Inner lumen proteins stabilize doublet microtubules in cilia and flagella

Mikito Owa,Takayuki Uchihashi,Hiro Iguchi,Masahide Kikkawa,Toshio Ando,Takashi Yamano,Hideya Fukuzawa,Haru-Aki Yanagisawa,Ken-Ichi Wakabayashi

doi:10.1038/s41467-019-09051-x

Abstract

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.

Highlights

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes
We focused on FAP45, an uncharacterized protein whose peptides were most frequently found in the proteomic analysis (Supplementary Table 1, total unique peptide and Axo columns)
FAP45 is a coiled-coil protein composed of 501 amino acids, has a predicted molecular weight of ∼59 kDa, and is conserved among organisms with motile cilia

Summary

Results

Identification of proteins essential for DMT stabilization. To identify proteins that stabilize DMTs, we searched the Chlamydomonas flagellar proteome database[27]. For further visualization of missing structures in fap[45] and fap[52], we applied Student’s t-tests to compare wild type and mutant density maps[33] (Fig. 3e and f, right). A comparison between the wild type and fap[52] DMT revealed that an arch-like density is missing in the B-tubule of the fap[52] mutant. In the wild type DMT, there are a large arch-like density and a small spike-shaped density in a 16-nm repeat inside of the B-tubule on the protofilament B9 and B10, called MIP3a and MIP3b, respectively (Fig. 3a and d, highlighted in yellow and orange; Supplementary Fig. 5a)[21]. When DMTs are adsorbed on the mica surface, AFM images were classified into three types as shown in Supplementary Fig. 7; Class 1: The 24 nm periodicity of ODAs was visualized on the top of the DMT, suggesting that the B-tubule and the radial spokes were immobilized on the mica; Class 2: The heads of the radial spokes in 96 nm periodicity were visualized on the top of the DMT, fap[45] a 90°

B7 B8 B9

B10 IJ A13 A1

Methods