The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

Abstract

Microtubules are cytoskeletal structures involved in structural support, microtubule-based transport and the organization of organelles in the cells. The building blocks of the microtubule, the α- and β-tubulin heterodimers, polymerize into protofilaments, that associate laterally to form the hollow microtubule. There exists a specific type of microtubule structures in the cilia, termed doublet microtubules, where high stability is required for ciliary beating and function. The doublet microtubule, consisting of a complete A-tubule and a partial B-tubule maintains its stability through unique interactions at its outer and inner junctions, where the A- and B-tubules meet.Using cryo-electron microscopy, we present the answer to the long-standing question regarding the identities, localizations and structures of the Chlamydomonas doublet microtubule inner junction proteins. Using a combination of sequence bioinformatics and mass spectrometry, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction associated protein FAP126. We show that inner junction proteins PACRG and FAP20, together with FAP52, previously unidentified FAP276, FAP106 and FAP126, form an interaction hub at the inner junction, which involves tubulin sites for post-translational modifications. We further compare the Chlamydomonas and Tetrahymena doublet microtubule structures to understand the common and species-specific features of the inner junction.

Local computational methods to improve the interpretability and analysis of cryo-EM maps

“…We have shown in our experiments that our LocSpiral approach improves map connectivity showing fewer fragmented and broken densities and better coverage of the atomic model. In fact, our LocSpiral approach has been applied on several published publications (Ichikawa, Khalifa et al 2019, Yang, Chen et al 2019, Gutmann, Schafer et al 2020, Jahagirdar, Jha et al 2020, Khalifa, Ichikawa et al 2020, enabling molecular modelling on maps with flexibility and light anisotropic resolution.…”

Crystal structure of human PACRG in complex with MEIG1

“…The structure of the Chlamydomonas axonemal doublet by cryo-electron tomography by Bui and colleagues [33] enabled us to build a homology model of human PACRG:MEIG1 at the inner junction of an axonemal doublet microtubule ( Figure 3). The Chlamydomonas doublet structure notably reveals that PACRG and FAP20 alternate at the inner junction, with each protein interacting with two protofilaments, on the A-and B-tubules.…”

“…However, it is unclear how PACRG interacts with tubulin and how MEIG1 positions itself within the axonemal doublet. In an accompanying manuscript, the structure of the axonemal doublet microtubule from C. reinhardtii was solved by cryo-electron tomography to a resolution of 3.6 Å [33]. High-resolution electron density was observed at the inner junction, which allowed model building of PACRG, FAP20 and a number of other axonemal proteins.…”

“…The structure of the human PACRG-like protein was built with the automated I-TASSER server [61], using the structure of human PACRG as a template (this work; PDB 6NDU). A homology model of the human axonemal doublet was built by superposing the human PACRG:MEIG1 complex onto the Chlamydomonas PACRG coordinates derived from cryo-electron tomography [33]. Mammalian alpha and beta tubulin models were generated by superposing the coordinates of pig tubulin monomers (PDB 1TUB) [34] on the corresponding coordinates of Chlamydomonas tubulin subunits.…”

“…Structural model of PACRG:MEIG1 in the axonemal doublet microtubule. (A)Chlamydomonas reinhardtii axonemal doublet microtubule structure solved by cryo-electron tomography[33]. PACRG and FAP20 are colored at the inner junction between the A-and B-tubules.…”