FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia

Abstract

Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat. IDA8 encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes.

Mutations in PIH proteins MOT48, TWI1 and PF13 define common and unique steps for preassembly of each, different ciliary dynein

“…A semi-quantitative estimation of the amount of dyneins in the isolated axonemes [ 28 , 50 , 51 ] was conducted using the spectral counting analyses on some LC-MS/MS spectrometers at University of Massachusetts Medical School Mass Spectrometry Facility.…”

Mutation of CFAP57, a protein required for the asymmetric targeting of a subset of inner dynein arms in Chlamydomonas, causes primary ciliary dyskinesia

“…In humans, WDR49 is expressed in the fallopian tubes and lung and FAM74A is highly expressed in the testes based on the GTEx project [75]. FBB7 is increased in our four mutant samples as well as in the dataset of Lin et al [52].…”

“…The ciliary beat frequency extracted from the trajectory was unchanged compared to wild-type cells ( Fig 5B). Using the FAP57 gene [52], we transformed fap57-050. Two independent transformants were analyzed and both showed wild-type swimming.…”

Absolute proteomic quantification reveals design principles of sperm flagellar chemosensation

“…Thus, V t = V ma + V a + V l , and the volume accessible to small molecules is V asm = V ma = + V l (Fig F and F′). We measured these volumes using cryo‐electron tomography reconstructions of rapidly frozen flagella for two evolutionary distant species, i.e., from the unicellular green algae Chlamydomonas reinhardtii and from sperm of the sea urchin Strongylocentrotus purpuratus (Carbajal‐Gonzalez et al , ; Lin & Nicastro, ; preprint: Fu et al , ; Lin et al , ) (Fig A–D).…”

“…For the measurement of flagellar diameters and axonemal protein volumes, the following previously published cryo‐electron tomography and subtomogram averaging data were used: the three‐dimensional (3D) reconstructions of intact wild‐type Chlamydomonas reinhardtii and sperm flagella from the sea urchin Strongylocentrotus purpuratus (Lin & Nicastro, ) for flagellar diameter, the 3D structures of the averaged DMT‐associated (Lin et al , ) and central pair complex repeats (preprint: Fu et al , ) of wild‐type Chlamydomonas , as well as the averaged DMT‐associated repeats (Lin & Nicastro, ), and central pair complex repeats of sea urchin sperm flagella (Carbajal‐Gonzalez et al , ).…”