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 that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.

Electron Cryo-Tomography Structure of Axonemal Doublet Microtubule from Tetrahymena thermophila

“…Lastly, we examined the inner junction region. Previous studies showed structural differences between Tetrahymena and Chlamydomonas in this region (Khalifa et al, 2020;Ma et al, 2019). Our modeling study confirmed this.…”

“…These differences are likely underlain by their unique molecular composition and structure (Satir et al, 2008). Previous studies have revealed marked differences in the cilia structure among different model organisms (Carvalho-Santos et al, 2011;Khalifa et al, 2020;Lin et al, 2014;Nicastro et al, 2011;Pigino et al, 2012). However, many comparisons were descriptive and were limited by the resolution of the structures.…”

“…Remarkably, the axoneme of the motile cilium/flagellum is one of the most conserved structures during the evolution of the eukaryotic branches of the phylogenetic tree of life (Bornens, 2018;Carvalho-Santos et al, 2011;Cavalier-Smith, 2002;Mitchell, 2016;Satir et al, 2008;Satir and Christensen, 2007). In addition to comparative genomic and proteomic analyses in different organisms (Avidor-Reiss et al, 2004;Fabritius et al, 2020;Li et al, 2004;Ostrowski et al, 2002;Pazour et al, 2005;Smith et al, 2005), structural comparisons of cilia among different organisms have been carried out previously, showing both evolutionary conservation and diversity of this organelle (Carbajal-González et al, 2013;Khalifa et al, 2020;Lin et al, 2014;Nicastro et al, 2011;Pigino et al, 2012). However, most studies were limited to morphological descriptions, partly due to the limited resolution in the structures.…”

“…For example, Fap53, Fap127, and Fap161 are near the seam of the A-tubule (pf A09~A10), which might provide critical structural roles. PACRG, Fap20, Fap52, and Fap106 are components of the inner junction and essential for its structural stability (Dymek et al, 2019;Khalifa et al, 2020;Ma et al, 2019;Owa et al, 2019;Yanagisawa et al, 2014). Fap67, a nucleoside diphosphate kinase that binds to the DMT via its DM10 domain, has been postulated to have a role in maintaining the local GTP concentration that is essential for axonemal homeostasis (Ma et al, 2019).…”

“…Besides α/β tubulins, it has a large number of proteins decorating the luminal wall of the A-and B-tubule, known as the microtubule inner proteins (MIPs) (Nicastro et al, 2011(Nicastro et al, , 2006Sui and Downing, 2006). Recent structure studies, in particular by cryoEM single particle analysis (SPA) and tomography (cryoET), have revealed the molecular architecture of the DMT in great details (Ichikawa et al, 2019(Ichikawa et al, , 2017Khalifa et al, 2020;Ma et al, 2019;Song et al, 2020). These results showed that the MIPs nonuniformly decorate the luminal wall of the DMT.…”