Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas . These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes.Although it is known that microtubules in cilia are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dynein and intraflagellar transport. To address this question, we identified a new class of microtubule-associated proteins, named FAP45 and FAP52, in Chlamydomonas.These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we established that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These inner protrusions are located near the inner junctions of doublet microtubules and lack of FAP45, FAP52, and FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrated that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly.

Outer arm dynein (OAD) in cilia and flagella is bound to the outer doublet microtubules every 24 nm. Periodic binding of OADs at specific sites is important for efficient cilia/flagella beating; however, the molecular mechanism that specifies OAD arrangement remains elusive. Studies using the green alga Chlamydomonas reinhardtii have shown that the OAD-docking complex (ODA-DC), a heterotrimeric complex present at the OAD base, functions as the OAD docking site on the doublet. We find that the ODA-DC has an ellipsoidal shape ∼24 nm in length. In mutant axonemes that lack OAD but retain the ODA-DC, ODA-DC molecules are aligned in an end-to-end manner along the outer doublets. When flagella of a mutant lacking ODA-DCs are supplied with ODA-DCs upon gamete fusion, ODA-DC molecules first bind to the mutant axonemes in the proximal region, and the occupied region gradually extends toward the tip, followed by binding of OADs. This and other results indicate that a cooperative association of the ODA-DC underlies its function as the OAD-docking site and is the determinant of the 24-nm periodicity.C ilia and flagella of eukaryotic cells are organelles that generate fluid flow on the cell surface and/or sense chemical or mechanical stimuli from the external environment (1). Cilia/flagella beating is driven by outer arm dynein (OAD) and inner arm dyneins. The arrangement of dyneins on the axoneme has an overall periodicity of 96 nm, within which OAD binds every 24 nm; this 24-nm periodicity is completely conserved in essentially all eukaryotic organisms with "9 + 2" axonemes (2, 3) and even occurs in insect sperm flagella containing multiple rows of doublet microtubules arranged in a spiral configuration (4, 5). OAD is best characterized in Chlamydomonas. It is a very large protein complex of ∼2 MDa, comprising 3 heavy chains, 2 intermediate chains, and 11 distinct light chains. Most of the subunits are conserved from protists to mammals (6). OAD is the most abundant and most powerful axonemal dynein, generating about twothirds of the total propulsive force of ciliary beating (7,8). Human diseases due to ciliary motility defects [termed primary ciliary dyskinesia (PCD)] are caused most commonly by defects in OAD assembly (9-11). The assembly process and the in situ structure of the OAD complex in the axoneme have been well studied (3, 12, 13). However, the mechanism underlying the periodic binding of OAD to the doublet is poorly understood.The outer dynein arm-docking complex (ODA-DC) has been identified as a complex that mediates OAD binding to the doublet (14, 15). In the flagella of Chlamydomonas mutants (e.g., outerdynein-arm deficient oda6) retaining the ODA-DC but not OAD, the ODA-DC is observed by electron microscopy as a small projection linearly arrayed every 24 nm along the outer doublet (3,(14)(15)(16)(17). It is composed of three subunits: DC1, ∼83 kDa, encoded by ODA3; DC2, ∼62 kDa, encoded by ODA1; and DC3, ∼21 kDa, encoded by ODA14 (18-20), which assemble in the cell cytoplasm and are transported into the fla...

Outer arm dynein (OAD) is bound to specific loci on outer-doublet-microtubules by interactions at two sites: via intermediate chain 1 (IC1) and the outer dynein arm docking complex (ODA-DC). Studies using Chlamydomonas mutants have suggested that the individual sites have rather weak affinities for microtubules, and therefore strong OAD attachment to microtubules is achieved by their cooperation. To test this idea, we examined interactions between IC1, IC2 (another intermediate chain) and ODA-DC using recombinant proteins. Recombinant IC1 and IC2 were found to form a 1:1 complex, and this complex associated with ODA-DC in vitro. Binding of IC1 to mutant axonemes revealed that there are specific binding sites for IC1. From these data, we propose a novel model of OAD-outer doublet association.