Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02453.
Departments of Cell Biology and Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390.
Mol Biol Cell. 2018 Oct 15;29(21):2566-2577. doi: 10.1091/mbc.E18-06-0405. Epub 2018 Aug 22.
Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic micro-tubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical, and imaging techniques, we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins colocalize to Tetrahymena cilia and basal bodies but assemble independently. Cryoelectron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4, and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.
二联体和三联体微管是中心体、基体、纤毛和鞭毛的基本且高度稳定的核心结构。与动态细胞质微管不同，它们的内腔表面覆盖有规则排列的微管内蛋白（MIP）。然而，MIP 的蛋白组成和生物学功能仍知之甚少。我们使用遗传、生化和成像技术，鉴定出四膜虫 RIB72A 和 RIB72B 蛋白为纤毛 MIP。标记的 RIB72A 和 RIB72B 的荧光成像表明，这两种蛋白都共定位于四膜虫纤毛和基体，但独立组装。RIB72A 和/或 RIB72B 敲除株的冷冻电子断层扫描显示，纤毛 A 微管的主要结构缺陷涉及 MIP1、MIP4 和 MIP6 结构。单个突变体的缺陷在双突变体中是互补的。所有突变体的游动速度和纤毛拍打频率都降低了，高速视频成像显示在动力冲程期间，鞭毛出现异常的高度弯曲。我们的结果表明，RIB72A 和 RIB72B 对于纤毛 A 微管 MIP 的结构组装至关重要，并且对于适当的纤毛运动很重要。
