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.

ABD) preferentially engages actin in the presence of mechanical load across actin filaments (''mechanoaccumulation''), while vinculin's ABD does not. Simultaneous optical trapping and confocal microscopy experiments demonstrate that a load of 1pN across actin activates a-catenin ABD binding. Atomic-resolution cryo-EM structures of the metavinculin ABD-actin (2.9Å ) and a-catenin ABD-actin (3.2Å ) complexes demonstrate both ABDs undergo major conformational changes upon actin engagement, prominently at their N-and C-termini, and their C-terminal regions differentially refold to bind distinct sites on the filament surface. A C-terminal truncation of a-catenin's ABD constitutively binds actin regardless of force, and a chimeric protein of vinculin's ABD featuring a-catenin's flexible termini gains mechanoaccumulation activity, suggesting the a-catenin C-terminus-actin interaction is necessary and sufficient for mechanically regulated binding. This work, for the first time, establishes a force-regulated actin-binding mechanism in structural detail, and lays the groundwork for the rational design of therapeutics targeting cytoskeletal mechanotransduction pathways.

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.

Proteomic exploration of the central pair Cilia and flagella are highly organized structures of eukaryotes which propel the cell motions or generate liquid flows around the cells. The central pair complex is located at the center of cilia and flagella. It is known to be essential for the regulation of beating of cilia and flagella, but its protein composition and architecture were poorly understood. By exploiting comprehensive mass spectrometry and several mutant strains of Chlamydomonas, we identified novel central pair proteins and mapped these proteins to the structure. Our model can be used as a foundation to understand the functions of the central pair complex.

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 Tetrahymenathermophila native doublet microtubule and identify 38 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.