α- and β-Tubulin Lattice of the Axonemal Microtubule Doublet and Binding Proteins Revealed by Single Particle Cryo-Electron Microscopy and Tomography

Abstract

Microtubule doublet (MTD) is the main skeleton of cilia/flagella. Many proteins, such as dyneins and radial spokes, bind to MTD, and generate or regulate force. While the structure of the reconstituted microtubule has been solved at atomic resolution, nature of the axonemal MTD is still unclear. There are a few hypotheses of the lattice arrangement of its α- and β-tubulins, but it has not been described how dyneins and radial spokes bind to MTD. In this study, we analyzed the three-dimensional structure of Tetrahymena MTD at ∼19 Å resolution by single particle cryo-electron microscopy. To identify α- and β-tubulins, we combined image analysis of MTD with specific kinesin decoration. This work reveals that α- and β-tubulins form a B-lattice arrangement in the entire MTD with a seam at the outer junction. We revealed the unique way in which inner arm dyneins, radial spokes, and proteins inside MTD bind and bridge protofilaments.

Subnanometre-resolution structure of the doublet microtubule reveals new classes of microtubule-associated proteins

“…The α- and β-tubulin assignment is consistent with a cryo-electron tomography study of the doublet partially decorated with kinesins15. As previously suggested, both the A- and B-tubules have B-lattice staggers15.…”

“…As previously suggested, both the A- and B-tubules have B-lattice staggers15. The microtubule lattice seam in which α- and β-tubulin subunits interact laterally is identified between PFs-A9 and A10 (Fig.…”

“…It does not break under significant stress during rapid ciliary/flagellar beating with a frequency range of between ∼16 to 70 Hz1314. The outstanding stability of the doublet is likely linked to microtubule inner proteins (MIPs), which bind firmly to the inner wall of the doublet microtubule101115. The densities of MIPs have been shown to be mostly similar across several species711.…”

“…The axoneme and the doublet are currently resolved only to low resolution (∼20–40 Å)561115, which does not allow insights into the biophysical properties of the doublet and the roles of MIPs in stabilizing and forming periodic units on the doublet. Low resolution hinders our understanding of the molecular architecture and interactions of tubulins within the lattice and the MIPs with tubulins.…”

Flagellar-associated Protein FAP85 Is a Microtubule Inner Protein That Stabilizes Microtubules

“…Only a small number of FAPs have been characterized biochemically and/or biophysically. Recent progress in electron tomography has enabled the elucidation of minor flagellar structures such as proteins within the DMTs, known as microtubule inner proteins (MIPs) (Heuser et al, 2012;Ichikawa et al, 2017;Maheshwari et al, 2015;Nicastro et al, 2011Nicastro et al, , 2006Pigino et al, 2012;Sui and Downing, 2006). However, the composition, biochemical properties, and functions of these MIPs have not been well characterized.…”