Abstract
Doublet microtubules (DMTs) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMTs, collectively named the microtubule inner proteins (MIPs). We used cryoET to study axoneme DMT isolated from Tetrahymena We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP RIB72A/B binds to the luminal wall of DMT by multiple DM10 domains. We found FAP115, an MIP-containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of A-tubule. It contacts both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. We observed substantial structure heterogeneity in DMT in an FAP115 knockout strain, showing extensive structural defects beyond the FAP115-binding site. The defects propagate along the axoneme. Finally, by comparing DMT structures from Tetrahymena and Chlamydomonas, we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.
Highlights
The motile cilium is an evolutionarily conserved cellular organelle that has a wide range of biological functions
Many non-tubulin proteins on the luminal wall of Doublet microtubules (DMTs) can be discerned based on their overall shapes
These include RIB72 and FAP115 in the lumen of A-tubule, FAP20, and PACRG forming a filament at the inner junction, and FAP52, whose two WD40 domains each with characteristic seven β-propeller repeats can be resolved (Fig 1C)
Summary
The motile cilium is an evolutionarily conserved cellular organelle that has a wide range of biological functions. In protozoa, such as ciliates and green algae, motile cilia, known as flagella, are essential for the cell’s locomotion and feeding (Bornens, 2018). Motile cilia are found in the oviduct of female reproduction organs, in the male sperm and at the node of the gastrulation site during embryo development. Defective motile cilia cause a spectrum of human diseases, such as primary cilia dyskinesia, situs inversus, and infertility (Reiter & Leroux, 2017)
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