TY - JOUR
T1 - Tetrahymena RIB72A and RIB72B are microtubule inner proteins in the ciliary doublet microtubules
AU - Stoddard, Daniel
AU - Zhao, Ying
AU - Bayless, Brian A.
AU - Gui, Long
AU - Louka, Panagiota
AU - Dave, Drashti
AU - Suryawanshi, Swati
AU - Tomasi, Raphaël F.X.
AU - Dupuis-Williams, Pascale
AU - Baroud, Charles N.
AU - Gaertig, Jacek
AU - Winey, Mark
AU - Nicastro, Daniela
N1 - Funding Information:
For cryo-ET, frozen grids were loaded into a cryo-holder (Gatan, Eindhoven, Netherlands) and inserted into a Tecnai F30 transmission electron microscope (FEI, Hillsboro, OR) equipped with a field We thank Chen Xu for training and manage-ment of the electron microscopes in the Louise Mashal Gabbay Cellular Visualization Facility at Brandeis University (Waltham, MA). This work was supported by funding from the National Institutes of Health (GM083122 to D.N., GM074746 and GM127571 to M.W., and GM089912 to J.G.) and the National Science Foundation (MBC-033965 to J.G.). P.L. was supported by a predoctoral fellowship from the American Heart Association. The three-dimensional averaged structures have been deposited in the Electron Microscopy Data Bank (EMDB) under accession code EMD-7806, EMD-7811, EMD-7805, and EMD-7807.
Publisher Copyright:
© 2018 Stoddard et al. This article is distributed by The American Society for Cell Biology under license from the author(s).
PY - 2018/10/15
Y1 - 2018/10/15
N2 - Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic microtubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly under-stood. 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.
AB - Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic microtubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly under-stood. 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.
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U2 - 10.1091/mbc.E18-06-0405
DO - 10.1091/mbc.E18-06-0405
M3 - Article
C2 - 30133348
AN - SCOPUS:85054894785
SN - 1059-1524
VL - 29
SP - 2566
EP - 2577
JO - Molecular Biology of the Cell
JF - Molecular Biology of the Cell
IS - 21
ER -