ structure of the central apparatus in mouse sperm using cryo-electron tomography, builds an atomic model with AlphaFold, and reveals the structural and functional roles of the bridge protein CFAP47, linking its disruption to novel mutations identified in asthenozoospermic patients.

Cilia and flagella are tiny, hair-like structures found on the surface of many eukaryotic cells, from single-celled protists to the cells lining the human airway and reproductive tracts. They move fluid, propel cells, or sense the environment, depending on their type and location. Most motile cilia share a common structure, nine outer doublet microtubules arranged around two central microtubules, known as the 9 + 2 axoneme. Ciliary beating comes from dynein arms, motor proteins that generate sliding forces between microtubules, causing the cilium to bend. This motion is tightly regulated by the central apparatus (CA), which consists of two singlet microtubules, C1 and C2. The CA modulates dynein arm activity and plays a key role in shaping the ciliary beat. When this system fails, it can lead to diseases known as ciliopathies, disorders caused by defective cilia structure or function.1 One such condition is asthenozoospermia, a form of male infertility marked by poor sperm motility due to defective or immotile flagella.2

Understanding the molecular basis of ciliopathies requires detailed knowledge of the structure and composition of cilia. Recent advances in single-particle cryo-electron microscopy have significantly expanded our understanding of ciliary architecture, particularly the doublet microtubules resolved across a range of organisms and cell types.3,4,5 These high-resolution reconstructions enable precise protein identification through backbone tracing and side chain density mapping. While the doublet microtubules are now well-characterized in many species, the diverse structures of the CA remain more elusive. To date, only the CA structure of the protist Chlamydomonas has been resolved at near-atomic resolution, enabling modeling of ~40 proteins.6,7 Many of the identified proteins are Chlamydomonas-specific. Furthermore, large and multi-domain proteins such as HYDIN and CFAP47 remain partially modeled. A major challenge in CA structural analysis is its inherent flexibility, both in the relative motion of the two central microtubules and in the long radial projections extending from them. Comparative structural studies by cryo-electron tomography (cryo-ET) suggest significant structural differences between the CAs of protists, mammalian motile cilia, and sperm, necessitating diversity in structural models.8 Cryo-ET offers the advantage of visualizing intact CA structures in situ without the damage caused by CA isolation, but its lower resolution limits the ability to generate pseudo-atomic models. To truly understand the diversity of CA architecture and its role in ciliary diseases, high-resolution models across multiple species and cell types are needed.

In a recent paper published in Cell Research, Zhu et al.9 reveal the in situ structure of the CA of mouse sperm using cryo-ET (Fig. 1a–c). Unlike previous studies that isolated the CA from the axoneme, the authors used cryo-focused ion beam milling to prepare thin lamellae from vitrified intact sperm, maintaining the CA in its native context. They collected nearly 2000 tomograms, an impressive dataset that enabled them to reconstruct the CA at resolutions reaching up to 6.6 Å, sufficient to visualize tertiary structural features. This approach allowed them to resolve the bridge, a structure that had previously been too flexible to be observed at high resolution.

Fig. 1: In situ structure of the mouse sperm CA reveals the function of CFAP47.

a Normal sperm display regular motility. b A cartoon of the 9 + 2 cross-section of the sperm flagella. The dashed square indicates the CA. c Depiction of the CA structure indicates that CFAP47 is an important part of the bridge linking the two microtubules. d Knockout of CFAP47 leads to unstable CA. e Defects in CFAP47 cause asthenozoospermia.

To interpret the density maps, the authors used an integrative visual proteomics approach, combining mass spectrometry data, structural information from Chlamydomonas, and DomainFit, a tool that fits AlphaFold-predicted models into their maps.10 In total, they identified 39 CA-associated proteins, including eight previously uncharacterized ones. Notably, they resolved structures of HYDIN and CFAP47, establishing them as the two main components of the bridge. Both proteins are composed of repetitive ASH domains: HYDIN, the longest CA protein, contains 33 ASH domains and over 5000 residues, while CFAP47 contains 20 ASH domains. HYDIN forms a semi-circular ring that partially encircles the C2 microtubule, with its N-terminus spanning the bridge. CFAP47 binds both HYDIN and the C1 microtubule seam, extending toward C2 microtubule to anchor the two microtubules.

To validate CFAP47’s role, the authors generated a Cfap47-knockout (KO) mouse. Cryo-ET showed that Cfap47-KO sperm lack the bridge structure, confirming CFAP47’s function (Fig. 1d, e). Functionally, Cfap47-KO sperm had an asthenozoospermia phenotype with normal morphology, showing greatly reduced motility, highlighting the importance of the bridge for ciliary beating. Whole-exome sequencing of 320 infertile male patients revealed two individuals with CFAP47 mutations, providing a clinical link to the CFAP47 structure. The structural data provided a molecular explanation for their infertility.

A surprising additional finding was the binding of SPACA9 in the lumen of both C1 and C2 microtubules. Previously identified as a microtubule inner protein in the B-tubules of doublets and in singlets, where it shows an 8-nm repeat, SPACA9 displayed different periodicities in the CA: 32 nm in C1 and 8 nm in C2, posing interesting questions regarding its assembly.

This comprehensive structural and mechanistic insight provides a fundamental framework for understanding CA-related fertility disorders and other human ciliopathies, while also showcasing an advanced integrated research methodology combining clinical data, cryo-ET, and animal models for elucidating molecular pathogenesis in a native state. This paves the way to the possibility of using in situ structural biology for the diagnosis of human patients in the future.