Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins

Abstract

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 42 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.

An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes

“…ENKUR is a MIP and is known to be positioned within the B tubule of the MT doublet 44 , and accordingly, our XL/MS data linked ENKUR directly to another B-tubule MIP, CFAP45, but not to other nearby MIPs, such as CFAP52 or PACRG (Figure 7A). Consistent with our interactome data, knockdown of ENKUR elicited a robust loss of CFAP45 localization in motile axonemes in Xenopus MCCs, providing the first known defect in ENKUR deficient axonemes.…”

“…We therefore explored deeper into the ENKUR/CFAP45 protein interaction neighborhood. Our XL/MS demonstrate that the association involves the N-terminus of CFAP45 and C-terminus of ENKUR, regions otherwise unresolved in the recently published structure of the native Tetrahymena doublet microtubule (DMT) 89 . Moreover, the structure raises the possibility that like other MIPs 86 , ENKUR and/or CFAP45 may penetrate the MT doublet wall.…”

Effect of alpha-tubulin acetylation on the doublet microtubule structure

“…We first examined a 48-nm repeat cryo-EM density map from native DMTs of Tetrahymena WT ( CU428 strain) (Fig. 1A) (S. Kubo et al, 2023). We identified and modeled all visible αK40 loops of α-tubulins in the map, which are known to be almost 100% acetylated (Akella et al, 2010; Gaertig et al, 1995).…”

“…We identified and modeled all visible αK40 loops of α-tubulins in the map, which are known to be almost 100% acetylated (Akella et al, 2010; Gaertig et al, 1995). Most Tetrahymena MIPs have been localized and identified in this map (S. Kubo et al, 2023). In contrast to the acetylated singlet microtubule structure (Eshun-Wilson et al, 2019), we observed many fully structured αK40 full (missing no more than two residues from residues 37 to 48) and partial structure loops (missing 3-5 residues) in DMT (Fig.…”

“…In addition, to eliminate the downstream effect of the lack of acetylation i.e. destabilization of DMT, we combined the mass spectrometry results in this study with the mass spectrometry studies of the RIB72A/B-KO and RIB72B-KO mutants (S. Kubo et al, 2023). The RIB72A/B-KO mutant lacks both RIB72A and RIB72B, which leads to a significant number of MIPs missing and slower swimming speed (Stoddard et al, 2018).…”