Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA
Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Health Research Institute of Asturias (ISPA), Oviedo, Spain.
Life Sci Alliance. 2021 Dec 30;5(3). doi: 10.26508/lsa.202101225. Print 2022 Mar.
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 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 knockout strain, showing extensive structural defects beyond the FAP115-binding site. The defects propagate along the axoneme. Finally, by comparing DMT structures from and , 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.
双联微管 (DMT) 为运动纤毛的轴丝组装提供了支架。除了α/β 微管蛋白外，DMT 还包含大量位于 DMT 管腔内壁的非微管蛋白，统称为微管内蛋白 (MIP)。我们使用 cryoET 研究了从 中分离出的轴丝 DMT。我们展示了 DMT 在纳米和亚纳米分辨率下的结构。这些结构证实，MIP RIB72A/B 通过多个 DM10 结构域与 DMT 的管腔内壁结合。我们发现 FAP115 是一种含有多个 EF 手结构域的 MIP，位于 A 微管管腔中四微管二聚体的界面处。它与横向和纵向微管界面都有接触，并在 DMT 的稳定性中起着关键作用。我们在 缺失菌株中观察到 DMT 存在大量结构异质性，表明在 FAP115 结合位点之外存在广泛的结构缺陷。这些缺陷沿着轴丝传播。最后，通过比较 和 的 DMT 结构，我们鉴定出了许多保守的 MIP 以及每个生物体特有的 MIP。DMT 结构的这种保守性和多样性可能与其特定功能有关。我们的工作提供了对 MIP 在运动纤毛组装和功能中的作用以及它们与人类纤毛病的关系的理解所必需的结构见解。
