The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications
Abstract
Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of Chlamydomonas reinhardtii and Tetrahymena thermophila, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub involved tubulin post-translational modification. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.
Data availability
Cryo-EM maps have been deposited in EM data bank (EMDB) with accession numbers of EMD-20855 (48-nm averaged Chlamydomonas doublet), EMD-20858 (16-nm averaged Chlamydomonas IJ region) and EMD-20856 (16-nm averaged Tetrahymena IJ region). The model of IJ of Chlamydomonas is available in Protein Data Bank (PDB) with an accession number of PDB: 6VE7.The mass spectrometry is deposited in DataDryad (doi:10.5061/dryad.d51c59zxt). Available at:https://datadryad.org/stash/share/bkrXp5Ww0iQUis6ocuEya2ivHWQ_YiTFO-VLeIjkQcM
-
48-nm repeat unit of the doublet microtubule from Chlamydomonas reinhardtiiElectron Microscopy Data Bank, EMD-20855.
-
16-nm averaged Chlamydomonas IJ regionElectron Microscopy Data Bank, EMD-20858.
-
16-nm repeat of the doublet microtubule from Tetrahymena thermophilaElectron Microscopy Data Bank, EMD-20856.
Article and author information
Author details
Funding
Canadian Institutes of Health Research (PJT-156354)
- Khanh Huy Bui
Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-04954)
- Khanh Huy Bui
Natural Sciences and Engineering Research Council of Canada (RGPIN-2018-04813)
- Javier Vargas
Canada Institute For Advanced Research (Arzieli Global Scholar Program)
- Khanh Huy Bui
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom
Publication history
- Received: October 15, 2019
- Accepted: January 16, 2020
- Accepted Manuscript published: January 17, 2020 (version 1)
- Version of Record published: January 31, 2020 (version 2)
Copyright
© 2020, Khalifa et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,433
- Page views
-
- 509
- Downloads
-
- 49
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Structural Biology and Molecular Biophysics
Formation of membraneless organelles or biological condensates via phase separation and related processes hugely expands the cellular organelle repertoire. Biological condensates are dense and viscoelastic soft matters instead of canonical dilute solutions. To date, numerous different biological condensates have been discovered; but mechanistic understanding of biological condensates remains scarce. In this study, we developed an adaptive single molecule imaging method that allows simultaneous tracking of individual molecules and their motion trajectories in both condensed and dilute phases of various biological condensates. The method enables quantitative measurements of concentrations, phase boundary, motion behavior and speed of molecules in both condensed and dilute phases as well as the scale and speed of molecular exchanges between the two phases. Notably, molecules in the condensed phase do not undergo uniform Brownian motion, but instead constantly switch between a (class of) confined state(s) and a random diffusion-like motion state. Transient confinement is consistent with strong interactions associated with large molecular networks (i.e., percolation) in the condensed phase. In this way, molecules in biological condensates behave distinctly different from those in dilute solutions. The methods and findings described herein should be generally applicable for deciphering the molecular mechanisms underlying the assembly, dynamics and consequently functional implications of biological condensates.
-
- Structural Biology and Molecular Biophysics
Single-molecule tweezers, such as magnetic tweezers, are powerful tools for probing nm-scale structural changes in single membrane proteins under force. However, the weak molecular tethers used for the membrane protein studies have limited the observation of long-time, repetitive molecular transitions due to force-induced bond breakage. The prolonged observation of numerous transitions is critical in reliable characterizations of structural states, kinetics, and energy barrier properties. Here, we present a robust single-molecule tweezer method that uses dibenzocyclooctyne (DBCO) cycloaddition and traptavidin binding, enabling the estimation of the folding 'speed limit' of helical membrane proteins. This method is >100 times more stable than a conventional linkage system regarding the lifetime, allowing for the survival for ~12 h at 50 pN and ~1000 pulling cycle experiments. By using this method, we were able to observe numerous structural transitions of a designer single-chained transmembrane (TM) homodimer for 9 h at 12 pN, and reveal its folding pathway including the hidden dynamics of helix-coil transitions. We characterized the energy barrier heights and folding times for the transitions using a model-independent deconvolution method and the hidden Markov modeling (HMM) analysis, respectively. The Kramers rate framework yields a considerably low speed limit of 21 ms for a helical hairpin formation in lipid bilayers, compared to μs scale for soluble protein folding. This large discrepancy is likely due to the highly viscous nature of lipid membranes, retarding the helix-helix interactions. Our results offer a more valid guideline for relating the kinetics and free energies of membrane protein folding.