Tubulin lattice in cilia is in a stressed form regulated by microtubule inner proteins

Abstract

Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around are composed of radially bundled doublet microtubules. In this study, we present a near-atomic resolution map of the Tetrahymena doublet microtubule by cryoelectron microscopy. The map demonstrates that the network of microtubule inner proteins weaves into the tubulin lattice and forms an inner sheath. From mass spectrometry data and de novo modeling, we identified Rib43a proteins as the filamentous microtubule inner proteins in the protofilament ribbon region. The Rib43a–tubulin interaction leads to an elongated tubulin dimer distance every 2 dimers. In addition, the tubulin lattice structure with missing microtubule inner proteins (MIPs) by sarkosyl treatment shows significant longitudinal compaction and lateral angle change between protofilaments. These results are evidence that the MIPs directly affect and stabilize the tubulin lattice. It suggests that the doublet microtubule is an intrinsically stressed filament and that this stress could be manipulated in the regulation of ciliary waveforms.

Structural insights into the architecture and assembly of eukaryotic flagella

“…FAP129 interacts with FAP127 on the inside and with ODA-DC on the exterior side, which may provide a molecular rationale for why FAP127 mutants show defects in ODA-DC assembly [ 67 , 68 ]. The structure of DMTs presented by Ma et al thus demonstrates that the distinction between inner and outer DMT-binding proteins is not so clear-cut and that MIPs may indeed protrude through ‘windows' between tubulin subunits to affect the exterior of DMTs [ 69 ].…”

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 nullTetrahymenacells reveals functional MIPs

“…Some MIPs have been shown to reach across the tubulin lattice and it is possible that these projections into the B-tubule could serve as a platform for B-tubule specific MIP localization (Ichikawa et al, 2017) (Ma et al, 2019). A-tubule MIPs also make connections with dynein arms and radial spoke proteins (Ichikawa et al, 2019;Khalifa et al, 2020). The propagation effect of lost connections from the inside of the axoneme to the accessory structures that are necessary for beating and ciliary waveform can explain the ciliary beating defects associated with MIP loss.…”

“…Rib72A/B are still present in the FAP115 KO, but Fap115, along with most other A-tubule MIPs, is absent in RIB72A/B KOs. There is evidence that local curvature of the doublet microtubules is affected by MIP structures (Ichikawa et al, 2017(Ichikawa et al, , 2019Khalifa et al, 2020), but specifically how the curvature is created and how it may affect recruitment and binding of additional MIPs is yet to be understood. Structural specificity of microtubules and direct binding to tubulin or other MIPs are likely mechanisms for early assembly of the MIP architectures.…”

“…While some studies have provided clues to the functional significance, e.g. microtubule lattice spacing and angles and intolerance to mechanical stress (Ichikawa et al, 2017(Ichikawa et al, , 2019Owa et al, 2019), there is still a need for additional information about the roles of individual MIPs and MIPs as a group. In addition, some MIP proteins have yet to be identified and characterized, and MIPs are likely to vary among species and systems.…”

Intraflagellar transport trains and motors: Insights from structure

“…For simplicity, in this review, we use human subunit names, except for the IFT proteins, where we use the well-established Chlamydomonas reinhardtii nomenclature (even when discussing orthologues in other species) [ 17 , 18 ]. Exciting progress has also been made in understanding the structures of the cargo-binding BBSome complex and the microtubule doublet, for which the reader is referred to recent articles [ [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] ].…”